The Johns Hopkins Medical Journal 33 (1922)

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The Johns Hopkins Medical Journal - Volume 33 (1922)

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The Johns Hopkins Medical Journal 33 (1922)

The Johns Hopkins Hospital Bulletin


Vol XXXIII — No. 371

BALTIMORE, JANUARY, 1922

Contents - January

  • Secondaiy Aiunuia of Infants. A Study of So-Called Infantile Splenic Anemia or Anemia Infantum PseiidoleuKemica. By Frank A. Evaxs and William M. Happ
  • Pregnancy and Labor in Young Primipar*. (Illustrated.) Bv .JoHx W. Harris
  • The Protective Power of Serum in Pernicious Anemia and Other Conditions Against Hemolysis by Saponin and by Sodium Oleate. By Ehka H. ZiXLK, Hallik M. Clark and Frank A. Evans
  • The Establishment of Collateral Circulation Following Ligation of the Thoracic Duct. (Illustrated.) By Fereinand C. Lee . . . .
  • Studies on Experimental Rickets. XV. The Effect of Starvation on the Healing of Rickets. (Illustrated.) By E. V. McCoLLUM, Nina Sim.monds, P. G. Shipley and E. A. Park


PREGNANCY AND LABOR IN YOUNG PRIMIPAR

By John W. Harris {From the Department oj Obstetrics, The Johns Hopkins Hospital and University)


In spite of the fact that most text-books of obstetrics contain little infonuation upon the subject, there is a prevalent opinion that pregnancy and labor are attended by greater danger in young girls than in older women. Williams ' differs from this view and states that labor in the girl of 16 or less is no more serious than in the women of more mature years. In order to test the correctness of this view I have collected and analyzed all labors in girls of 16 years of age or less which have occurred in the obstetrical service of The Johns Hopkins Hospital.

Vamier ' has compared the average duration of labor in 100 primiparce of less than 20 years of age with that in a similar number of patients between the ages of 20 and 30.


He found the average duration was 13 hours, 5 minutes in the former as compared with 13 hours, 28 minutes in the latter. In all of the patients in Ixith groups the pelvis was normal and the child presented by the vertex.

Gache,' of Buenos Aires, analyzed 91 cases of labor occurring in girls between the .ages of 13 and 16. In 84 patients the pelvis was normal. Of the 91 patients, 78 were delivered spontaneously and 13 by operative measures. He estimated that the average duration of labor was 24 hours and that the children averaged 3039 grams in weight, and concluded that, so far as he could ascertain, the age of the patient is practically a matter of indifference.


Bondj' ' has reported 69 labors between the ages of 14 and 16, of wliich 12 resulted in the birth of premature children. In the 57 patients delivered at term he believed that labor was prolonged in 19 instances.

From the first 10,000 admissions to the obstetrical service of The Johns Hopkins Hospital I have collected the details of 500 labors occurring in girls between the ages of 13 and 16 inclusive. Since most of the abortions are admitted to the gynecological service, the few that appear in our records have not been considered, as they would give no accurate picture of the frequency of this complication.

Table 1 presents an analysis of the age at which delivery took place in each of the two races, and shows that there are more than twice as many blacks as whites in the series. As less than 45 per cent of all patients admitted to the service are blacks, this unusual predominance of colored young priniiparffi may be regarded as an index of the incidence of precocious sexual relations in that race.

TABLE 1


Age


White


Black


Total



12



2


2



13


1


6


7



14


IS


28


46



1^


45


95


140



16


96


209


305


Total .


160


340


500


Pelvis. — In every patient in the series the pelvis was measured both externally and internally and the results are shown in Tables 2 and 3.

In the 160 white patients contracted pelves were noted in 21, a percentage of 13.12. The generally contracted and the funnel were the most frequent types noted, making up 16 of the 21 cases.

Williams," in a series of 1313 wloite women, irrespective of age, found that the incidence of contracted pelvis was 13.33 per cent, and stated that the funnel and the generally contracted were the two types most frequently encountered, the two making up 125 of his 175 cases. On comparing our figures with his, it is seen that the incidence of contracted pelvis is practically the same in both series, and that the types of pelvic contraction most frequently noted were identical. In other words, the white girl of 13 to 16 years of age has as large a pelvis as her older sister.

In the 340 blacks in our series the incidence of pelvic contraction was 60.58 per cent. The two most frequent types encountered were the generally contracted and the generally contracted rhachitic, the two making up 140 of our 206 cases. Williams' found 312 contracted pelves in 902 colored women of all ages, a percentage of 40.93. Of these the generally contracted and the generally contracted rhacliitic made up 258 of his 312 cases. Thus it is seen that in colored young


primiparas contractions of the pelvis occur 50 per cent times more frequently than in colored women of more mature years. This is to be expected when it is remembered that casual observation seems to indicate that the black woman in Baltimore reaches physical maturity later than does the white. Our figures are in accord with those of Williams that the two types of contracted pelves most frequently noted in the black are the generally contracted and the generally contracted rhachitic.

Abnormal Pregnancy. — Table 4 shows the complications of pregnancy met with in the series of 500 cases.

TABLE 2

Classification of Pelves in 160 White Young Peimipaii«;



Normal pelves


Contracted pelves



Age


Generally contracted


Simple flat


Funnel


Gen. cont. funnel


Total cont. pelves


Total


13 14 15 16


1 14 37

87


1 5 3





2 2 3


1 1

1



4 S 9


1 18 45 96


Total.


139


9


2


7


3


21


160


TABLE 3 Classification of Pelves in 340 Black Yotjnq Fbimifaba^




Contracted pelves




TS

Age


Normal pelves



tc



1



§1


s


is

^ >


Total





s


gti


5'-s


c


S3



go,





c


m


a


b


b.


a



H



12



2


2


2


13



3



3








6


6


14


8


11



4


°


2


3



20


28


15


41


16



16


1


9


12



54


95


16


85


51


1


34


1


12


24


1


124


209


Total.


134


83


1


57


2


23


39


1


206


340


TABLE 4 Abnormal Pregnancies



Zj


a


« 



5






es


"S


•-* 6



^







e




z


« 



H


s


H


White


2


9


1


1






13


Black


8


7



25


1


1


1


43


Total


10


16


1


26


1


1


1


56


At first glance it appears that there was an abnormally high incidence of preeclamptic toxaemia and eclampsia, 10


14


[Xo. 371


of the former and 16 of the latter. However, five of the eases of the former and 14 of the latter v^ere not treated in the prenatal clinic but were referred to the service because of the existence of the disease. Consequently, no such incidence of these complications should be expected to obtain in the average run of young primipariB.

It is noted that there were 26 cases of syphilis in the series, 25 being in black women and 1 in white. This is a much lower incidence of the diseai?e than that reported from this clinic by Williams.' The discrepancy can be explained not only on the grounds that in the young girl syphilis would not be expected to be present so frequently, but also because of the fact that more than half of the patients in the series passed through our hands before the introduction of the routine Wassermann determination and, therefore, it may be assumed that many cases of infection escaped detection.

Premature Labor. — Of the entire series of 500 cases 58, or 11.6 per cent, terminated in premature labor. When divided according to race, it is seen that this termination occurred in 17 of the 160 whites and in 41 of the 340 blacks, an incidence of 10.52 and 12.05 per cent, respectively.

In the majority of cases in both races the cause of premature termination could not be determined. In the blacks syphilis was the most important ascertainable etiological factor, and it seems safe to surmise that, had the more recent methods of diagnosis been applied throughout the series, many of the cases classified as undetermined would have shown that syphilis was the causative factor.

TABLE 5 Prematuke Labors





m


M fi



1




1


s



H


White .


11


1


2



2


1





17


Black..


24


11


1


2


1



1


1


41


Total . .


35


12


3


2


3


1


1


1


58


Duration of Labor. — Of the 442 patients delivered at term the duration of labor was accurately recorded in 430 — 138 whites and 292 blacks. The 12 remaining patients were delivered by Coesarean section or accoueliement force before the cervix had become fully dilated.

Tables 6 and 7 show the average duration of term labor in the two races. From Table 6 it is seen that the average duration of labor for the entire series of 138 white girls is 15 hours, 44 minutes. However, when the pelvis is normal the average falls to 15 hours, 10 minutes ; on the other hand, when the pelvis is contracted, labor is 4 hours, 43 minutes, longer than when it is normal.

The average duration of labor in the 292 blacks is 16 hours, 40 minutes. This increase may be assumed to be due to the greater frequency of contracted pelves in that race; as, when the pelvis is normal, the average duration is 14 hours, 40


minutes, as compared with an average of 18 hours, 7 minutes, when the pelvis is contracted.

It is usually stated that the average duration of labor in primiparffi, irrespective of age, is 18 hours. G. Veit ' places it at 20 hours. Accepting the lower figure as correct, it is seen that the young primipara has a labor shorter by 2 hours, 16 minutes in the white and 1 hour, 20 minutes in the black race.

TABLE 6 Average Duration of Labor — White


Age


Total


Normal pelvis


Contracted pelvis


No. cases


Average duration


No. eases


Average duration


^•— jd^atr,


13 14 15 16


1 15 40 82


lOh. 12m. 20h. 02m. 15h. 1.5m. 15h. 16m.


1

11 33 76 .


lOh. 12m. 18h. 48m. 15h. 27m. 14h. 35m.


4 231i. 24m. 7 |14h. 15m. 6 24h. 06m.


Total


1.38


15h. 44m.


121


1.5h. lOra.


17 19h. 52m.


TABLE 7 A^•ERAGE Duration op Labor — Black



Total


Normal pelvis


Contracted pelvis



No. cases


Average duration


No. cases


Average duration


NO- cases 1 ,^J-^f„


12 13 14 15 16


2

5

25

82 178


18h. 03m. 17h. 20m. 191i. 44m. 16h. 48m. lOh. 09m.




8

39



181i. 50m. 131i. 53ra. 14h. 43m.


5 17

43 103


18h. 03m. ITli. 20m. 20h. 10m. 19h. 27m. 17h. 15m.


Total


292


16h. 40m.


122 141). 40ra.


170 ISh. 07m.


ilorc precise information as to the duration of labor may be gained by the " modal " method. This is shown in Tables 8 and 9. The shaded blocks represent the total number of term labors divided into groups of two hours each, while the lines represent the labors occurring in patients with normal pelves. Inspection of these tables shows that in both races the most frequent duration of labor in the entire series, as well as in those with normal pelves, is between 10 and 12 hours. Unfortunately, no comparable statistics concerning the duration of labor in older women are available, but it is not believed that they would show a shorter duration tlian that observed in young primipane.

It is interesting to note that, in spite of the much higher incidence of contracted pelvis in the blacks, the most frequent duration of labor is the same as for the whites. The explanation is, as will be shown later, that the babies of the former are smaller, and, tlierefore, are less likely to be disproportionate to the size of the pelvis.



Delivery. — Of the 448 deliveries at term, 379 were spontaneous and 63 were operative, an incidence of 85.74 and 14.36 per cent respectively. Of the latter 18 were in whites and 45 in blacks. Notwithstanding the apparently greater frequency of operative interference in the blacks, the determination of the percentage frequency shows that the incidence is approximately the same in both races, being 12.95 per cent for the whites and 14.85 per cent for the blacks.

TABLE 8


YOUnS WHITE PRIMIPARAE



rJI...


cviAi , i;^ 1 1 1 n


£2 ^6 30 3a 3d 4a 46 M 34 56 6e 66 70

Houes


TABLE 9


voty/vs Bu\a< pmiPAgAC



u_n

34 J8 -JZ 46 50 54 56 6^ 66 10 Ti IS di Sf HOURS


Opemfions. — The types of operations are shown in Table 10. While the incidence of pelvic contraction was 60.58 per cent in the blacks, only 17 of the 45 operations were done because of pelvic dystocia. Of these, 10 were Cesarean sections, 4 pubiotomies and 3 craniotomies. In the whites only two operations were necessitated by the pelvis, one Caesarean section and one high forceps.

Weight of the Child. — In casting about for an explanation for the relatively short labor in very young primipars it seemed possible that it might be afforded by the smaller size of the children. Accordingly the material was studied from


that point of view. Tables 11 and 13 show the average weight of the children in both races. In the series of 143 whites delivered at term the average weight of the children was 3181 grams. When classified according to the pelvis of the mother, the average in the 126 patients with normal pelves was 3200 grams, while in the 17 with contracted pelves it was 3045 grams, a difference of 155 grams in favor of the child bom of a motlier with a normal pelvis. The average weight of

TABLE 10 Operations •



S



o.


i.


•og




1




"5




^


3


.a


•^ o



« c


1


J3


3-I

-art


3




s


n


>


o


&<


o



1




S


^




3 rt


ii'O


S


^



H


>>


6


•<


Q ®


S"


£


o


White


3


2


i


2



1


1


13


Black


3


4


4


1


4


1


1


18


Total


6


6


S


3


4


2


2


31


on the third day after the the spontaneous delivery of a term child. The other three patients were delivered of premature children, one by manual dilatation of the cervix and version and extraction because of prolonged labor and intrapartum infection. She died suddenly two hours later, with a clinical diagnosis of hemorrhage and shock, but the findings at autopsy were negative. Of the other patients one died on the fifth day of the puerperium from streptococcus peritonitis and the other on the tenth day from pyelonephritis which had existed for weeks before delivery.


Fcetal Mortality. — Of the 442 deliveries at term, 31 children were stillborn or died within the first two week.s of life, a percentage of 7.0. The causes of death are shown in Table 13.

Conclusions

Based upon the study of the 500 patients comprised in this report, it seems permissible to conclude that pregnancy and labor are attended by no gi-eater danger in the young primipara than in the older woman. On the other hand, the duration of labor is actually shorter. As our figures show that the size of the children is not inferior to that noted in older women, and that abnormal pelves occur quite as frequently, this result must be attributed to the greater elasticity of the soft parts. Consequently, speaking from a purely obstetrical point of view, the ages under consideration appear to be the optimum time for the occun-ence of the first labor.

BIBLIOGRAPHY

1. Williams, J. Whitridge: Text-book of Obstetrics. D. Appleton and Co., 1917, 250.

2. Varnier : Combien de temps dure I'accouchement. L'Obstetrique journaliere, 1900, 174-181.

3. Gache : La grossesse et raccouchement chez les primipares de 13, 14, 15, et 16 ans. Annales de gyn. et d'obst., 1904, n. s., 1, 723-736.

4. Bendy: Die Geburt in den Entwicklungsjahren. Ztschr. f. Geb. u. Gyn., 1911, LXIX, 213-246.

5. Williams, J. Whitridge: The Funnel Pelvis. Am. Journ. Obst., 1911, LXIV, 106-124.

6. Idem: The Significance of Syphilis in Prenatal Care and in the Causation of Foetal Death. Johns Hopkins Hosp. Bulletin, 1920, XXXI, No. 351, 141-145.

7. Veit: Beitrage zur geburtshiilflichen Statistik. Monatsschr. f. Geburtsh., 1S54, V, 344-381 ; 1855, VI, 101-132.

8. Riggs: A Comparative Study of White and Negro Pelves, etc. Johns Hopkins Hopsital Reports, 1904, XII, 421-454.




THE ESTABLISHMENT OF COLLATERAL'CIRCULATION FOLLOWING LIGATION OF THE THORACIC DUCT

By Ferdinand C. Lee (From the Anatomical Laboratory oj The Johns Hopkins University)


The general physiology of the lymphatic system has been studied from various angles. The discovery of the lymphatic system by Asellius^ in 1627 was due to an observation correlating digestion with the activity of lymphatic vessels. Clinical pathological findings stimulated individuals to wound the thoracic duct in the chest and study the subsequent phenomena (Lower'); while others (Flandrin," Colin *) ligated the thoracic duct in thetneck and observed the influence of this procedure on the animal economy. Later when the knowledge of chemistry became advanced, thoracic duct fistulse were made with a view toward studying the fluid in the duct under ordinary conditions and as modified by the digestion of various foods, e. g., fats. Again, through the work of Heidenhain' and Starling," the effects of lymphagogues of various classes were investigated. Finally, the thoracic duct was studied in regard to its power of supplying blood cells, chiefly small h-mphocytes, to the blood stream (Biedl and v. Decastello ') .

In this report an attempt has been made to review the literature of the experimental ligation of the thoracic duct, and also to give a description of a new method for ligating that vessel.

Review of Literature

Probably the first one to attempt the ligation of the thoracic duct was Duverney' in 1675. He ligated the subclavian vein near the entrance of the duct, in some cases applying the ligature proximally and in others distally to the entrance. The dogs lived for a fortnight.

Sixteen years later in 1691, Flandrin,' a well-known veterinarian of his time, chose to repeat the work of Duverney, but preferred the horse, since he considered the lymph vessels in the smaller animals as too inconstant in their location. In the first experiment the animal died at the end of three days ; subsequent dissection showed nothing of particular interest. In the second case a young horse was used. It experienced no ill effects immediately following the operation, nor in the subsequent 15 days during which it was allowed to live. " Perfect suppuration" took place in the wound, and with the possible exception of a distension of the thoracic duct at its point of ligation, the autopsy revealed no marked difference


in the appearance of the lymphatics. Similar results were obtained with 10 more horses; the majority of the animals were killed 15 days following ligation, and the only abnormal finding was a distension of the thoracic duct at its point of ligation, with occasional extension of this distension down into the chest. One animal was kept for two and a half months after operation, and at autopsy showed a cicatrized thoracic duct. It is also interesting to note that Flandrin was among the first to introduce a cannida into the thoracic duct for the purpose of studying the properties of the lymph. He concluded that the lymph reaches the blood-stream through other channels than the thoracic duct.

In 1798 Sir Astley Cooper" reported the results of some experimental work involving the ligation of the thoracic duct in dogs. He was stimulated to undertake the experiments because he had observed three cases of obstruction to the thoracic duct in man. In two of these cases he found evidence of collateral circulation ; in the third case no such evidence was found. In the first experiment the dog died 48 hours after the duct had been tied; dissection showed the viscera obscured by an effusion of chyle; particularly was tliis the case with the pancreas and the kidney; a small quantity of chyle had extravasated into the peritoneal cavity; the lacteals were empty; the thoracic duct was twice its normal size. These findings Cooper explained on the basis of a ruptured cisterna chyli. The second experiment was a great deal like the first, only the dog lived 3 days longer. The autopsy findings were the same; the cisterna chyli had burst and the extravasation had hidden entirely the aorta and the vena cava. In a third experiment the thoracic duct was cut and allowed to drain into the tissues; the animal died on the fourth day; dissection showed a pouch of lymph in the wound. These experiments were repeated always with the same results; no animal survived the tenth day. In one case Cooper noticed that a branch of the thoracic duct went over to the right trunk; also that the amount of extravasation bore a direct relation to the amount of food which the animal had just previously taken. Cooper did not give any evidence, e. g., by injections, to support his belief that the cisterna had ruptured.


22


[No. 371


The only other investigator besides Cooper to report rupture of the cisterna chyli following ligation was Mayer." Contrariwise, Eogers " observed no ill effects from ligation.

In 1821, Magendie " reported the exp€rinients which Dupuytren made on several horses. Dupuytren ligated the thoracic duct in the neck, and obsei^ved that some animals died at the end of five to six days ; while others did not seem to be affected by the operation. Subsequent injections showed that in the animals that died there was a complete obstruction of the thoracic duet; whereas, in those that survived — and one case was allowed to go for six weeks — a collateral circulation was always demonstrable. Accordingly, it was argued that the death of those animals that lived only five to six days, and in which the duct was tied, was due to the deprivation of nourishment furnished via the thoracic duct; and the only reason that the other animals lived longer was because collaterals to the thoracic duct had formed and had thus transmitted the necessary food substances to the blood stream.

Leuret and Lassaigne " tied the thoracic duct in the neck of a dog and found that the animal lived perfectly well, even gained weight. At the end of 58 days it was sacrificed at the height of digestion; dissection showed that the canal had been satisfactorily ligated. The only findings of importance were that the duct, the cisterna and the mesenteric lymphatics contained a small amount of chyle; and that the mesenteric veins seemed larger than normal. They believed that the chyle took an abnormal course to reach the blood, and tliat tliis path was from the cisterna chyli to the portal vein. They proved this assertion to their own satisfaction when they ligated the portal vein and found blood in the cisterna chyli and at the conunencement of the thoracic duct. They also found that when the blood and chyle were taken from the thoracic duct and allowed to stand, two distinct clots were formed : the one, red, remained below ; while the other, pink, evidently chylous, remained above.

Colin ^ in 1873 reviewed briefly and analyzed to some extent the work of the above mentioned investigators and then related liis own experiences with ligations of the thoracic duct in various animals. All ligations seem to have been done in the neck. His first experiment was on a bull. The animal made an uneventful recovery and was sacrificed at the end of six or seven weeks when it was found that the thoracic duct was obliterated to the extent of five or six centimeters below the point of ligation, but that collaterals had formed at this point and had extended to the regular entrance of the thoracic duct into the veins. He repeated these experiments on two cows, taking care to include in his ligations all branches of the thoracic duct, and found at the end of 24 hours that there was a marked distension of the lymphatic system, as well as an extensive extravasation into the mesentery. He believed that the animals would have died from the operation if they had been kept long enough. On a bull 18 months old ligation resulted in death at the end of nine days ; lymph did not reach the veins. After the completion of these oljservations on these large animals he made use of dogs for further experiments. One dog died from chylothorax on the dav


following the operation. Two other dogs died at the end of 20 and 25 days respectively, having refused food for almost the entire time. In both cases the thoracic duct was thickened and obliterated at its upper end and injections did not reach the veins. These animals " died the death of starvation." Colin concluded that ligation of the thoracic duct was not always followed by the same results. In those cases in which the duct was double, or anastomosed with the right thoracic duct, lymph reached the blood stream, and the animal recovered ; whereas, if the duct were single and when ligation had been complete, death resulted promptly. He called the attention of future investigators to the fact that the entrance of the thoracic duct into the veins is so variable that in some cases it is absolutely impossible to prevent the lymph and chyle from reaching the blood stream.

However, Schmidt-Miihlheim " devised an operation which seemed to take care of all possible variations of the entrance of the thoracic duct into the veins. The problem which Ludwig gave Schmidt-iliihlheim was to establish whether the digestion products of albumen reached the blood stream via the thoracic duct or not. From this it can be readily seen what an exaggerated importance physiologists up to that time attached to the thoracic duct. He used dogs for his experiments and operated without an atesthetic or curare. He must be credited, however, with using strict aseptic technique during the operation, and, accordingly, ranks among the first to observe aseptic measures on experimental animals in the solution of biochemical problems. The operation consisted in isolating and tying off successively, on the right side as well as on the left, the v. jugularis externa, v. jugularis interna, v. axillaris and v. anonyma. Also the two thoracic ducts, right and left, were cut between ligatures. The operation did not affect the general condition of the animals in any particular; but since they were sacrificed at the end of six or seven days, no report of the effect of long continued ligation of the thoracic duct on the health of the animal is available. The complete occlusion of the entrance of the thoracic duct into the veins of the neck was demonstrated immediately after death by the injection of Berlin blue into the cisterna chyli at a pressiire of 40-50 mm. of mercury; this was followed by dissection of the duct to the point of ligation. Again, the superior vena cava was also opened and a search made for evidence of the dye. Only those cases in which complete obstruction had been established were considered satisfactory; and following careful chemical analysis SchmidtJ\liihlheim concluded that in complete obstruction to the chyle-flow into the blood vascular system, the digestion and absorption of albumens, as well as tlieir conversion into urinary products, are the same as when the chyle is unobstructed in its passage to the blood stream. Tliis result was an advance in the field of physiological chemistry.

The autopsy findings in these exj^erimental dogs differed particularly in one essential respect from the observations of Cooper.' Cooper, it will be remembered, found rupture of the cisterna chyli follomng ligation with extensive extravasations ; however, he failed to verify the rupture by injections.


January, I'Jg-a]


?;j


Schmidt-JIuhlheim, on the other hand, convinced himself by injecting directly into the cisterna with a solution o£ Berlin blue at a pre-ssure of 40-50 mm. of mercury, and keeping this pressure up for hours that, in spite of wide extravasation, the cisterna chyli was intact; in no case was any dye found in the perivascular tissue. Marked distension of the lymphatics, chiefly abdominal, as well a.s enlargement of the lymph glands, was observed constantly. Also, it was noted, that a retrograde injection of the large hnnphatic trunks emptying into the cisterna was possible to the extent of 3-4 cm., because the valves had l)ecome incompetent ; particularly was this true of lymphatics coming from the liver. On the contrary, a retrograde injection of smaller lymphatics was never possible. He added that the mucosa of the intestine always had a normal appearance.

In 1883 Boegehold '" reviewed the important work clone on the thoracic duct. He reviewed not only the anatomy with its numerous variations, the physiologv', the pathology, and clinical features involving the thoracic duct, but also gave an account of the experimental work done on the thoracic duct, adding the results of some of his own experiments. He was interest-ed in studying the effects of wounds of the thoracic duct, and to that end cut the thoracic duct partially or entirely in the chest of dogs. He found that cutting about one-quarter of the periphery of the duct led to fibrin formation and clotting at the damaged area ; cutting completely across the duct caused ehylothorax and death ; wounding the duct slightly, after it had been tied off effectually in the neck according to the method of Schmidt-Miihlheim, resulted at the end of three days in a small amount of fibrin fonnation at the point of injury. Accordingly, he argued that absolute integrity of the thoracic duct was not essential to life. This was shown also by a number of clinical cases that suffered no apparent ill effects from complete obstruction to the duct. Also it seemed as if collaterals were rapidly develoi>ed subsequent to obstruction; but no proof of this was given.

A new departure in ligation of the thoracic duct was effected in 1898 by Stiiler." This author, after referring in uncomplimentary terms to the hypothesis of v. Schwerdt " regarding the pathology of Morbus basedowii with respect to the formation of subcutaneous collateral lymphatics, gave the results of his experimental work on the ligation of the duct in rabbits. Under aseptic precautions and ether anesthesia he ligated the thoracic duct high in the abdominal cavity, just below the diaphragm. Four experiments in all were made. In the first case the aorta was damaged and the animal bled to death ; in the second case the diaphragm was ruptured at operation and the animal later died of peritonitis. The fourth animal also died of peritonitis. The third animal, however, survived and gained weight. A piece of the supposed thoracic duct removed at the time of operation showed microscopically that it actually belonged to the thoracic duct. The author intended to report later the findings in this animal, l)ut no subsequent record is available.

Lucibelli " published the results of his work on the effects of ligation of the thoracic duct. He used two large dogs as


his subjects and gave them milk four or five hours before the operation as an aid in identifying the duct. With chloroform or Richet's solution as anesthetics and with sterile precautions, the operation was performed in the left side of the neck. In the first dog the operative wound became infected and continued to discharge pus for from 20 to 25 days after operation. At the end of this time the dog ceased losing weight and began to gain; his general condition also improved. At the end of two montlis from the time of operation the animal was in fair condition and the various examinations of the different body fluids were made. These examinations were very elaborate and included a complete urine analysis, isotonicity of the blood, red, white and differential blood counts. As a control for these findings an entirely different dog of the same weight, Init living under the same conditions, was used. The first dog gradually became worse and died 3 months and 11 days after the operation. Autopsy showed a spleen five times normal size, mesenteric glands hypertrophied, glands of neck large, large heart, liver and pancreas. However, no injections were made to see whether the ligation in the neck was complete. The second dog did not have any post-operative infection. At the end of three months the same extensive examinations of the urine and blood were made, and immediately thereafter the dog was killed. Autopsy showed no loss of weight as in the previous case; slight engorgement of lymph glands; spleen four times normal size ; small amount of fluid in pleural cavities ; nothing unusual otherwise was observed. There also followed an exhaustive description of the histological findings of the tissues removed from the two animals. From these data, Lucibelli concluded that complete ligation of the thoracic duct caused death in the dog; that partial ligation caused little disturbance because collateral lymphatics established themselves soon; that complete ligation caused changes in all the organs and impaired their secretory function; that changes in the histological picture were due to the toxicity of stagnant lymph ; and that of various causes for cedema, change in the lymphatics was one.

The most recent work on the lymphatic system that involved ligation of the thoracic duct, is that by Bunting and Huston." These authors, in considering the fato of the lymphocyte, resorted to Mgation of the left thoracic duct and jugular vein just before the duct entered, and in some experiments the neck lymphatic tranks on the right side were also tied. All their work was done on the rabbit. They found that this procedure, coupled with splenectomy, produced a marked but temporary decrease in the absolute number of lymphocytes in the circulating blood. They also noted the numerous anomalies of the thoracic duct, and in one case observed anastomosing lymphatic vessels leading through the thymus to the right side.

In the foregoing brief review of the literature regarding the ligation of the thoracic duct as it affects the general economy of the animal, it can be seen that a gradual change in the opinion concerning the importance of the thoracic duct has taken place. At first it was held that ligation of the duct was fatal because the necessary alimentary juices no longer


24


[No. 371


reached the blood stream (Cooper"). Then it was argued that only those cases in which the ligation was complete were fatal; whereas, when all the branches of the thoracic duct were not included in the ligation and chyle could thus get into the blood stream, the animal survived (Magendie '^). Finally, it was believed, even complete ligation of the duct had no evil effect on the animal (Leuret and Lassaigne," Schmidt-Miihlheim ") .

Discussion of Litekatuke

In considering the literature quoted above, it is evident that no conclusive work has been done on the question regarding the effects on the animal of complete ligation of the thoracic duct and its branches, as well as noting the effects of such a ligation after a comparatively long period of time. Duvemey' obviously accomplished nothing with the ligations of the subclavian vein either proximal or distal to the entrance of the thoracic duct. Flandrin' anticipated Leuret and Lassaigne" by 30 years; yet he gave no evidence to support the complete ligation of the duct. Sir Astley Cooper ° and Mayer" claimed that ligation of the thoracic duct is lethal. Cooper claimed that rupture of the cisterna chyli accounted for the extreme extravasations ; however, Schmidt-^Miihlheim " showed by injections that with similar extravasations the cisterna was not ruptured. Indeed Cooper did not describe the rupture to any extent, but considered it a matter of fact. Magendie" believed that death of the animal at the end of 5 or 6 days was due to the effects of the ligation and nothing else. In all probability these deaths were due to infection. Again, Magendie used injections to trace out the lymphatic paths, and in this respect his work was better than that of many investigators who worked a hundred years later. Leuret and Lassaigne on the basis of one experiment found that Ligation had no evil consequences. However, they operated only on one side of the neck, satisfied themselves by dissection without the aid of previous injection that the duct really was tied off; postulated that the chyle took a different course, namely, from the cisterna chyli to the portal vein, and proved this again to their own satisfaction, by simply ligating the portal vein and finding, as a result, some blood in the cisterna chyli. Colin ' after numerous experiments on various domestic animals came to the same conclusion as ^Magendie ^ 50 years before. In his first animal he obviously did not get all the branches of the duct in the neck successfully ligated. In the case of the two dogs that lived respectively 30 and 25 days after operation, infection was also probably the cause of death, since the animals refused all food and behaved so differently from other dogs in which the duct was really tied without infection. In this respect the careful work of Schmidt-Miihlheim " is noteworthy. Up to this time all operative work had been done without antiseptic or aseptic precautions, and although " perfect suppuration " took place, nevertheless, this very fact vitiated practically the entire experiment. Schmidt-Miihlheim made the first serious attempt to prevent the chyle from reaching the blood stream ; and furthermore, he used injections to make sure that the ligations were complete. It would have


been interesting to know what would have taken place if the animals had been kept longer than seven days. Unfortunately, all the animals were sacrificed within a week from the time of operation. However, to tie off practically the entire superior vena cava is a considerable operation and a great shock to the animal; nevertheless, the animals recovered from the operation very satisfactorily. Even this operation would not necessarily always shut off the chyle from the blood stream, since it is possible that branches of the thoracic duct could enter the axillary vein distally to the point of ligation and eventually reach the heart through the collateral venous circulation which obviously would be established. The advantages of this operative procedure were at once recognized and frequently used by biochemists (Munk and Friedenthal "°) . Boegehold " also stated that ligation of the thoracic duct exerted no deleterious effect on the animal, basing his argument on clinical cases as well as on the result of his last experiment, in which he ligated the thoracic ducts according to the method of Schmidt-Miihlheim. In this animal he made a small injury to the duet, and three days after this found a small clot at the site of injury. Here the same objections can be raised as were advanced with Schmidt-Miihlheim; besides this, injection of the lymphatics was not made to ascertain whether the ligation was complete. The experiments of Stiller " are as novel as they are inconclusive. All that can be gathered from his work is that a complete ligation of the thoracic duct was attempted in the abdomen; that a large lymphatic trunk, probably the thoracic duct, was ligated; thdt the animal recovered and gained weight; and that the subsequent findings in the animal were promised but are not available. Lucibelli " tried to determine what effects ligation of the duct would have on the animal economy, particularly when studied several months after the operation. His animals were killed at the end of three months, which is the longest time any animals with ligated thoracic ducts were ever allowed to survive. Unfortunately, his work is not without serious objections. In the first place he ligated the duct on one side of the neck only, although previous work, particularly that of Selimidt-Miihlheim," had showTi how frequent it was that branches of the thoracic duct went to the right side, and how essential it was to ligate not only the thoracic duct on one side, but also to ligate the large veins in the base of the neck on both sides, as well as to ligate the right thoracic duct. That the dog is no exception in having many anomalies in the entrance of the thoracic duct into the veins was previously emphasized by Kuft'erath."^ In the second place, one of the two animals had a postoperative infection lasting nearly a month. This infection alone discounted the value of the results obtained by careful chemical analysis of the urine and blood. Again, he used as controls, not the same animal before operation, but preferred an entirely dilferent dog of about the same weight as tlie experimental animal and kept under the same conditions. Also, judging from the urinaiy findings, the first animal had what LucibeUi called a chronic interstitial nephritis. Accordingly, no animal with a kidney lesion, even though the thoracic duct be securely tied, could be compared


Jandaky, 1933]


25


with a normal animal, particularly in so I'ar as the urinary findings are concerned. The presence of the infection vitiated also all conclusions regarding the histological changes purported to be consequent to a thoracic duct ligation. There is, furthermore, no evidence supported by post-mortem procedures to substantiate the conviction that the ligations were successful ; and the absence of confirmatory examination alone makes the whole work faulty. It is needless to discuss the authoi-'s contention that his two experiments support the view that oedema is due to stagnant lymph formation. Finally, in the work of Bunting and Huston " there was no evidence to support the belief that the thoracic duct was completely ligated. Their work did show that the rabbit is also no exception to the general rule which holds that the entrance of the thoracic duct into the veins of the neck may be extremely varied.

Before proceeding to the experimental work, it may be added that numerous investigators (Kunkel, Fleischl," Kufferath,'* Harley," Josue,"' Wertlieimer and Lepage,'" Davis and Carlson") also ligated the thoracic duct and studied the effect of simultaneous ligation of the biliary duct, or subsequent changes in the number of lymphocj'tes in the blood. None of these authors operated in a way to preclude all possibility of anomalous collateral circulation, nor did all inject their specimens post-mortem to be sure of the course of the lymph.

The Russian literature also contains reports of work done on the ligation of the thoracic duct. Some of the work is experimental (Khlopin^), in which the absorption of fats was studied; others (Yitlin^°), reported a case of injury to the duct, and reviewed the literature on other cases of injury to the duct. It is interesting to note that Vitlin ^° and Temkin '° took the same case from the clinic of Professor Better in Berlin as a stimulus for their respective articles.

At the suggestion of Dr. Cunningham, and with his frequent advice, experiments were undertaken to determine what effect on the general economy of the animal ligation of the thoracic duet would have ; also what collateral circulation, if any, would be established. It is a pleasure to thank Dr. Sabin and Dr. Cunningham for their interest and help during this investigation.

Experimental Eesults

In all the experimental work the cat was used exclusively, chiefly because it is a convenient and very satisfactory laboratory animal, and also because the lymphatics of the cat have been described to a considerable extent. The animals were for the most part yoiing adult male cats, seemingly in good health. Food was withheld for 24 hours before the operation which was performed under ether anesthesia and with strict aseptic precautions. During the operation, the animal received the benefit of hot-water bottles and after operation it was placed in a wann cage. All the animals were weighed immediately before the operation and at various times after the operation.

Operative Procedures. — At first, attempts were made to ligate the thoracic duct in the neck. In these cases the animals were fed cream five hours before operation in order to make


the duct more conspicuous; at times this advantage was heightened by adding Scharlach R to the cream. Several of these operations were successful in so far as injections from below showed that the duct had been effectively ligated. But it was soon evident that this procedure clid not produce enough positive results to make it reliable for work which required absolute ligation of the duct, for frequently the duct had numerous entrances into the veins of the neck, and it was not possible to operate without feeling doubtful about the ligation of all the branches.

Accordingly it was decided to try to ligate the duct high up in the chest, before all the anomalous branches were given off. Because of the consequent pneumothorax following opening of the chest, intratracheal ether was given. A calibrated No. 13 F soft rui)ber catheter with accessory openings at its end was inserted into the trachea. Air for the apparatus was supplied by a foot bellows which was connected to a mercury safety valve adjusted to 20 mm. of mercury, and led over ether in. a WouLfe bottle, which was further provided with a by-pass direct to the catheter. With this apparatus all gradations from pure air to heavily saturated ether vapor could be administered at physiological pressures. The operation consisted in going into the second or third intercostal space, spreading the ribs apart, isolating the carotid artery and vagus nerve, inclosing as much of the surrounding tissue as possible in a ligature, and closing the cavity. This operation also was not successful, because isolation of the vagus nerve and carotid artery was often difficrdt, and besides branches of the duct to the right side were completely missed by this undertaking.

The Operation. — Finally it was decided to operate lower down in the chest, and this procedure gave the desired results. The animal was placed on its right side, with the left front leg drawn a little upward and forward. The left side of the chest was shaved over an area extending from the third to the tenth intercostal spaces, and from the midline in the back to the parasternal line. Iodine technique to the skin was employed. A transverse incision about 4.5 cm. long was made over the sixth or seventh intercostal space, extending forward from a point about 4 cm. from the midline of the back. Dissection was carried down to the latissimus dorsi muscle, the fibers of which were split longitudinally. By palpation the desired intercostal space was detemiined and dissection was carried down through the serratus muscle to the superior border of the rib bounding the inferior portion of the intercostal space ; this was done to avoid damaging the intercostal vessels which course superiorly. One was sure of good exposure when it was necessary to cut a few lateral fibers of the sacrospinal muscle. The intercostal muscles were then severed along the superior border of the rib, care being taken to cut to a uniform depth. After the pleura was reached, the tip of a pair of blunt-pointed scissors was thrust into the pleural cavity and the opening enlarged. A small pair of self-retaining rib retractors were inserted and the opening widened to about 3 cm. The lungs collapsed considerably and did not interfere with further work, particularly when the intratracheal pressure was kept low.


26


[No. 371


The thoracic duct was usually not seen because it contained no chyle. The adventitia over the anterior surface of the aorta was grasped with forceps and dra^-n slightly toward the opening. The adventitia inferior to the forceps was then carefully dissected from about the aorta as much as possible and an aneurysm needle with a fine silk thread was passed around the aorta ; this thread served as a traction suture on the aorta. A right-angled aneurysm needle was then inserted from the left to the right side of the aorta and under it, and turned through an angle of 180°, so that the point rested on the right side of the vertebral eolumu near the midline. The point was allowed to pass along the vertebral column to the left side until it appeared at the sympathetic chain. The tissues included in this ligature were the thoracic duct and sometimes the azygos vein. The aneun'sm needle was then passed under the aorta from its right to its left side, the tissues of the previous ligature were again included, but chiefly was it desired to get the tissue between the vertebral column and the esophagus. Sometimes it was necessary to include more tissue about the esophagus in a third ligature ; the ligation was thus completed. The ribs were approximated with a double medium silk suture; interrupted sutui'es for the muscle; the usual subcuticular stitch for closure. N"o dressing was applied but the iodinized area was carefully washed with ether to avoid any iodine irritation with consequent damage to the incisions. The entire operation from incision to closure did not take more than half an hour.

Post-Operative. — The animals made a satisfactorj' recovery following operation. They frequently ate on the day after operation. There was no evidence of diarrhoea, cedema or respiratory distress; slight abdominal tenderness was frequently obsened. The animals were weighed at intervals following the ligation ; the results were not unifonn. Some of the animals gained weight, some lost, whereas the one wHch was sacrificed 66 days following the operation, and showed absolute ligation of the duct, evidenced little change in weight. On a series of animals leucocyte counts and differential blood counts were made to study chiefly the lymphocyte change following the operation. This work will be reported later.

The cats were sacrificed from 24 hours to 77 days after ligation. Under ether anesthesia the abdomen was opened and the mesenteric lymphatics of the small intestine were injected vrith a saturated aqueous solution of Berlin blue. The ordinary hypodermic syringe with a Xo. 28 needle was found to be preferable to larger Record syringes, since the pressure of injection could be regulated better. The chief point in injecting the mesenteric IjTnphatics was to avoid any possible injection of veins, for if the dye reached the blood stream other than through lymphatico-venous communications, the experiment would be inconclusive. Thus, if the sul)serous layer or muscular coats of the intestines were injected, then the dye could possibly enter the veins as well as the lymphatics. The same objection held for the mesenteric IjTuph gland, since a direct injection of a lymph gland would also entail the possibility of .striking veins as well as lymphatics. During the injection of the last cubic centimeters of the dye.


the animal was killed, and the injection was continued until the ventricle stopped beating. This was done to insure a sufficient amount of dye at the Ij'mphatico-venous communications. The blue pigment was usually seen in the lungs after the chest was opened and the course of the injection with respect to the thoracic duct studied. Frequently the animal died during the course of the injection from a pulmonary embolus caiised by the injected material. Material for sections was cut from various organs ; .the remainder of the animal, except the head and extremities, was fixed in formalin and saved for further dissection.

Types of Collateral Circulation Focxd after Ligation of Duct

As a result of these dissections, two general types of collateral circulation were established. The one type consisted of a collateral circulation to the right thoracic duct, the other tj^pe comprised those cases in which the lymph entered the azygos vein or its branches.

The first type is illustrated- by Fig. 1 which shows the collateral circulation established to the right thoracic duct. It may thus be justly said that tliis case illustrates absolute ligation of the thoracic duct and the periaortic lym])hatic plexus. The animal from which the figure was drawn was sacrificed one week after operation. The structures of interest were taken out en bloc, dehydrated and cleared in oil of wintergreen. Attention is called to the absolute ligation of all lymph drainage to the left side of the neck. Below the point of ligation is a small but significant lymphatic plexus supplied by a large branch coming off from the thoracic duct. From the superior aspect of this plexus a small branch is seen to course on the azygos vein to a small gland at the mouth of the subclavian vein, and from there two trunks finally join and empty into the junction of the jugulo-cephalic trunk and the internal jugular vein.

Attention is also called to the general appearance of the abdominal lymphatic vessels soon after ligation of the thoracic duct. An cedema developed about the large lymphatic vessels, particularly around the cisterna chyli and its large trunks. This a?dema depended in its appearance on the nature of the fluid in the lymphatics. If these were laden with chyle, the cfidema was chylous in nature; if no chyle were present, the perivascular tissues were bathed in a limpid, slightly yellowtinged, clear fluid which seemed to contain small fatty droplets. In no case did this cedema resemble ascites to any degree. Likewise, the cisterna chyli was never found ruptiired. This cedema in some animals became well established at the end of 24 hours, and persisted for one week, as was shown in a case in which a marked constriction, but not occlusion, of the thoracic duct was secured.

The lymph glands imderwent marked hypertrophy. They had a fatty appearance, and showed the small follicles on the surface. They were very oedematous, and on section a considerable amount of clear, colorless fluid escaped. The increase in the size of lymph glands was more impressive in regious where they were ordinarily small and scarce, e. g., between


January, 192-2]


27


the large mesenteric gland and the small intestine. Occasionally, glands of the size of almonds were seen in this region. In no case did the spleen seem larger than normal. The thoracic duct above the point of ligation was iisually small and contracted.

In none of the dis.sections was the vagus nei-ve included in the ligature; occasionally, the left sympathetic chain was taken. Nor did the pigment reach the lungs via the direct lymphatic communication between the lungs and the cistema ehyli ( Cunningham " ) .

The second type of collateral circulation is shown in Fig. 2, which was drawn from a specimen in which the subject was sacrificed 66 days after ligation. The thoracic duct with the entire lymphatic plexus was ligated. The azygos vein was included in the ligature and thus precluded any collateral vessel developing, as illustrated in Fig. 1. It is seen that an extensive lymphatic plexus has developed at the cistema chyli and the azygos vein. Specimens of the contents of the azygos vein removed near the lymphatico-venous junction as well as a little further along the course of the vein, and examined under the microscope, revealed the Berlin blue pigment.

In one animal that was sacrificed three weeks after complete ligation, the lymphatico-venous connection was in the ninth left intercostal vein just before the latter entered the azygos vein. The communicating vessel took a tortuous path, commencing at a point on the thoracic duct at the level of the entrance of the ninth intercostal vein into the azygos vein, turned inferiorly and extended 7 mm. along the left side of the aorta, giving off small branches which entered the walls of tb-at vessel ; made several small shai-p turns and extended about 4 mm. superiorly and laterally ; then went medially and slightly inferiorly, making an acute angle with the ninth intercostal vein which it entered about 6 mm. from its junction with the azygos vein. This communicating vessel was almost the size of the intercostal vein, and was undoubtedly taking over the function of the thoracic duet.

In another case, a lymphatico-venous communication probably existed between a branch of one of the left lumbar veins and the cistema chyli, since the blue pigment was found in the distal part of the lumbar vein, but in much greater quantity at the entrance of the lumbar vein into the inferior vena cava. As the connection was not actually seen, the absoluteness of the jmiction cannot be claimed.

Eetrogeade Injection of Lymphatics Before entering into the discussion of the above findings, attention is called to the retrograde injection of lymphatics as found in the majority of the dissections. In studying the lymphatic distribution in embryoe by the injection method, advantage is constantly taken of the fact that specimens up to 5 cm. in length have the valves in their lymphatic vessels incompletely developed or entirely absent, and accordingly allow extensive retrograde injection of their lymphatic vessels (Sabin,"'°° Heuer," Cunningham"). In observing the relation of lymphatic vessels to connective tissue in pig foetuses by means of injections, MacCallum "^ noticed that the valves were


not very competent. In the adult, retrograde injections of lymph vessels entering the cistema chyli and regional visceral lymph glands have been described by Schmidt-Miihlheim " and Bartels." While injecting the mesenteric lymphatics, as above described, frequently one could see contributing branches of the vessel which was being injected centripetally gradually sufl'er retrograde injection through the successive dilatation of the intervalvular segments and the incompetence of their respective valves. In several cases such retrograde injections were carried to the subserous coat of the small intestine; while other cases showed a retrograde injection through the lymph glands draining the kidney with the afferent lymphatics injected as far as the hilum of that organ. Likewise, injections of small lymphatic vessels in the adventitia of the aorta and of vessels on the surface of the psoas muscle have been seen. Again, the large lymphatic plexus draining the liver and gall-bladder has been observed in a markedly dilated though uninjected state. In no case, however, have the parenchymatous lymphatic vessels been filled; and this in spite of the fact that these vessels are supposed to have either no or only a few valves (Bartels,°° p. 71). The problem seems to lie in emptying lymphatic capillaries before injections are made; this condition may possibly be achieved by introducing hypertonic solutions into the blood stream, thus emptying the lymphatic capillaries and allowing the injection mass to enter. Waldeyer,^' in 1867, called the attention of pathologists to the possibility of retrograde lymphatic transport of metastases.

Discussion

A new method has thus been described for ligating the thoracic duct. While ligations of the duct have been performed in the neck and in the abdominal cavity by other workers, no attempts, it seems, have been made to secure ligation in the chest, although the duct has been wounded frequently in that cavity for experimental purposes (Boegehold "). The operation can be performed without extensive dissection and without ligation of large veins (Schmidt-Miililheim ") ; it can be performed speedily and with a considerable guarantee of success. The method has provided experimental animals with absolute ligation that have lived apparently unaffected as long as 66 days after the operation. This period of survival of the animal in this study may be compared to the report of Lucibelli, in which it was stated that the longest period of survival after ligation of the duct was three months and eleven days, but in this work of Lucibelli there is much reason to believe that the ligation was not absolute. However, from the above experimental results, one can conclude that the integrity of the thoracic duct is not essential to the life of the animal.

Furthermore, the method of injecting the mesenteric lymphatics avoids all possibility of injecting the venous system inadvertently, and has provided excellent examples of retrograde injections of the lymphatic vessels. Naturally, previous ligation of the thoracic duct may have played a considerable role in facilitating these injections.

It is interesting to note to what extent the present knowledge of the embryology of the lymphatic system may be util


28


[No. 371


ized to explain the experimental findings. However, although it is not the purpose of this article to enter into the discussion of the various views regarding the origin of the lymphatic system (Huntington,'* Sabin,'" McClure," Lewis," Pensa"), it can be stated that at one time in embiyonic life a large lymphatic plexus exists extending from the veins at the base of the neck on either side, down around the aorta, and terminating in the cisterna chyli ; also, that the lower part of the embryonic thoracic duct is bila,tcrally sjTiimetrical. The


branch leading from this plexus to the right thoracic duct may also be considered a part of the embryonic plexus; the fact that it enters a gland first before proceeding to the veins suggests that it was originally one of the smaller and more remote portions of the plexus. In another animal which was sacrificed 77 days after ligation, this small connecting vessel had a counterpart in two huge trunks, each of which was almost the size of a normal thoracic duct; the corresponding gland was also markedly enlarged. This response of embryological


Superior vena ca\/


Juqulo-cephalic

trunk


Subclavian vein


7)h. Intercostal vein


Azyqos vein



Internal jugular vein.

Inferior thyroid vein. Common juqular vein. Subclavian vein Lett innominate vein.


Superior vena cava.


Point of ligation


Point ot ligation.



Lumbar vein


Entrance of lymptiatic into az,yqos vein.

Riqhf lumbar lymph node


Cisterna ctiyli.


Cisterna chyli


Fig. 1.


origin of the thoracic duet in the pig has been described by Baetjer." With these embiyological data as a basis, Davis" described and explained various types of thoracic duct anomalies, and it is interesting to note to what extent Fig. 1 fits in with some of the schematic representations of embryonic lymph channels that he depicted. Thus the small plexus developed below the point of ligation may well represent a portion of the embryological lymphatic plexus which normally does not function, but which, in response to the ligation, may have been forced to enlarge and dilate. Again, the small


Renal arfer_y. Fig. 2.

non-functioning lymphatic capillaries in a physiological capacity, if true, recalls in a measure the important work of Krogh " in relation to blood capillaries.

On the other hand, it is impossible to explain with our present knowledge of embryology, the connection between the thoracic duct and azygos vein, as illustrated in Fig. 2. It seems as if the earliest stages in the development of lymphatic vessels are as yet unknown. The recent work of Sabin ** on the development of blood vessels would suggest that the ditferentiation of angioblasts as well as the sprouting of the vascular


Januakt, 1923]


29


endothelium could lead to the formation of lymphatics at other places along the venous system than from the anterior cardinal veins or from the veins of the Wolffian body. Thus, they could arise from the azygos vein, form a plexus with those lymphatic vessels arising from veins at the usual places, and gradually become small and functionless "until physiological exigencies, produced, for example, by ligation of the thoracic duct, caused them to enlarge and function. Again, that large venous network in the early embryo which is associated with the azygos vein and which later disappears may be a factor in producing this lymphatico-venous connection.

E. E. Clark, in the dascription of a very curious anomaly of the thoracic duct, considered the embryology of the lymphatic system in the explanation of the condition. Likewise, clinical cases of lymphatico-venous fistulse have been discussed on this basis (Halsted"').

Again, the establishment of collateral lymphatic circulation may occur through the regeneration of lymphatics. However, the studies in the regeneration of lymphatics have not been extensive enougli, nor do they carry the necessary conviction (Meyer ") . As much as one is inclined to believe in the regeneration of lymphatics — and this could easily explain the result as illustrated in Fig. 1 — nevertheless, the necessary experimental proof seems lacking.

Since several cases of lymphatico-venous communications have been found (see Fig. 2), the subject of these connections is brought up for consideration. A host of writers (see Bartels "), as early as 1G62, had described unusual lymphaticovenous connections; and numerous other investigators have attempted to disprove the evidence. Boddaert," in 1899, described in the rabbit a connection of the thoracic duct with the azygos vein. Leaf " in 1900 maintained that the azygos vein received many lymphatic vessels. However, Bartels,^' in 1909, questioned the correctness of any such lymphatico-venous communications. More recent work had thrown new light on the subject. Silvester'" described the presence of permanent communications between the lymphatic and the venous systems at the level of the renal veins in adult South American monkeys; Baum " described cases where vasa efferentia of lymph glands emptied directly into veins; Job""' reported lymphatic conununications with the inferior vena cava, ileolumbar, renal and portal veins in rats. It seems, then, well established that the lymph does not necessarily have to enter the venous system at the base of the neck.

The presence, then, of these lymphatico-venous connections enters into the problem of fat absorption. The entire anatomy of the chyliferous portion of the lymphatic system suggests that the newly absorbed fat avoids direct entrance into the liver. Biochemists, even with refined methods for determining fats in body fluids (Bloor""), can recover from the thoracic duct only 60-70 per cent of the previously ingested food fats, but they are at a loss to explain the disposal of the remaining 30-40 per cent. Munk and Friedenthal ^ found that the blood fat increased after the thoracic duct had been tied. D'Errico " maintained that the percentage of fat in the portal vein was normally greater than that in the jugular vein, also that.


following ligation of the thoracic duet, the fat content of the portal vein with respect to the solid residue decreased but still remained higher than that of the jugular vein. He also believed that ligation of the duct accentuated lymphaticovenous communications. It is obvious that lymphatico-venous connections have been a generally unrecognized factor in all these studies of fat absorption involving ligation of the thoracic duct; and consequently these investigations are open to criticism.

Albrecht v. Ilaller, Albin and Hebenstreit are reported by Boegehold " to have seen the thoracic empty into the azygos vein. Wutzer,"* while demonstrating the course of the thoracic duct in the body of a woman 37 years old, noticed that there were three connections between the duct and the azygos vein; also, that above these connections, the duct became attenuated and fibrotic. This case seems to show that in regard to this lymphatico-venous connection there is an analogous relationship between the human subject and the laboratory animal.

However, in spite of this analogy, one cannot claim with absolute siirety that ligation of the thoracic duct in the human subject would prove equally inocuous. Yet it is clear, from the current surgical text-books as well as from the individual reports of injury to the thoracic duct, that there is no accepted method of treatment. Usually one of the four procedures is employed when the duct is injured : (1) Eepair of the wounded duct and provision for subsequent ligation if necessary; ( Gushing "'); (3) tamponade; (3) ligation of the vessel or (4) implantation of the severed duct into a vein (Harrison °). Convalescence is the rule. Gushing considered that in case of ligation of the thoracic duct the lymph current would be reversed, and finally all the lymph would be taken up by the right thoracic duct — a postulate supported in a measure by these experiments. A record of numerous clinical cases involving injury to the thoracic duct with a discussion of their treatment has been given by Zesas." " However, it is very questionable whether all the injuries to the thoracic dact really constituted injuries to the main trunk. Large lymphatic vessels from the head and neck often join the duct before its entrance into the vein and may be mistaken for the duct itself. Again, the presence of chyle does not guarantee the vessel to be the main trunk, because there frequently are several branches of the duct before its entrance into the veins.

In short, then, with the aid of the above experimental data, treatment of thoracic duct injuries would resolve itself into: 1, Eepair of the injury if possible ; or, 2, ligation. There is every reason to believe that similar treatment would apply to injury of other large and important lymph vessels ; e. g., the intestinal trunk.

Summary

After reviewing the literature on the experimental ligation of the thoracic duct, an intra-thoracic method has been described for complete ligation of the thoracic duct in the cat. It seems that the integrity of the thoracic duct is not essential to the life of the animal. In some cases in which the ligation was absolute, collateral lymph circulation was established to the right thoracic duct; while in other cases which showed


30


[No. 371


complete ligation, lympliatico-venous connections were found to exist between the thoracic duct and the azygos vein. The embryology of the lymphatic system may explain partly, but not entirely, these findings which also have a bearing on studies of fat absorption as well as on the clinical aspect of injuries to the thoracic duct.

BIBLIOGRAPHY

1. Asellius, G.: De lactibus sive lacteis venis Quarto Vasorum Mesaraicorum genera Novo Invento Gasparis Aselli Cremonensis Anatomici Ticinensis Dissertatio, Mediolani, 1627. Quoted from Bartels.°°

2. Lower: Tractatus de corde, item de motu, colore et transfusipne sanguinis et de chyli in eum transitu. Leyden. Editio sexta. MDCCXXVIII. Quoted from Schmidt-MuhUieim."

3. Flandrin: Experiences sur I'absorption des vaisseaux lymphatiques dans les animaux. Jour, de med., 1791, LXXXVII, 221.

4. Colin, G.: Traite de physiologie comparee des animaux. Deuxieme edit., Paris, 1873, II, 238.

5. Heidenhain, R.: Versuche und Fragen zur Lehre von der Lymphbildung. Arch. d. gesamm. Physiol., 1891, 209.

6. Starling, E.: The Influence of Mechanical Factors on Lymph Production. Jour, of Physiol., 1894, XVI, 224.

7. Biedl and v. Decastello : Ueber Aenderungen des Blutbildes nach Unterbrechung des Lymphzuflusses. Pfliigers Arch., 1901, LXXXVI, 259.

8. Duverney: Memoires de I'Academie des sciences, 1675. (Quoted from Colin.)

9. Cooper, Sir Astley: Three Instances of Obstruction of the Thoracic Duct, with some Experiments showing the Effects of Tying that Vessel. Medical Records and Researches selected from the papers of a private medical association. London, 1798, 86.

10. Mayer: (See Nockher: Dissertation: De Morbis Ductus Thoracici. Bonn, 1831, 35).

11. Rogers: Salzb. raed.-chir. Zeit., 1823, I, 112. (Quoted from Nockher.")

12. Magendie: Memoire sur le mecanisme de I'absorption chez les animaux a sang rouge et chaud. Jour, de Physiol., 1821, I, 21.

13. Leuret et Lassaigne : Recherches physiologiques et cliniques pour servir a I'histoire de la digestion. Paris, 1825, 178.

14. Schmidt-MUhlheim, A.: Gelangt das Verdaute Eiweiss durch den Brustgang ins Blut? Arch. f. Anat. u. Physiol. (Physiol. Abth.), 1877, 549.

15. Boegehold, E.: Ueber die Verletzung des Ductus Thoracicus. Arch. f. Chir., 1883, LXXX, 443.

16. Stiller: Die intraabdominale Ligatur des Ductus thoracicus beim Kaninchcn. Zugleich eine Kritik der Arbeit von Schwerdt ueber Morbus Basedowii. Cor.-Bl. d. allg. artzl. Ver. v. Thiiringen, 1898, XXVII, 412.

17. (a) V. Schwerdt: Der Morbus Basedowii, ibid., 365. (b) Idem: Erwidenmg auf die Arbeit des Herm Dr. Stiller. Ohrdruff: "Die intraabdominale Ligatur des Ductus thoracicus beim Kaninchen." ibid., 416.

18. Lucibelli, C: Sugli effetti dell' allaciatura del condotto toracico. N. Riv. Clin. Terap., NapoU, 1902, V, 401.

19. Bunting, C. H., and Huston, John: Fate of the lymphocyte. Jour, of Exper. Med., 1921, XXXIII, 593.

20. Munk and Friedenthal : Ueber die Resorption der Nahrungsfette und den wcchselnden Fettgehalt des Blutes nach Unterbindung des Ductus thoracicus. Zentralbl. f. Physiol., 1901, XV, 297.

21. Kufferath: Ueber die Abwesenheit der Gallensiiuren im Blute nach dem Verschluss des Gallen und des Milchbrustganges. Arch. f. Anat. u. Physiol. (Physiol. Abth.), 1880, 92.

22. Kunkel: Ber. d. Gesellsch. d. Wissensch. in Leipzig, 1873. (See Harley.")

23. V. Fleischl: Ibid., 1874. (See Harley.")

24. Harley, V.: Leber und Galle wahrend dauemden Verschlusses von Gallen- und Brustgang. Arch. f. Anat. u. Physiol. (Physiol. Abth.). 1893, 291.

25. Josue, A.: Ligature du canal thoracique; presence de microbes dans le bout inferieur, tandis que le sang est aseptique. Compt. rend. Soc. de biol., Paris, 1895, S. 10, II, 25.

26. 'Wertheimer and Lepage: Sur les effets de la ligature simultanee du canal choledoque et du canal thoracique. Jour, de physiol. et de path, gen, 1899,1,259.

27. Davis, B.F., and Carlson, A. J.: Contributions to the Physiology of the Lymph. IX. Notes on the leucocytes in the neck, lymph, thoracic lymph, and blood of normal dogs. Am. Jour. Physiol., 1909, XXV, 173.

28. Khlopin, N.: (On the influence of hgating the thoracic duct in dogs upon the assimilation of fats.) St. Petersburg, 1892.

29. Vitlin, I.: (Injuries to the thoracic duct in cases of chylothorax and chylous ascites in connection with anatomical and experimental data on the chyhferous system.) Ivharkov. Med. Jour., 1909, VII, 87.

30. Temkin, S.: Dissertation: Zur Frage iiber die Verletzung des Milchbrustganges (Ductus thoracicus) and die Fiille von Chylothorax und Ascites chylosa im Zusammenhang mit dem anatomischen und experimentellen Angaben ilber das Chylusgefiisssystem. Berlin, 1909.

31. Cunningham, R. S.: On the development of the lymphatics of the lungs of the embryo pig. Contrib. Embryol. (Carnegie Inst.), Wash., 1916, IV, 47.

32. Sabin, F. R.: On the origin of the lymphatic system from the veins and the development of the lymph hearts and thoracic duct in the pig. Am. Jour. Anat., 1902, I, 367.

33. Sabin, F. R.: On the development of the superficial lymphatics in the skin of the pig. Amer. Jour. Anat., 1904, III, 183.

34. Heuer, G. J.: The development of the lymphatics in the small intestine of the pig. Amer. Jour. Anat., 1909, IX, 91.

35. MacCallum, W. G.: Die Beziehung der Lymphgefasse zum Bindegewebe. Arch. f. Anat. u. Phys. (Anat. Abth.), 1902, 273.

36. Bartels, P.: Das Lymphgefiisssystem, in v. Bardeleben: Handbuch der .\natomie des Mensehen, Bd. III. Abt. IV, 69.

37. Waldeyer, W.: Ueber die Entwicklung der Carcinome. Virchows Arch., 1887, XLI, 470.

38. Huntington, G. S.: The anatomy and development of the systemic lymphatic vessels in the domestic cat. Phila., 1911. (Wistar Inst. Anat. & Biol., Phila., Memoirs, No. 1.)

39. Sabin, F. R.: Der Ursprung und die Entwickelung des Lymphgefasssystems. Ergebnisse der Anatomie und Entwickelungsgeschichte, 1913, XXI, 1.

40. McClure, C. F. W.: The development of the thoracic and right lymphatic duets in the domestic cat (Felis domestica). Anat. Anz., 1908, XXXII, 534.

41. Lewis. F. T.: The development of the lymphatic sj'stem in rabbits. Am. Jour. Anat., 1906, V, 95.

42. Pensa, A.: Studio suUa morfologia e sulla topografia della cisterna chili e del ductus thoracicus nell' uomo ed in mammiferi. Richerche di anat. norm. d. r. Univ. di Roma, 1908, XFV, 1.

43. Baetjer, W. A.: On the origin of the mesenteric sac and thoracic duct in the embryo pig. Amer. Jour. Anat., 1908, VIII, 303.

44. Davis, H. K.: A statistical study of the thoracic duct in man. Am. Jour. Anat., 1915, XVII, 211.

45. Krogh, A.: The supply of oxj'gen to the tissues and the regulation of the capillary circulation. Jour. Physiol., 1919, LII, 457.

46. Sabin, F. R.: Studies on the origin of blood-vessels and of red blood corpuscles as seen in the living blastoderm of chicks during the second day of incubation. Contrib. Embryol. (Carnegie Inst.), Wash., IX, 213.

47. Clark, E. R.: An anomaly of the thoracic duct with a bearing on the embr\'ology of the lymphatic s>'stem. Contrib. Embrj'ol. (Carnegie Inst.), 'Wash., 1915, II, 45.

48. Halsted, W. S. : Congenital arterio-venous and lyinphaticovenous fistulae. Unique clinical and experimental observations. Contrib. Med. and Biol. Research. Ded. to Sir Wm. Osier, 1919, 1, 560.

49. Meyer, A. W.: An experimental study on the recurrence of lymphatic glands and the regeneration of lymphatic vessels in the dog. Johns Hopkins Hosp. Bull., 1906, XVII, 185.

50. Boddaert, R.: Etude sur une communication exceptionelle entre le canal thoracique et la veine azygos chez le lapin. Ann. de la Soc. de med. de Gand, 1899, LXXVIII, 123.

51. Leaf, C. H.: On the relation of blood to lymphatic vessels. Lancet, 1900, I, 606.

52. Silvester, C. F.: On the presence of permanent communications between the lymphatic and venous sj'stem at the level of the renal veins in adult South American monkeys. Am. Jour. Anat., 1912, XII, 447.

53. Baum, H.: Konnen Lymphgefasse direkt in Venen einmiinden? Anat. Anz., 1911, CXL, 593.

54. Job, T. T. : The adult anatomy of the Ij'mphatic sj'stem in the common rat (Epimys norvegicus). Anat. Record, 1915, IX, 447.

55. Idem: Lymphatico-venous communications in the common rat and their significance. Am. Jour. Anat., 1918, XXIV, 487.

56. Bloor, W. R.: Fat assimilation. Jour. Bio-Chem., 1916, XXIV, 447.

57. d'Errico, G.: Contributo alio studio delle vie di assorbimento del grasso alimentare. Arch, di fisiol., 1907, FV, 513.

58. Wutzer, C. W.: Einmiindung des LKictus thoracicus in die Vena azygos. Mviller's Arch., 1834, 311.

59. Gushing, H.: Operative wounds of the thoracic duct. Report of a case with suture of the duct. Ann. Surg., 1898, XXVII, 719.

60. Harrison, E.: On the treatment of wounds of the thoracic duct. Brit. Jour. Surg., 1917, IV, 304.

61. Zesas, D. G.: Die operativ enstandene Verletzungen des Ductus thoracisus. Ihre Bedeutung. Ihre Behandlung. Deutsche Ztschr. f. Chir., 1911-12, CXIII, 197.

62. Idem: Die nicht operativ enstandene Verletzungen des Ductus thoracicus. Ibid., 1912, CXV, 49.


STUDIES ON EXPERIMENTAL RICKETS XV. THE EFFECT OF STARVATION ON THE HEALING OF RICKETS

By E. V. McCoLLUM and Nina Simjionds Department of Chemical Hygiene, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore

and

P. G. Shipley and E. A. Park,

Department of Pediatrics, The Johns Hopkins University, Baltimore


We have shown elsewhere ^ that certain types of faulty diets induce bone changes identical with those seen in rickets in human beings. Cod-liver oil exerts a pronounced effect in preventing the development of these changes, or, once they are established, tends to cause the bones to return toward a normal condition.

The most effective way which we have found to demonstrate the therapeutic value of cod-liver oil is through the application of our " line test," which has been fully described in another paper. Young rats are restricted to our diet ( Lot 3143) which has the following compo.«ition for a period of 35-40 days.

Lot 3143

Wheat 33.0

Maize 33.0

Gelatin 15.0

Wheat gluten 15.0

NaCl 10

CaCOs 3.0

Any other diet having identical dietary properties would serve the same purpose.

This diet contains proteins of good quality and in great abundance (about 33 per cent), and about twice the calcium necessary for optimal growth and nutrition. It is, however, low in fat-soluble A and below the optimum in its content of phosphorus. One hundred grams contains 0.3019 grams of phosphorus.


If young rats are fed on this diet they develop a condition of the skeleton which is identical with the most severe rickets seen in the child. In the bones of these animals (Fig. 1) the cartilage is overgrown and abnormally persistent. The shaftward margin of the cartilage is irregular and the cartilage is invaded by blood vessels from the shaft of the bone. Lime salts are not deposited in the cartilage, so that no zone of provisional calcification is formed. A wide zone of tissue is formed between the cartilage and the shaft proper of the bone. This is made up of blood vessels, connective tissue and marrow elements, and of cartilage cells in all stages of degeneration, reversion, and of metaplasia into osteoid tissue. This is the rachitic metaphysis. The cortex of the shaft of the bone and the trabeculae of the spongiosa are surrounded by wide zones of osteoid tissue (uncalcified bone). No signs of abnormal resorption of bone tissue are to be found. These bones are very soft and at autopsy the animals show deformities of the skeleton which we have discussed in detail in a preceding communication.'

When a young rat has been restricted for 35-40 days to this diet, experience has shown that the cartilage and adjacent portion of the metaphysis in the long bones will be entirely free from calcium salts. If now cod-liver oil is administered to the amount of 3 per cent of the diet, calcium salts are deposited in the cartilage on the epiphyseal side of the metaphysis. This amounts to the reappearance of the provisional zone of calcification. From the work of Schmorl and others we know this phenomenon to be the beginning of the healing of the lesion. The deposited salts appear in longitudinal sections


32


[No. 371


of the bone as a fine line crossing the proliferative zone of the epiphyseal cartilage transversely.

We have found that the administration of cod-liver oil to the amount of 2 per cent of the diet causes the line of calcification to appear in the cartilage within five days.

We have recently observed that when young rats with rickets are made to fast for periods of three to five days (distilled water only being oifered). liealing begins in exactly the same way as it does when suitable amounts of cod-liver oil are administered.

Ten animals, as indicated in Table I, were fed on our diet No. 3143. At the expiration of tlie preparatory period they were placed in special cages and starved for 3-5 days.* They were then killed with chloroform and autopsied. The long bones were bisected longitudinally, immersed in a 1 per cent solution of silver nitrate, and examined under the microscope after exposure to light for the presence of a newly deposited provisional zone of calcification. The results of this gross examination were checked by the study of celloidin or frozen sections.

TABLE I Data DESCEn'xmi; op Rats Employed for Showino the Effects op

Fasting on the Initution of the Healing Process in Rickets


Number of rats


Age in days when put on diet


Days on diet


Age in days at death


Days

starved


Sex


Weight at death


1183


60


70


130


5


d


82


1221


4.i


64


109


5*


d


87


1341


45


49


94


2*


9


101


1345


55


49


104


3*


9


85


1358'


60


55


115


3*


d


125


1364


65


30


95


3<^


d


80


1365


65


30


95


3*


9


80


1366


65


30


95


3


9


68


1368


60


35


95


3


9


75


1369


60


35


95


3


9


75


Found dead on the morning of the day indicated. The results of starvation were controlled by the examination of animals which had been raised in the same cages on diet No 3143 at the same time, but had not been staiTed.j Not one of the starved animals failed to show reformation of the provisional zone of calcification, and other evidences of the healing of the rachitic process. None of the controls had a vestige of calcium in the cartilage or metaphysis.

Since we have found that even on diet No. 3143 rats wliich were exposed daily to sunlight in the summer from the begin

The cages employed for the fasting rats had wide mesh wire bottoms, so that the feces passed through, and it was impossible for the animals to cat their stools. No bedding of any kind was available for the rats to eat.

t We have sections from the bones of nearly 100 animals which were maintained on diet No. 3143 under the same conditions as our test animals. These served as an additional control for the test.


ning of the experiment did not develop rickets,' three methods are now known of inducing the deposition of calcium salts in the cartilage of the bones of rachitic rats, viz., through starvation, exposure to light, and through the administration of cod-liver oil.

It is difficult to think of any single explanation for the common effect produced by cod-liver oil, sunlight and starvation on the skeleton of the rat rendered rachitic by means of a faulty diet. The possibility has been suggested that the effect of codliver oil may be attributable to physical force of one kind or another. This view we have discussed more fully in another place. But even if the effect of cod-liver oil should be the result of a physical force rather than a chemical reaction it does not follow that one could generalize regarding the other vitamins and accept the view that they necessarily act in an analogous manner. The analogy between the effects on the body of vitamin preparations and of the hormones is so striking that we are not justified in hastily abandoning the view that the vitamins are organic substances. Two at least of the hormones, thyroxin and adrenalin, are definite organic compounds.

Starvation causes healing of the rickets in a rat just as do cod-liver oil and sunlight, and the mechanism of the deposition of calcium salts in the proliferative cartilage may be the same. But cod-liver oil and sunlight improve the general condition of the animal. This is indicated by increased growth, longevity, muscular activity, storage of fat, and sexual potency. Starvation, on the other hand, causes the death of the rat in a few days. Its effects on the animal are favorable only in that they cause the healing of rickets. The mode of action of the three agents must be entirely different. It seems necessary to think that sunlight and cod-liver oil act by enabling the cells to carry on, even when their food supply is unfavorable. In other words, they aid the organism to adapt itself to an adverse environment. Starvation, on the other hand, must produce healing of the rickets at the expense of other body tissues. We cannot disprove at present the possibility that the cell functions more effectively when it is relieved of the load which a faultj' diet imposes on it. However, during starvation certain tissues are destroyed for the upkeep of the supply of nutriment for those whose integrity must be safe-guarded to preserve life.

We have shown that the administration of cod-liver oil is followed by healing of rickets in rats even when they are receiving a diet with a faulty calcium-phosphate ratio. It may be that during the disintegration of protoplasm, which is consequent upon stan^ation, some substance is liberated which permits the animal to readjust the salt ratio within itself. But although it is possible that starvation liberates from the tissues an organic factor or factors, such as are in cod-liver oil, it is not necessary to account for healing in this way. Providing calcium and phosphorus are present in the proper proportions, normal calcification of the skeleton will occur. When the defects in the diet are such as they happen to bo in the diet which we employed in these experiments (diet No. 3143), it would only be necessaiy for phosphorus to be liberated into the blood stream from disintegrated protoplasm (muscle for ex


THE JOHNS HOPKINS HOSPITAL BULLETIN, JANUARY, 1922



Fig. 1.- -l>lH.iM,,,iri animal whicli li:nl n i fication of tin ciniili


i|ili of -d >\'(.-tiun friuii tlie rachitic rd diet No. 3143. There is no calciil cartihige or of the metaphysis.



Fig. 2.— Photomicrograph .-^howiii:: ;i,i i . -lurination ol a pre \i?ional zone of calcification (Ca) in the bone of a rachitic aniiiu which had been star\-ed for .5 days.


Januaey, 1922]


33


ample) in order to cause calcium deposition to occur in the cartilage. Animals on diet No. 3143, like rachitic children, have much less than the normal amount of phosphorus in the circulating blood. The administration of cod-liver oil or radiation from the mercury vapor quartz lamp causes the phosphorus in the blood to rise and it is not improbable that the healing of rickets which results from starvation is the immediate result of an equivalent increase of this element in the circulating blood. Just as soon as the load of a defective diet is removed and the body is forced to draw on its own tissues for maintenance of life and function, stored foodstuffs are released into the blood stream as the result of a process of selective tissue decomposition.

These experiments furnish the first anatomical proof of the beneficial effect of starvation on the animal body. The good effects of fasting are given a new meaning, because the organism is able to adapt itself to pathogenic distortions of normal metabolic ratios when the burden of carrying on exogenous metabolism is removed. Since the starving body is capable of readjusting abnormal relations within itself it is easy to understand the benefit derived by a diabetic from occasional hunger daySj and why it is that the wasted athreptic infant does not develop rickets.


Eickets has certain of the characteristics of a deficiency disease because a certain substance contained in cod-liver oil and elsewhere corrects an anatomical condition which develops when the calcium and phosphorus in the diet are present in wrong proportions. Yet rickets has a feature entirely distinct from beri-beri, scun'v, and xerophthalmia. The relation between two inorganic elements determines the extent of the animals' need for the organic factor wliich cod-liver oil furnishes. No such relationship between a vitamin and any other food substance has been clearly demonstrated in any other condition.

BIBLIOGRAPHY

1. E. V. McCollum, Nina Simmonds, P. G. Shipley, and E. A. Park. Studies on Experimental Rickets. VIII. The Production of Rickets by Diets low in Phosphorus and Fat-soluble A. Jour. Biol. Chem., 1921, XLVII, 507.

2. P. G. Shipley, E. A. Park, E. V. McCollum, Nina Simmonds, and H. T. Parsons. Studies on Experimental Rickets. II. The Effect of Cod-Liver Oil Administered to Rats with Experimental Rickets. Jour. Biol. Chem., 1921, XLV, 343.

3. E. A. Park, G. F. Powers, P. G. Shipley, E. V. McColhim, and Nina Simmonds. Studies on Experimental Rickets. XIV. The Prevention of the Development of Rickets in Rats by Sunhght. Jour. Amer. Med. Asso., 1921.


NOTES AND NEWS


Dr. Joseph Akerman is Associate Professor of Obstetrics in the Medical Department of the University of Georgia, Augusta, Ga.

Dr. John H. Baird is Associate in Surgery, The Medical College of Virgmia, Richmond, Va.; also Associate in Oral Surgery at the same institution.

Dr. Stanhope Bayne-Jones is Associate Professor of Bacteriology and Pathology, The Johns Hopkins Medical School.

Dr. Smiley Blanton is Professor of Mental Hygiene, University of Wisconsin, and Attending Specialist at U. S. Public Health Service Hospital No. 37, Waukesha, Wis.

Dr. E. Bates Block is now Professor of Neurology and Psychiatry, Medical Department, Emory University.

Dr. IMontague L. Boj'd is Urologist to the Georgia Baptist Hospital, Atlanta, to the Grady City Hospital, Atlanta, and to the Wesley Memorial Hospital, Atlanta.

Dr. Eveleth W. Bridgman is Associate in Clinical Medicine, Johns Hopkins Medical School, Assistant Visiting Physician, Johns Hopkins Hospital, and Dispensary Physician, Johns Hopkins Hospital.

Dr. N. Worth Brown is Visiting Physician to the Toledo Hospital, Toledo, Ohio.

Dr. Nathaniel H. Brush is Attending Neuro-Psychiatrist to the Santa Barbara Cottage Hospital, Santa Barbara, Cal.; and Chief of the Neuro-Psychiatric Service in the Out-Patient Department of the same Hospital.

Dr. C. Sidney Burwell, formerly of the Massachusetts General Hospital, is Resident Physician at The Johns Hopkins Hospital, and Instructor in Medicine in The Johns Hopkins Medical School.

Dr. George L. Carrington is on the surgical service of the house staff of the New Haven Hospital, New Haven, Conn.

Dr. David W. Carter, Jr., is Visiting Physician, B.iylor Hospital, Dallas, Texas, Visiting Physician, Parkland Hospital, Dallas, and Associate Professor of Medicine, Baylor University College of Medicine.


Dr. Howard L. Cecil is to be associated this year with Baylor Hospital and University, Dallas, Texas.

Dr. Malvern B. Clopton is now holding the following positions: Visiting Surgeon, St. Luke's Hospital, St. Louis; Associate Surgeon, St. Louis Children's Hospital, St. Louis; Assistant Surgeon, Barnes Hospital, St. Louis; and Associate in Clinical Surgery, Washington University Medical School, St. Louis.

Dr. Frank D. Conroy is Resident of the Contagious Department of the Cincinnati General Hospital, and Instructor in the College of Medicine of the University of Cincinnati.

Dr. Ernest K. Cullen is Attending Gynecologist, Harper Hospital, Detroit, and Professor and Director of the Department of Gynecology, Detroit College of Medicine and Surgery.

Dr. Edward C. Davidson is at present on the staff of the surgical department of the Heniy Ford Hospital.

Dr. Wilburt C. Davison is Assistant Dispensary Pediatrician, Johns Hopkins Hospital.

Dr. Clyde L. Deming is an Assistant Professor in Surgery, in charge of the Urological Sei-vice, at Yale University.

Dr. Alphonse R. Dochez is Associate Professor of Medicine, College of Physicians and Surgeons, Columbia University, and Visiting Physician, Presbyterian Hospital, New York City.

Dr. John C. Donaldson is Assistant Professor of Anatomy, School of Medicine, University of Pittsburgh.

Dr. Roger G. Doughty is a Fellow at the Cleveland Clinic, Cleveland, Ohio.

Dr. Edgar E. Duncan is Resident at the Woman's Hospital, Baltimore.

Dr. Ethel C. Dunham is Instructor in Pediatrics in the Yale University Medical School, New Haven.

Dr. William C. Duffy is Resident Surgeon, New Haven Hospital, and Instructor in Surgery, Yale University.


34


[No. 371


Dr. Franklin G. Ebaugh is Assistant Physician, New Jersey State Hospital, Trenton, also Visiting Psychiatrist, State Home for Boys, Jamesbury, N. J.

Dr. Arthur B. Emmons, 2d, is Director of the Harvard Mercantile Health Work and Instructor in the Practice of Industrial Medicine, Harvard Medical School.

Dr. Augustus R. Felty is Assistant in Medicine, Columbia University — Presbyterian Hospital, New York City.

Dr. Morris Flexner is Instructor in Clinical Microscopy in the Medical Department of the University of Louisville.

Dr. James C. Fox, Jr., is Assistant in Medicine, Yale University School of Medicine, and member of the staff of the New Haven Hospital.

Dr. Thomas O. Gamble is Associate Visiting Obstetrician, Albany Hospital, and Dispensary Obstetrician, South End Dispensary, Albany, N. Y.

Dr. Hildegarde C. Germann is teaching Anatomy and Physiology in the Blessing Ho.spital Training School for Nurses in Quincy, Illinois. Dr. Germann is also in practice in Quincy.

Dr. Herbert Z. GifBn is Chief of Section in Division of Medicine in the Majo Clinic, Professor of Medicine on the Mayo Foundation for Medical Education and Research, Graduate School, University of Minnesota.

Dr. Calvin H. Goddard is now an Assistant Director, in charge of the Out-Patient Department, Johns Hopkins Hospital.

Dr. Emil Goetsch is Professor of Surgery, at the Long Island College Hospital, and Surgeon-in-Chief, Long Island College Hospital, Brooklyn, N. Y.

Dr. L. W. Gorham is Clinical Professor of Medicine, Albany Medical College, and Assistant Attending Physician, Albany Hospital.

Dr. Jacob P. Greenhill is Chief Resident of the Chicago Lying-in Hospital, and Demonstrator in Operative Obstetrics at the Northwestern Medical School, Chicago.

Dr. Cb'de G. Guthrie is Chief of the Medical Service, Johns Hopkins Hospital Dispensary, Associate Physician, Johns Hopkins Hospital, and Associate Professor of Medicine, Johns Hopkins Medical School.

Dr. Margaret I. Handy is Junior Chief in Pediatrics at the Delaware Hospital, Wilmington, Del.

Dr. Edward M. Hanrahan, Jr., is now Assistant Resident Surgeon, Union Memorial Hospital, Baltimore.

Dr. George Harrop is Resident Physician at the Presbyterian Hospital, New York City, and Instructor in Medicine at Columbia University.

Dr. R. F. Hastreiter is on the staff of the Clara Barton Hospital, Los Angeles, Cal.

Dr. David K. Henderson is now Phy.sician-Superintendent of the Royal As>-lum, Gartnavel, Glasgow, Scotland.

Dr. William H. Higgins is Associate in Medicine, Medical College of Virginia.

Dr. Frank Hinman is Visiting Urologist, San Francisco City and County Hospital; Urologist, University of California Hospital; Assistant Clinical Professor of Urology, University of California Medical School.

Dr. A. D. Hirschfelder is Professor and Director of Pharmacology, University of Minnesota, Minneapolis.

Dr. James M. Hitzrot is Associate Attending First Surgical Division, New York Hospital, New York City; Professor of Clinical Surgery at Cornell Medical School, New York City.

Dr. Walter R. Holmes is Associate in Gynecology, Emoiy University, Atlanta, Assistant Visiting Surgeon, Georgia Baptist Hospital, Atlanta, and Assistant Visiting Surgeon, Piedmont Hospital, Atlanta.

Dr. John G. Huck is now Assistant in Medicine, Johns Hopkins Hospital, and Instructor in Medicine, Johns Hopkins Medical School.


Dr. Henry T. Hutchins is Surgeon, Massachusetts Women's Hospital, Boston, and Consulting Gynecologist, Memorial Hospital, Pawtucket, R. I.

Dr. Albert Keidel is Associate in Clinical Medicine, and Physician in Charge, Syphilis Department, John Hopkins Medical School.

Dr. Norman M. Keith is at present Assistant Professor of Medicine of the University of Minnesota (Mayo Foundation), Rochester, Minn.

Dr. Ferdinand C. Lee is Instructor in Anatomy, Johns Hopkins Medical School.

Dr. Charles S. Levy is Chief Surgical Resident at the Hebrew Hospital, Baltimore.

Dr. D. S. Lewis is Assistant Physician at the Royal Victoria Hospital, Montreal.

Dr. Frederick H. Linthicum is now in private practice in Los Angeles, Cal.

Di'. Hans Lisser is Assistant Clinical Professor in the Department of Medicine, University of California Medical School, and Chief of the Ductless Gland Clinic, University of Cahfornia Out-Patient Department, San Francisco, Cal.

Dr. Herbert M. Little is Assistant Professor in Obstetrics and Lecturer in Gynecology, McGill University, Montreal, and Associate Gynecologist, Montreal General' Hospital, and Obstetrician, Montreal Maternity.

Dr. Arthur J. Lomas is now Superintendent of the University Hospital, Iowa City, Iowa.

Dr. David R. Lyman, who has been Medical Superintendent of Gaylord Farm Sanatorium for some time, is now Clinical Lecturer on Tuberculosis, Yale Medical School.

Dr. Irving P. Lyon is Associate Professor of Medicine, University of Buffalo, Attending Physician to the Buffalo General Hospital, the Buffalo City Hospital, the Diagnostic Clinic (Buffalo City Hospitals and Dispensaries), and Consulting Physician to the New York State In.stitute for the Study of Malignant Diseases.

Dr. L. K. McCafferty is Clinical Assistant, Skin and Sj'philis, Vanderbilt Chnic, Columbia University, and Assistant in the OutPatient Department, Skin and Sj'philis, Presbyterian Hospital, New York City.

Dr. William S. McCann, formerly of the Russell Sage Institute, Bellevue Hospital, is now Associate Physician, Johns Hopkins Hospital.

Dr. James C. McClelland is Demonstrator in Surgery in the Toronto General Hospital, which also includes an appointment on the Univeisity of Toronto Staff.

Dr. William B. McClure is Junior .attending Pediatrist, Evanston Hospital, Evanston, Illinois, Assistant Attending Physician, The Children's Memorial Hospital, Chicago, Assistant Director, The Otho S. .A.. Sprague Memorial Institute, Laboratory of the Children's Memorial Hospital, Chicago, and is doing graduate teaching in the Children's Memorial Hospital.

Dr. Albert McCown is engaged in private practice in Tacoma, Washington.

Dr. Ralph H. Major is Professor of Internal Medicine at the University of Kansas, Rosedale, Kans.

Dr. Verne R. Mason is engaged in private practice in Los Angeles, Cal.

Dr. Kenneth F. Maxcy is Assistant Surgeon in the Regular Corps of the Service, and stationed at Field Headquarters, Memphis, Tenn.

Dr. Milo K. Miller holds the position of Pediatrician, in The Clinic, and Visiting Physician, Children's Free Dispensaiy, South Bend, Ind.

Dr. Joseph E. Moore is Instructor in Clinical Medicine, Johns Hopkins Medical School, Baltimore.


January, 1923]


35


Di'. Hugh J. Morgan is now Assistant Resident Physician at the Hospital of The Rockefeller Institute for Medical Research, New York City.

Dr. I. William Nachlas is at present Assistant in Orthopedic Surgery, Johns Hopkins Medical School, Assistant Dispensar>' Orthopedist, Johns Hopkins Hospital, Adjunct in Orthopedic Surgery, Hebrew Hospital, and Orthopedic Surgeon, Robert Garrett Hospital, Baltimore, Md.

Dr. Patrick I. Nixon is Gynecologist, Robert B. Green Memorial Hospital, San Antonio, Texas.

Dr. Charles C. Noms is Assistant Gynecologist to the Hospital of the University of Pennsj'lvania, Gynecologist and Obstetrician to the Philadelphia General Hospital, Gynecolgical and Obstetrical Radiologist to the Philadelphia General Hospital, Gynecologist to the Children's Hospital, Philadelphia, and Gynecologist and Obstetrician to the Henr\- Phipps Institute, Philadelphia.

Dr. Edward Novak is Dispensary Physician, Johns Hopkins Hospital, and Attending Phj'sician, South Baltimore General Hospital.

Dr. John R. Oliver is Assistant Dispensaiy Psychiatrist, Phipps Clinic, Johns Hopkins Hospital. Chief Medical Officer to the Supreme Bench of Baltimore City, Lecturer on Medical Jurisprudence at the Law School of the University of Marjdand, and Associate Editor of the Journal of the American Institute of Criminal Law and Criminology.

Dr. John K. Ormond is Assistant Surgeon-in-Chief at the Henry Ford Hospital. Detroit, Mich.

Dr. Walter W. Palmer is now Bard Professor of Medicine, College of Ph.vsicians and Surgeons, Columbia University, and Director of Medicine of the Presbyterian Hospital, New York City.

Dr. Katherine Pardee is engaged in post-graduate study as Fellow in Internal Medicine on the Mayo Foundation at Rochester, Minnesota.

Dr. Kenneth A. PlielpS is Instructor in Oto-Larj'ngology and Ophthalmology, at the University of Minnesota, Minneapolis.

Dr. Winthrop M. Phelps is taking a combined house-officership in orthopedics in Boston — in the Children's Hospital until May, 1922. and in the Massachusetts General Hospital from Maj- to November, 1922.

Dr. Lawrence Post is Chnical Assistant in Ophthalmology, Washmgton Universitj- Medical School, and instructor in specially arranged post-graduate courses.

Dr. Mary Putnam is Dispensary Physician at the Children's Hospital, Boston, Massachusetts.

Dr. Arnold R. Rich, who has a leave of absence for one year, is Associate in Pathology, Johns Hopkins Medical School, Baltimore.

Dr. G. Canby Robinson is Acting Professor of Medicine, Johns Hopkins Medical School, Baltimore, and Acting Physician-in-Chief, Johns Hopkins Hospital.

Dr. Stephen Rushmore is Visiting Obstetrician, Evangeline Booth Hospital, Consulting Gynecologist, Symmes Arlington Hospital, Associate Professor of Gynecology, Tufts College Medical School, Boston, Mass,

Dr, S, W. Schaefer is an Instructor in the Colorado School of Tuberculosis in Colorado Springs, Colo.

Dr. Ralph B. Seem, was granted in April, 1921, one year's leave of absence from the Albert Billings Memorial Hospital, University of Chicago, to be Acting Superintendent of the Union Peking Memorial College Hospital, Peking, China,

Dr, Albert B. Siewers is Assistant in Neuropsychiatry in the Medical School, Syracuse University and in the Syracuse Free Dispensary.

Dr. J. Morris Slemons is engaged in private practice in Los Angeles, California.

Dr. Joseph T. Smith, Jr., is Associate in Obstetrics and Instructor in Gynecology, Western Reserve University, Meilical School, Cleveland.


Dr. Walter R. Steiner is Visiting Physician, The Hartford Hospital, and Consulting Physician, The Manchester Memorial Hospital, the New Britain General Hospital, the Bristol Hospital, and the Middlesex Hospital (Middletown), and Lecturer on Medicine, Kennedy School of Missions, Hartford Theological Seminary, Hartford, Conn.

Dr. Heniy A. Stephenson is Assistant Clinical Professor of Obstetrics and Gynecology, Stanford University, San Francisco.

Dr. A. Raj'mond Stevens is Clinical Professor of Genito-Urinary Surgery, New York University, New York City.

Dr. Emerson L. Stone is Assistant in Obstetrics and Gynecology, Yale Medical School, and Assistant Resident in Obstetrics and Gynecology, New Haven Hospital, New Haven, Conn,

Dr, William S. Thayer is now Visiting Physician, Private Wards, Johns Hopkins Hospital.

Dr. Thomas H. Thomason is Assistant in Surgeiy, Johnson and Beall's Hospital, Fort Worth, Texas.

Dr. Clara M. Thompson is at present engaged in a rotating interneship at the New York Infirmary for Women and Children New York City.

Dr. Martillus H. Todd is at present Chief Surgeon of the plant of the United States Coal and Coke Company at Lynch Mines, Kentucky.

Dr. Margaret Tyler is Instructor in Obstetrics and Gynecology in the Y'ale Medical School, New Haven, Corm.

Dr. Karl H. Van Norman is Superintendent of the Charles T. Miller Hospital, St. Paul, Minnesota, having assumed his duties on December 1.

Dr. Allen Voshell is now connected with the University of Virginia Medical School, and the University Hospital, University, Va., with the titles of Adjunct-Professor of Surgery in charge of Orthopedics, and Orthopedist to the University Hospital.

Dr. Leslie T. Webster is Assistant in the Division of Pathology an<l Bacteriology. Rockefeller Institute for Medical Research.

Dr. Winford O. Wilder is Consulting Urologist at the Mercy Hosjiital, Springfield, Mass., and at the Cooley Dickinson Hospital, Northampton, Mass.

Dr. Lawson Wilkins is Assistant Dispensary Physician, Harriet Lane Dispensary, Johns Hopkins Hospital, and practicing Pediatrician in Baltimore.

Dr. Le Grand Woolley is Obstetrician, Junior Staff, Dr. W. H. Groves, L.T. S. Hospital. Salt Lake City, and Instructor in Gross Anatomy, University of mah Medical School, Salt Lake City, Utah.

Dr. Paul G. Woolley is Director of the Laboratories of the Herman Kiefer Hospital. Detroit, and Associate Professor of Pathology in the Detroit College of Medicine and Surgen,', Detroit.


NOTES ON NEW BOOKS

The Principles and Practice oj Medicine. 9th Edition. By the late Sir Willum Osub and Thomas McCrae, M. D. (New York and London: Appleton and Company, 1920.) Osier's Principles and Practice of Medicine has stood out for so many years as a remarkably concise and accurate presentation of the facts of internal diseases that comment on the basic characteristics of the book is .superfluous. One is ever anew impressed by the genius which has gathered into brief sections the essential matter of a broad and ill-defined domain. Sir William always took great interest in the book — in a sense his magnvmi opus — and expressed the desire and hope that its existence might be continued. In the preface to the ninth edition Dr. McCrae outhnes the more immediate purpose of the present revision which adds to the previous material the recent discoveries and advances in medicine. The engaging descriptions, however, of the natural history of disease will always be the most valuable feature of the book. A., L. B,


3G


[No. 371


THE JOHNS HOPKINS HOSPITAL REPORTS


VOLUUE I. 423 pa^es, 99 plates.

VOLUME II. 670 pages, with 28 plates and figures.

VOLUME III. 766 pages, with 69 plates and figures.

VOLUME IV. 604 pages, 33 charts and Illustrations.

VOLUME V. 480 pages, with 32 charts and illustrations.

The Malarlnl Fevers of Baltimore. By W. S. Thaieb, M. D., and

J. Hewetson. M. D. A Study of some Fatal Cases of Malaria. By Lewellts P. Barker. M. B.

Studies in Typhoid Fever. By William Osleb, M. D., with additional papers by G. Blumeb, M. D., Simon Flexner. M. D.. Walter Keed, M. D., and H. C. Parsons. M. D.

VOLUME VI. 414 pages, with 79 plates and figures.

VOLUME VII. 637 pages with illustrations.

VOLUME VIII. 652 pages with illustrations.

VOLUME IX. 1060 pages, 66 plates and 210 other illustrations.

Contributions to the Science of Medicine, Dedicated by his Pupils to William Henrt Welch, on the twenty-fifth

anniversary of his Doctorate. This volume contains 38 separate

papers. VOLUME X. 616 pages, 12 plates and 26 charts. VOLUME XI, 666 pages, with 38 charts and illustrations. VOLUME XII. 548 pages, ^2 plates and other illustrations. VOLUME XIII. 605 pages, with 6 plates, 201 figures, and 1 colored chart. VOLUME XIV. 632 pages, with 97 figures.

VOLUME XV. 642 pages, with 87 illustrations.

Twelve papers on pneumonia. By Des. Chatard. Fabtan, Emerson,

Marshall, McCrae, Steiner, Howard and Hanes. A Study of Diarrhoea in Children. J. H. Mason Knox, Jr., M. D., and

Edwin H. Schorer, M. D. Skin Transplantation. By John Staigb Davis. M. D. Epidemic Cerebrospinal Meningitis and Serum Therapy at The Johns

Hopkins Hospital. By Frank J. Sladen, M. D.

VOLUME XVI. 670 pages with 161 figures.

Studies in the Experimental Production of Tuberculosis In the Genitourinary Organs. By George Walker. M. D.

The Effect on Breeding of the Removal of the Prostate Gland or of the Vesiculce Scminales. or of Both ; together with Observations on the Condition of the Testes after such Operations on White Rats, By George Walker, M. D.

Scalping Accidents. By John Staige Davis, M. D.

Obstruction of the Inferior Vena Cava with a Report of Eighteen Cases. By J. Hall Pleasants. M. D.

Physiological and Pharmacological Studies on Cardiac Tonicity In Mammals. By Percival Douglas Cameron, M. D.

VOLUME XVII. 586 pages with 21 plates and 136 figures.

Free Thrombi and Ball Thrombi in the Heart. By Joseph H. Hewitt,

M. D. Benzol as a Leucotoxin. By Lawrence Selling, M. D. Primary Carcinoma of the Liver. By Milton C. Winternitz, M, D. The Statistical Experience Data of The Johns Hopkins Hospital, Baltimore,

Md., 1892-1911. Bv Frederick L. Hoffman. LL. D., P. S. S. The Origin and Development of the Lymphatic System. By Florence R.

Sarin. M. D. The Nuclei Tuberls Laterales and the So-called Ganglion Optlcum Basale.

Bv Edward P. Malone, M. D. Venous Thrombosis During Myocardial Insufficiency. By Frank J. Sladen.

M. D., and Milton C. Winternitz, M. D. Leukaemia of the Fowl : Spontaneous and Experimental. By Haret C.

SCHMEISSER, M. D.

VOLUME XVIII. 446 pages with 124 figures.

Fasciculus I. A Study of a Toxic Substance of the Pancreas. By B. W. Goodpasture,

M. D,, and George Clark, M. D. Old Age in Relation to Cell-overgrowth and Cancer. By B. W. GooD PASTDRE, M. D., and G. B. Wislocki, M. D. The Effect of Removal of the Spleen Upon Metabolism In Dogs; Preliminary Report. By J. H. King. M. D. The Effect of Removal of the Spleen Upon Blood Transfusion. By J. H.

King, M. D.. B. M. Bernheim. M. D.. and A. T. Jones, M. D. Studies on Parathyroid Tetany. By D. Wright Wilson, M. D.. Thornton

Stearns, M. D., J. H. Jannby, Jr., M. D., and Madge DeG. Thhelow,

M. D. Some Observations on the Effect of Feeding Glands of Internal Secretion

to Chicks. By M. C. Winternitz. M. D. Spontaneous and Experimental Leukaemia In the Fowl. By H. C

Schmeisser, M. D. Studies on the Relation of Fowl Typhoid to Leukaemia of the Fowl. By

M. C. Winternitz, M. D.. and II. C. Schmeisser. M. D. Hyaline Degeneration of the Islands of Langerhans In Pancreatic Diabetes.

Bv M. C. Winternitz. M. D. Generalized Miliary Tuberculosis Resulting from Extension of a Tubercular

Pericarditis Into the Right Auricle. By M. C. Winternitz, M. D.


Acute Suppurative Hypophysitis as a Complication of Purulent Sphenoidal

Sinusitis. By T. R. BoGGS. M. D., and M. C. Winternitz, M. D. A Case of Pulmonary Moniliasis in the United States. By T. R. BoGOS,

M. D., and .M. C. PiNCOi Fs, .M. D. Gauchers Disease (A Report of Two Cases In Infancy). By J. H. M.

Knox, M. D., H. R. Wahl, M. D., and H. C. Schmeisser, M. D. A Fatal Case of Multiple Primary Carcinomata. By E. D. Plass, M. D. Congenital Obliteration of the Bile-ducts. By James B. Holmes. M. D. Multiple Abscesses of the Brain in Infancy. By James B. Holmes. M. D. Gastric Carcinoma in a Woman of Twenty-six Tears. By R. G. Hdssei

M. D. Subdiaphragmatic Abscess with Rupture Into the Peritoneal Cavity Pol

lowing Induced I'neumothorax for Pulmonary Hsemorrhage. By R, G

HussEV, M. D. Heart Block Caused by Gumma of the Septum. By E. W. Briogema

M. D., and H. C. Schmeisser, .M. D. Analysis of Autopsy Records.

A. The Johns Hopkins Hospital. (Table Showing Percentage of

Autopsies. I

B. The City Hospitals, Bay View. (Table Showing Percentage of

Autopsies.) " The Monday Conferences."

Clinical Representatives on the Staff of the Department of Pathology. Donation.

Fasciculus II. The ROIe of the Autopsy in the Medicine of To-day. By M. C. Winternitz,

M. D, Experimental Nephropathy in the Dog. Lesions Produced by Injection

of B. bronchisepticus Into the Renal Artery. By M. C. Winternitz,

M. D.. and William C. Quinby, .M. D. Mesarteritis of the Pulmonary Artery. By M. C. Wintebnitz, M. D., and

H. C. Schmeisser. M. D. A Clinical and Pathological Study of Two Cases of Miliary Tuberculosis of

the Choroid. By Robert L. Randolph, M. D., and H. C. Schmeisseb,

M. D. The Blood-vessels of the Heart Valves. By Stanhope Bayne-Jones, M. D. Equilibria In Precipitin Reactions. By Stanhope Batne-Jones, M. D. Carcinoma of the Pleura with Hypertrophic Osteoarthropathy. Report of

a Case with a Description of the Histology of the Bone Lesion. By

Stanhope Bayne-Jones. M. D. The Interrelation of the Surviving Heart and Pancreas of the Dog in Sugar

Metabolism. By .Admont H. Clark, M. D. Congenital Atresia of the Esophagus with Tracheo-Esophageal Fistula

Associated with Fused Kidney. A Case Report and A Summary of the

Literature on Congenital Anomalies of the Esophagus. By E. D.

Plass. M. D. Ectopia Cordis, with a Report of a Case In a Fifteen-Month-Old Infant.

By James B. Holmes, M. D. Studies in the Mechanism of Absorption from the Colon. By Samuel

(ioLDSCiiMiDT. M. D., and A. B. Dayton. M. D. Report of Two Fatal Cases Following Percy's T/ow Heat Treatment of

Carcinoma of the Uterus. By V. N. Leonard, M. D., and A. B. Daytor,

M. D. The Relationship in Typhoid Between Splenic Infarcts and Perltonltl*

tinassociated with Intestinal Perforation. By A. B. Dayton. M. D. Left Duodenal Hernia. By A. B. Dayton. M. D.

Histological as Related to Physiological and Chemical Differences In Certain Muscles of the Cat. By H. Hays Biili.akd, M. D. A Method of Clearing Frozen Sections. By H. Hays Bollard. M. D. On the Occurrence and Significance of Fat in the Muscle Fibers of the

.\trlo- Ventricular System. By H. Hays Bollard, M. D. Studies on the Metabolism of Cells in vitro. 1. The Toxicity of a -Amino

Acids for Embyonic Chicken Cells. By Montrose T. Borrows, M. D.,

and Clarence A. Neymann, M. D. The Significance of the Lunula of the Nail. By Montrose T. Borrows,

M. D. The Oxygen Pressure Necessary for Tissue Activity. By Montrose

Burrows, M. D.

The Functional Relation of Intercellular Substances in the Body to Certain Structures In the Egg Cell and Unicellular Organisms. By

Montrose T. Borrows. M. D. Studies on the Growth of Cells in vitro. The Cultivation of Bladder and

Prostate Tumors Outside the Body. By Montrose T. Borrows, M. D..

J. Edward Burns, M. D.. and TosHio SrzoKL, M. D. The Study of a Small Outbreak of Poliomyelitis In an Apartment House,

Occurring in the Course of on Epidemic in a Large City. By Montbose

T. Borrows, M. D.. and Edwards A. Park. M. D. Papilloma of the I^arvux. Report of a Case Treated with Radium with

Resultant Chronic Diffuse Thyroiditis. By William C. Doffy, M. D. Analysis of Autopsy Records. Autopsy Statistics.

(a) Bay View.

(h) Johns Hopkins Hospital. Report of the Photographic Department. General Improvements, Donations.

VOLUME XIX. 358 pages with 29 plates. The Structure of Normal Fibers of Purkinje In the .\dult H

Their Pathological Alteration in Syphilitic M

STT.icnT. M. D.. and T. Wingate Todd. M. D. The Operative Story of Goitre. The Author's Operation

Halsted, M. D. Study of Arteriovenous Fistula with

Cai-landee. M. D.

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CONTENTS

  • Immunological Reactions of Bence-Jones Proteins. I. Differences Between Bence-Jones Proteins and Human Serum Proteins. (Illustrated.) By S. Batxe-.Joves and D. Wkight Wilson'
  • Yaws. An Analysis of 1046 Cases in tlie Dominican Republic. (Illustrated.) By W. L. MOS.S and G. H. Bigelow
  • Studies on a Case of Chromic Acid Xephritis. (Illustrated.) By Ralph H. Major
  • Adaptation of Bacteria to Growth on Human Mucous Membranes with Special Reference to the Throat Flora of Infants. By Arthur L. Bloomfield
  • Dermoid Cysts of the Ovary. A Report of Four Cases. (Illustrated.) By Karl H. Martzloff

Titles of Papers Appearing During the Year 1931, Elsewhere than in the Bulletin', by the Present and Former Members of The Johns Hopkins Hospital and Medical School Staff


IMMUNOLOGICAL REACTIONS OF BENCE-JONES PROTEINS

I. DIFFERENCES BETWEEN BENCE-JONES PROTEINS AND HUMAN SERUM PROTEINS

By S. Batne-Jones and D. Weight Wilson (From the Department of Pathology aiid Bacteriology and of Physiological Chemistry, The Johns Hopkins University, Baltimore)


In a previous paper/ presenting a preliminary account of our study of the immunological reactions of Bence-Jones proteins, we have reported that by the use of the precipitin, complement-fixation and anaphylactic reactions we have been able to show that differences exist between the so-called BenceJones protein and the proteins of human serum, and also between preparations of Bence-Jones protein from various sources. While the specimens of Bence-Jones protein which we studied were all typical as regards their phases of coagulation and solution by heat in an acid medium, they differed considerably in their physical properties and in their immunological reactions. These differences were indeed so great that we were forced to regard the so-called " Bence-Jones body " as a group of similar, but not identical, substances, and to refer to the group as the Bence-Jones proteins. One of these I)roteins (No. 4) crystallized spontaneously in the urine of the patient who excreted it. This property permitted purification of the substance by recrystallization. An account of this patient (E) with a description of the protein has been published by Walters ' from the Mayo Clinic, where the patient was under observation on the service of Dr. Eowntree. The other specimens of Bence-Jones proteins used by us were


isolated from urine by various methods of precipitation. They will be described in detail in our paper dealing with their specific immunological differences. In the present report, we shall give the evidence in support of the contention that it is possible by the use of immunological methods to differentiate Bence-Jones proteins from the proteins of human serum.

With the more frequent use of purified proteins in immunological studies, an increasing number of them are being found to possess individual antigenic specificity. It has been known for a long time that the proteins of the lens of the eye and of spermatozoa have this characteristic, and more recently Wells and Osborne,' using purified vegetable proteins. Woods * using the pigment from the uveal tract, and Dale and Dakin " with crystalline albumin from the eggs of hen and duck, have established the fact that chemical composition, rather than biological origin, is the ba.sis of tlie specificity of these substances. The chemical and physical peculiarities of Bence-Jones proteins and their absence from the blood and urine of normal persons have conveyed the obvious suggestion that it might be possible by immunological studies to draw a distinction between the blood-proteins and Bence-Jones proteins. This phase of the question has occupied the attention of a number of investi


38


[No. 372


gators. The results, however, of the experiments of all except two students of this problem have been reported as indicative of an identity rather than a difference between these proteins. ' ' ° From accounts of the way in which the experiments were conducted, and from the results of our work, it seems that the uncritical use of mixtures of proteins has led to these equivocal results. The most notorious example of this is the work of Abderhalden and Rostoski,' which has influenced many of the subsequent immunological studies of Bence-Jones proteins and the opinions iipon the nature of Bence-Jones proteinuria. The patient studied by Abderhalden had chronic nephritis as well as multiple myelomata. As a consequence of the two diseases he excreted in his urine Bence-Jones protein and proteins which Cameron and Wells," in studies of the urine in nephritis, have shown to be identical with the proteins of human serum. Abderhalden injected the whole urine of this patient into a rabbit, and obtained an immune senmi which precipitated both Bence-Jones protein and human serum. In drawing his conclusions, however, he neglected the possibility that he was dealing with a mixture, and developed his ideas about the identity of Bence-Jones protein and serum proteins as if he had been using single pure antigens. It now seems probable that unless a Bence-Jones protein can be separated from urine by crystallization, and purified by recrystallization, it is impossible to separate it by any method of fractional precipitation from other proteins which are usually present with it in the urine.

The investigators who have succeeded in finding immunological differences between Bence-Jones proteins and serum proteins are Massini " and Hektoen.^' By means of the complement-fixation test, Massini was able to show that an antiserum to the preparation used by him fixed complement in the presence of higher dilutions of the Bence-Jones protein than of human serum. Hektoen's recently published preliminary note reports that by absorption of precipitins specific reactions can be obtained which sharply differentiate Benoe-Jones proteins from the proteins of human serum, even when mixed antigens are iised.

Reports of Experiments

In our experiments to discover, if possible, immunological differences between Bence-Jones proteins and the proteins of liuman serum, we used precipitin and complement-fixation reactions with the sera of rabbits immunized to human serum and to several preparations of Bence-Jones proteins, and anaphylactic reactions of guinea-pigs sensitized to these substances.

PRECIPITIN REACTIONS

The preeipitin-sera were prepared as follows :

Anti-human senim. Rabbit No. 161 immunized to human serum by 7 intravenous injections at intervals of 2 to 6 days. The first dose was 0.5 c. c, the last 12 c. c. of serum. Ten days after the last injection, the animal was bled, when its serum gave a precipitate with a 1-1000 dilution of human serum.

Anti-Bence-Jones protein sera: Rabbit No. 144 was immunized to the crj'stalhne Bence-Jones protein (No. 4) by six intravenous injec


tions of a 1 per cent solution of No. 4 at intervals of 4 to 6 days. The first dose was 2 c. c, the last 20 c. c. Ten days after the last injection, when the rabbit's serum caused a precipitate in a 1-1,000,000 dilution of a 4 per cent solution of No. 4. the animal was bled.

Rabbit No. 153 was injected intravenously at intervals of 4 to 6 days with amounts of a 0.5 per cent solution of Dr. J. Ro.senbloom's preparation of Bence-Jones protein increasing from 3 c. c. to 20 c. c. Six days after the fifth injection, the rabbit was bled. Precipitin titer 1-100,000 (antigen dilution).

Rabbit No. 189 was immunized to a preparation of Bence-Jones protein designated as " Simpson," given us by Dr. C. G. Guthrie. After 8 injections of amounts of 2-6 c. c. of a 2 per cent solution of this protein, at intervals of 3 to 5 days, the precipitin titer of this animal was 1-2000 (antigen dilution).

Rabbit No. 195 was immunized to a preparation of Bence-Jones protein denoted as " Farrel." and given to us by Dr. C. G. Guthrie. At intervals of six days, 1 c. c, 5 e. c, and 7 c. c. of a 3 per cent solution of this protein were injected intra\-enously. Five days after the last injection, when the animal was bled, the titer of the serum was 1-8000 (antigen dilution).

With the exception of No. 4, which was the crystalline BenceJoues protein, the other Bence-Jones proteins were non TABLE I Precipitin Reactio.ns


Antihuman serum 161. Precipitation with antigen


Human serum

Bence-Jones protein (4 per

cent solution). No. 4 (crystalline)

No. 1

Coag. No. 2

Coag. R. urine

R. urine

Taylor

Farrel

Simpson (2 per cent)

Dine

Dine urine

X

X urine

Rosenbloom



Dilution <


t antigen



1-10


1-100


++++


+ + +


+ +






+ +





++


+



++


+



++


+


+



+


++


+


+



+


++


+


++


+



+++


++


+


+


+


0,


crystalline, and had been isolated from urine by various methods of precipitation.

The precipitin tests were done in clean sterile tubes, witli clear solutions of the sera and antigens. False results due to bacterial growth were eliminated. The antigens, in various dilutions, were layered upon the anti-sera, and the first reading was made by noting the presence or absence of precipitate at the plane of junction of the two fluids one hour after their apposition. The fluids were then mixed, placed in the incu


February, 1923]


39


bator at 3'i° C. for 24 hours, and a second reading taken of the sediment in the bottom of the tubes. The controls, which are omitted from the following tables, were made by mixing equal amounts of each component with normal salt solution and incubating these mixtures with the series of tests. Wlien precipitate occurred in the controls, the corresponding tests were discarded or repeated with fresh solutions. In the tables, the signs indicate the amount of precipitate formed ; denoting no precipitate, + + + + a very heavy precipitate. The results of the precipitin reactions with antihuman serum arc summarized in Table I.

The advantage gained from the use of a pure crystalline protein is at once apparent from the data presented in Table I. The antiserum to lumian serum proteins does not precipitate the crystalline ] reparation of Bence-Jones protein. On tlie other hand, antihuman serum forms a precipitate with all noncrystalline Bence-Jones proteins which have been salted-out

TABLE II Precipitin Reactions



Precipitation with


Dilution of antigen




1-10 1-100


1-1000


1-2000


1-100,000


144. Anti-BenceJones, No. 4.


Bence-Jones. No.4

(4 per cent). Human serum. ..




-1- \+++


•H--H


-h-(--H


-(--t--h



153. Anti-BenceJones, Rosenbloom.


Bence-Jones, Rosenbloom (4 per cent).




+ + + + ++


-f-H-l-t--l--f


-H--I -f


+



189. Anti-BenceJones, Simpson.


Bence-Jones,

Simpson (2 per

cent). Human serum...


+ +


+


-1--)


-t




195. Anti-BenceJones, Parrel.


Bence-Jones,

Farrel (4 per

cent). Human serum...


-I- + -H -t

-I-I--I--1--H- +

++ +


-i-f







or otherwise precipitated from urine. With the whole urine from patients with multiple myelomata, containing presumably more protein identical with the proteins of serum, the precipitation by antihuman sermn is greater than with the fractions of these urines containing the Bence-Jones protein.

In Table II are summaries of precipitin reactions with human serum, antisera to several preparations of Bence-Jones proteins and their homologous antigens.

From Table II it is seen that a very potent antiserum to the crystalline Bence-Jones protein does not precipitate human serum, while the antisera to the other less pure preparations of Bence-Jones proteins give "cross-reactions," precipitating both their homologous antigens and human serum.

As Hektoen " has pointed out, the method of absorption of precipitins can be used, when mixtures of proteins are unavoidable, to demonstrate differences between serum proteins and Bence-Jones proteins. There is, however, much to be learned about this method before the results obtained witli it can be correctly interpreted."' " In its use, we encountered a number of difficulties. In the first place, precipitating sera, such as those used by us, are no longer active when diluted more than 1 to 20, or 1 to 40. This places a narrow limit upon the


applicability of the metliod, as it may be necessary during the process of absorption to dilute the serum beyond its effective jirecipitin-concentration. When dilution of the serum is avoided, the so-called " prozone " may interfere with the formation of a precipitate during the phase of absorption. In many mixtures of undiluted serum and a concentrated solution of its protein antigen no precipitate occurs, yet when the mixture is diluted, the precipitate appears. In this case, of course, neither the antigen nor antibody is removable by centrifugation, and the delayed precipitation due to the first reaction often occurs when salt solution or the diluted antigens to be tested are added subsequently. It may be that the hydrogen-ion concentration of the fluids is a factor of prime importance in the precipitin reaction. A concentration of hydrogen-ions in the region of the isoelectric point of serum globulin may permit a precipitin reaction to progress to completion even in mixtures of undiluted immune serum and concentrated solutions of proteins. We have not investigated this point thoroughly. The results, however, of our experiments with the absorption of precipitins are unequivocal when the " ring-test " is used, readings of the precipitate at the junction of serum and antigen solutions being taken within one or two hours after the one has been layered upon the other. When allowed to stand for 18 hours or more, a slow precipitation

TABLE III Ab-sorption op Precipitins


Anti-BenceJones, Farrel, 105.


Human serum


Human serum

Bence-Jones.

No. 4. Simpson

Farrel

Human serum

Bence-Jones,

No, 4, Simpson

Farrel

Human serum

Bence-Jones,

No. 4. Simpson

Farrel

Human serum

Bence-Jones,

No. 4. Simpson

Fari^l

Bence-Jones,

Farrel. Human serum,

Bence-Jones,

Farrel. Human serum.


Dilution of antigen


s


o

g


-^


'-'


-1


-t


-1


-t

± -1-4

-l-l




+++


++





often occurs in all sera, including the controls, showing that the original reaction had not been complete but was continuing under the conditions permitted by the subsequent dilution.


40


[No. 373


The technic used in absorbing the precipitins was as follows : With precautions to prevent bacterial growth in the tubes,

1 c. c. of antiserum was mixed with 1 c. c. of a 2 or 4 per cent solution of a Bence-Joues protein. This mixture was incubated at 37° C. for 24 hours, placed in the ice-chest for 18 hours and then centrif uged until clear. The usual series of tests and controls were then made with this absorbed serum, diluted one to two as a consequence of the addition to it of an equal quantity of the antigen for the phase of absorption. A control to determine the effect of simple dilution and manipulation upon the antiserum was made by carrying a mixture of equal parts of immune serum and salt solution through the stages of incubation and absorption.

Antihuman serum No. 161 was absorbed in this manner with 4 per cent solutions of the crystalline Bence-Jones protein, and the Farrel preparation of Bence-Jones protein, and with a

2 per cent solution of the Simpson Bence-Jones protein. To show the effect of the reversal of this procedure, an antiserum to the Earrel specimen of Bence-Jones protein was absorbed with human serum. The results of these absorption tests are summarized iu Table III.

Several of the experiments summarized in Table III show again that antihuman serum does not precipitate the crystaUine preparation of Bence-Jones protein (Ko. 4) and, further, that this Bence-Jones protein does not absorb precipitin from antihuman serum. The data demonstrate that by absorption of antihuman serum with certain preparations of Bence-Jones proteins it is possible to remove from this immune servmi the precipitin responsible for the flocculatiou with solutions of these preparations, while only shghtly lowering the titer of the serum for its homologous antigen, human serum. Confirmation of the result is obtained when an antiserum to one of these preparations of Bence-Jones protein (Farrel) is absorbed with human serum. The conclusion seems inevitable that some of these non-crystalline preparations of Bence-Jones proteins contain traces of human serum proteins, while, in this respect, the crj'stalline Bence-Jones protein acts as a single antigen.

COMPLEMEXT-FIXATION RF.ACTIONS

For the complement-fixation reactions antihuman sermn was tested against the various Bence-Jones proteins, and antisera to the crystalline and Rosenbloom Bence-Jones proteins were tested against human serum. Preliminary titrations were made with all sera and solutions of the proteins to determine their anticomplementary action, and in the tests the anticomplementary controls contained twice the amount which did not inhibit hemolysis. This required the«use of sera diluted one to two, and 1 to 20 dilutions of the solutions of Bence-Jones proteins. These dilutions, as has been pointed out, are just on the limit of effective concentration of precipitin. The results of the complement-fixation tests, therefore, indicate broad relationships, as closer interactions are lost through the dilution. In the tests, 0.25 c. c. was the tmit volume of each component used. The usual antisheep amboceptor, with a titer of 1 to 3200, and guinea-pig serum as complement were employed. After the mixtures of antiserum, protein solution


and complement were made, they were incubated in the waterbath at 37° C. for one hour. At the end of that time, three units of amboceptor were added, the 2.5 per cent suspension of sheep cells placed in the tubes, and all returned to the waterbath. Readings were taken at the end of one hour, or when all the proper controls had cleared. The results of these tests are collected in Table IV, in which -|- -|- -I- -f indicates complete fixation (absence of hemolysis), and lesser degrees of fixation by proportionally fewer -|- signs.

The complement fixation reaction, according to this method, combines a differentiation based upon dilution of the antisera and antigens with the differences due to fixative power of

TABLE IV Complement Fixation Reactioxs


Serum


Antigen


Cone.


Dilut.


Result


Anti-human serum, Ifil. Diluted 1-2.


Human serum

Benee-Jonos protein:



1-40


++++



Xo. 4


i%


1-20




Xo. 1


4%


1-20




Coag. R. urine


4%


1-20






1 20


+ -1+




Tavlor


4% 4% 2%


1 20




1 20



Simpson


1-20




4% 4% i% 4%


1-20




X


1 20





1 20


+++ ++++



A n t i - Bence - Jones, Xo. 4. Serum 144. Diluted 1-2.


Bence-Jones, Xo. 4. . . Human serum


1-20

1-20


A n t i - Banee - Jones, Rosenbloom. Serum 153. Diluted 1-2.


Bence-Jones, Rosenbloom. Human serum


4%


1-20 1-20


++++



specific precipitates. Its effect, therefore, is to indicate only broad relationships. The results thus obtained, as presented in Table IV, confirm the results of the precipitin reactions in showing immunological distinctions between Bence-Jones proteins and the proteins of human serum.

AXAPHTLACTIC REACTIOXS

Anaphylactic reactions were studied upon the guinea-pig as a whole and on excised uterine horns of young virgin guineapigs. The animals in this series were actively sensitized, except where otherwise stated, by an intravenous injection of 0.25 c. c. of human serum or of a 4 per cent solution of a Bence-Jones protein. After an interval of approximately three weeks, the animals were tested by an intra jugular injection of serum or Bence-Jones protein. The data of these experiments are presented in Table V.


February, 1922]


41


Anaphylactic reactions with the crystalline Bence-Jones protein were not as sharp as we desired, as it was difficult actively to sensitize guinea-pigs to this preparation. Both large and small doses of the crystalline protein were given on the first intravenous injection, and the animals were tested by a second intravenous injection of larger amounts after intervals of 18 to 27 days. With one series, fairly satisfactory results were obtained. The guinea-pigs of this lot received as the sensitizing dose injected intravenously 0.25 c. c. of a 4 per cent solution of the crystalline Bence-Jones protein (No. 4) on January 27, 1921. On February 14, 21 days later, an intravenous injection of 1 c. c. of the 4 per cent solution of No. 4 produced a severe but not fatal shock, in guinea-pig No. 396. None of 10 guinea-pigs thus sensitized to Bence-Jones protein No. 4 showed any reaction to human serum.

TABLE V Anaphylactic Reactions


Human e Human e


Sensitizing dose


1 0.26 c. 1 0.25 c.


Beiice - Jones, Rosen bloom prep. 1.5 c. c, 17c sol.

Bence-Jones, Taylor's prep. 1 c. c. 1.5% sol


Human serum O.aS <

Hence -- Jones No.

. c. 0.57c sol. 10 minutes later : 2 <

human serum

pcrit.


Bence - Jones

. c. 17c sol. 10 minutes latei human serun


18 days

IS days 27 days 18 days 18 days


Intoxicating dose


ntri


No.


Bence - Jones, Kosenbloom prep. 1 c. c. 49'o sol.

Bence-Jones, Taylor'f prep, 1 c. c. 4% sol.

Human serum 0.25 c. e.


Human serum 0.25 c- c. Human serum 0.25 c. c


Typical anaphyla Death, one mir No reaction.


The anaphylactic reactions summarized in Table V and in the above paragraph demonstrate a complete difference between the crystalline Bence-Jones protein and the proteins of human serimi. On the other hand, guinea-pigs sensitized to human serum were also sensitive, though in less degree, to the Rosenbloom and Taylor preparations of Bence-Jones proteins, and guinea-pigs sensitized to these Bence-Jones proteins and to Bence-Jones protein No. 1 were extremely hypersensitive to human serum. From tlie results of the precipitin reactions, we are led to assume, in explanation of the crossed anapliylactic shocks, that the non-cry.stalline preparations of Bence-Jones proteins (No. 1, Taylor and Rosenbloom) contain traces of human serum proteins. These traces of serum proteins are sufficient to sensitize the animal to human serum, so that when a large amount of human serum is administered in the second injection, fatal anaphylactic shock occurs. Their content of serum proteins, however, is too small to produce a fatal reaction when they are injected in the amounts used by us into guinea-pigs primarily sensitized to human serum.


An analysis of these preparations of Bence-Jones proteins was permitted by the graphs obtained by the Schultz-Dale method." The a])paratus and solutions used in our studies of the anaphylactic reactions of isolated uterine muscle were essentially those described by Dale. Virgin guinea-pigs, sensitized to human serum or Bence-Jones proteins, were killed by a blow on the head about three weeks after the first injection. The uterus was removed, one horn was attached to the lever of a kymograph, and this strip of smooth muscle was then submerged in Ringer-Locke solution. The bath, 50 c. c. of RingerLocke solution, was kept at 37-38° C, and was supplied cou


Fk. 1. — Uterus of guinea-pig Xo. 1. Passively sensitized to Bence-Jones protein No. 4 by injection of 1 c. c. serum 144 into peritoneal cavity. Killed 18 hours later.

At A : 1 c. c. human serum.

At B: 1 c. c. 3% sol. Bence-Jones protein No. 4.

At C: 1 c. c. 3% sol. Bence-Jones protein No. 4.

tinuously with oxygen. Tracings of the movements of the strip of uterus were recorded on a smoked drum.

It was found more satisfactory passively to sensitize guineapigs to the crystalline Bence-Jones protein. This was easily accomplished by injecting intraperitoneally 1 c. c. of the serum of rabbit No. 144, which had been immunized to this BenceJones protein. The precipitin titer of this serum was 1 to 1,000,000, in terms of dilution of the antigen. Eighteen hours later, these guinea-pigs were found by tests to be hypersensitive to Bence-Jones protein No. 4.


-12


[No. 372


Kepresentative graphs, Figs. 1, 2 and 3, illustrate the results of some of the anaphylactic reactions with the isolated smooth muscle of the uterine horns of sensitized guinea-pigs. From Fig. 1 it is seen that the uterus of a guinea-pig sensitized to the crystalline Bence-Jones protein No. 4 did not react to human serum, and Fig. 2 shows that the uterus of a guinea-pig sensitized to human serum was not affected by the solution of this crystalline protein. Fig. 3 is of especial interest, as it is analogous to experiments on the absorption of antibodies. The uterus used in this test was from a guinea-pig actively sensitized to Eosenbloom's non-cry.-tnlliuo preparation of Bence


FiG. 2. — Uterus of guinea-pig No. 378, sensitized to human serum.

At A: 1 c. c. 4% sol. Bence-Joncs pi-otein No. 4. At B: 1 c. c. himian serum. At C: 1 c. c. hvmian serum.

Jones protein. When treated with a solution of this BenceJones protein, a contraction occurred. A subsequent application of the same protein failed to cause a contraction, showing that the muscle was desensitized to that antigen. It, however, remained hypersensitive to himian serum, as shown by the next phase of the curve, which records the contraction produced by the addition of human serum to the bath. After this second contraction, the uterus was specifically desensitized also to human serum. This analysis permits the definite conclusion that the Eosenbloom preparation of Bence-Jones protein is a mixture of a Bence-Jones protein and human serum proteins.


SUMMAEY AND CONCLUSIONS

In tlie possession of a crystalline Bence-Jones protein we had at our disposal an ideal substance for immunological studies. By crystallization, it could be freed from possible traces of serum proteins and thus permitted the use of a purified preparation to obviate the confused results which vitiate many immunological experiments. Its quality as an antigen was easily established, and the reactions dependent upon its antibodies were unequivocal. In contrast to this, tlie non-ci v.~tal


FiG. 3. — Uterus of guinea-pig sensitized to Bence-Jones protein (Rosenbloom).

At A: 1 c.c. 4% sol. Bence-Jones protein (Rosenbloom).

At B: 1 c. c. same solution.'

At C : 0.5 c. c. himian senun.

At D: 0.5 c. c. human serum.

line preparations of Bence-Jones proteiu.s, precipitated from the urine by fractionation with salts or heat, gave the " cross " reactions usually obtained with mixed antigens. Comparisons between the Bence-Jones proteins and the proteins of normal human serum were made by the use of precipitin, complementfixation and anaphylactic reactions. The precipitin reactions were extended by the method of the absorption of antibodies and the anaphylactic reactions were submitted to analysis by the Schultz-Dale method of the graphic record of the contrac


February, 1922]


43


tion of smooth muscle. The results of all these experiments were in accord, and allow the following conclusions to be drawn :

1. Tlie crystalline Bence- Jones protein acts as a single antigen.

3. The non-crystalline preparations of Bence-Jones proteins, isolated from the urine by salting-out or other precipitation methods, contain traces of serum proteins.

3. The Bence-Jones proteins are immunologically different from the proteins of normal human serum.

4. These differences between proteins from the same animal are further evidence in support of the conception that the specificity of proteins is not dependent upon their biological origin, but due to their chemical constitution.

BIBLIOGRAPHY

1. Bayne-Jones, S., and Wilson, D. W.: Proc. Soc. Exp. Biol, and Med., 1921, XVIII, 220-222.


2. Walters, W.: J. Am. Med. Assn., 1921, LXXVI, 641-645.

3. Wells, H. G., and Osborne, T. B.: J. Infect. Dis., 1913, XII, 341-358.

4. Woods, A. C: Tr. Sect. Opth. Am. Med. Assn., 1917, 133-161.

5. Dakin, H. D., and Dale, H. H.: Biochem. J., 1919, XIII, 248-257.

6. Abderhalden, E., and Rostoski, 0.: Ztschr. f. physiolog. Chem., 1905, XLVI, 125-135.

7. Rostoski : Munch, med. Wchnschr., 1902, XLIX, 740.

S. Boggs, T. R., and Guthrie, C. G.: Am. J. Med. Sci., 1912, CXLIV, 803-814.

9. Hopkins, F. G., and Savory-, H.: J. Physiol., 1911, XLII, 189-250.

10. Cameron, A. L., and Wells, H. G.: Arch. Int. Med., 1915, XV, 746-753.

11. Massini, R.: Deutsch. Arch. f. klin. Med., 1911, CIV, 29-43.

12. Hektoen, L.: J. Am. Med. Assn., 1921, LXXVI, 929.

13. Friedberger, E., and Jarre, H.: Ztschr. f. Immunitatsforsch. u. exper. Therap., 1920, Orig., XXX, 351.

14. Krumwiede, C, and Cooper, G. M.: J. Immunol., 1920, V, 547-562.

15. Dale, H. H.: J. Pharmacol, and Exper. Ther., 1912, IV, 167-223.


YAWS

AN ANALYSIS OF 1046 CASES IN THE DOMINICAN REPUBLIC

By W. L. Moss, Assistant Professor of Preventive Medicine and Hygiene, Harvard Medical School

AND G. H. BiGELOW,

Instructor in Preventive Medicine and Hygiene, Harvard Medical School


Hi.sTORicAL Sketch of Santo Domingo The island occupied by the Republics of Santo Domingo and Haiti is the second largest of the West Indian group lying between Cuba and Jamaica on the west and Porto Eico on the east. It lies between the 17th and 20th parallels of north latitude and between the 68th and 74th meridians.

Its 28,249 square miles of area are diversified by almost every variety of topography; fair and fertile valleys, broad savannahs, towering mountains, sinister desert. The northern half or two-thirds of the island is traversed by two almost unbroken mountain ranges, the Monte Christi and the Cibao or Cordillera, between which stretches from the sea on the west to Samana Bay on the east a beautiful valley, the " Vega Real," the eastern half of which is well watered and wonderfully fertile. It is in this valley and on the lower slopes of the adjacent mountains that most of the cacao and tobacco grow, which, with sugar, make up the chief exports of the island. Indeed the cacao crop of Santo Domingo is said to be the third largest of any country of tlie world. The southeastern part of the island is occupied by a broad coastal plain extending back from the Caribbean as much as 50 miles. This is occupied by broad savannahs, well watered and covered with a luxuriant growth of grass which would furnish excellent pasturage for vast herds of cattle. It is here that the great sugar estates are located which produce the largest single


article of commerce of the island. Coffee is grown commercially to some extent in the mountains of the southwestern part of Santo Domingo toward the Haitian border.

Tropical fruits — bananas, plantains, pine-apples, cocoanuts, oranges, lemons, limes, mangoes, aguacartes, bread-fruit — -flourish. Birds of brilliant plumage rival the gorgeous tropical flora in beauty. The fauna is curiously limited in some respects, the largest native mammal on the island being the agouti, a shy little beast somewhat resembling the guinea-pig in appearance and about the size of a rabbit.

The heat of the tropics is tempered along the coast by sea breezes, and by the altitude of the mountains in the interior, the highest peak, Loma Tina, rising 10,200 feet above sea level.

The commercial life of the island centers in the various ports scattered along the coast — Monte Christi, Puerto Plata, Samana, Sanchez, La Romana, Maccoris, Santo Domingo City and Barahona. The number of inhabitants in these towns varies from a few hundred in the smaller up to about 25,000 in Santo Domingo City, the capital.

The population of Santo Domingo, wliich occupies the eastern three-fourths of the island, is estimated at between 700,000 and 800,000 inhabitants, while Haiti, occupying the western fourth of the island, has a population of about two and a half million. Haiti is known as the Black Republic,


44


[No. 372


and takes pride iu this appellation as indicating pure blood. Tlie language is French. Santo Domingo, ou the other hand, does not boast of its negro blood, although it is probable that considerably more than 75 per cent of the population possess it. In the larger towns, e.specially on the coast, there is a small white population, which shades down through all gradations of color to pure black. Few whites live in the interior, except in the occasional inland towns, the largest of which is Santiago de los Caballeros, a flourishing city of about 20,000 inhabitants, beautifully situated in the Vega Real. The language of Santo Domingo is Spanish.

Life in the interior is simple and furnishes little incentive for industry. The requirements for shelter and clothing are simply met and nature furnishes an adequate and healthful diet, with little exaction beyond the effort required to take it.

Perhaps few of us realize or remember the important part played by this obscure island in the history of Western civilization. Discovered on December 6, 1492, by Christopher Columbus, its present capital, Santo Domingo City, is the oldest existing European settlement in the Western Hemisphere. At the time Columbus landed and took possession of the island iu the name of the King and Queen of Spain, calling it La Espaiiola, the native population consisted of Arawak Indians, estimated at between two and three million souls. Columbus described them as a mild and peaceful race, characterized by their sweetness of temper, the men of hideous visage but the women comely. They offered little resistance to the Spanish occupation, and their land was given by the King of Spain in huge grants to his favorites, who, attracted by the glowing descriptions of the wealth and beauty of the country, came over iu large numbers, built palaces and cathedrals, set up a vice-regal court and lived in an extravagance which almost rivaled that of the court of Spain.

Tlie Indians were easily subjugated, quickly reduced to a state of slavery, and under Spanish tyranny and cruelty were soon exterminated. The appalling rapidity with which this took place may be appreciated from the estimates of the population in the succeeding years. The two or three millions of Indians present in 1492 were reduced in a period of 15 years to 60,000 by 1507, and had been further reduced to 14,000 by 1514. Enrique, the surviving caique, was so enraged by the treachery of the Spaniards, which had led to the capture and execution of Queen Anacaona, that he fled with a small band of followers to the fastnesses of the mountains, where he successfully resisted all efforts to capture him. In 1533 the remaining Indians, 600 in number, under this chief were given lands and established at Boya, a small village within five miles of the camp at which the work we are to report was done.

With the Indians practically exterminated, the land with all its wealth of resources was of little value to its new possessors. To make it again profitable, the African slave trade was instituted as early as 1508. The cruelty and abuses practiced on the Indians were repeated on the Africans, and doubtless would have been attended by the same result, but for the fact that, as they were killed or died off, fresh impor


tations were brought in. With the decline of the Spanish power the greatest bliglit to tlie existence of the slaves was removed, and under climatic aud economic conditions, which were for the most part favorable, they multiplied so that the present population of Santo Domingo consists largely of descendants of the African slaves with a greater or less admixture of Spanish blood.

Although the liistory of the island abounds in interest we may not attempt even a brief sketch of it in this paper. In passing one may recall that it was from Santo Domingo that Cortez, Balboa, Pizarro and De Soto set out to make their brilliant explorations and discoveries. It was the rendezvous for many of the pirate craft that preyed on the British, Dutcli and French commerce with the new world. Here the term " fillibuster " originated. The long speedy boats used in this piracy were called fly-bote or freibote, and their crews were known as freiboters, freebooters or fillibusters. The British and French united to retaliate against the Spanish pirates and established a base on St. Christopher and later on the island of Tortuga, Just north of Santo Domingo, where they were Joined by the Dntch. In addition to their operations on the water, they made frequent incursions into the northern part of Santo Domingo for the purpose of killing the cattle. Those who engaged in this pursuit came to be known as buccaneers, from boucan, the spit on which they cooked their meat.

From Tortuga the French gaiued a permanent foothold on the western end of the island, the part now occupied by Haiti.

Recall that the entire island has been at one time under Spanish rule, that the Dutch and British have each gained a foothold on the island, that eventually independence from France and Spain was gained, and the Republics of Haiti and Santo Domingo were established, that each of these young republics at one time or another gained possession of the entire island, and that these various changes were not effected without blood-shed. Indeed, the history of the island from the time of its discovery by Columbus in 1492 up to its military occupation by the United States in 1916 has been one of external war and internal revolution. Even in the short history of the republics, the list of presidents is of wearying length, the common way of removing a president being by assassination, and of establishing a new government, by revolution.

The result of this constant state of turmoil was that the resources of the island remained undeveloped, the people impoverished ; a relatively large national debt was acquired, lawlessness prevailed, education and sanitation were sadly neglected.

Even in the few years during which the United States Military Government has been iu control, splendid progress has been made in bettering these conditions. Peace has been maintained, law and order prevail, schools have been established, agriculture has been fostered, highways (one of the greatest essentials to the development of the country) constructed, and great progress has been made in sanitation. The credit for this work is due to the splendid group of U. S. Naval officers, who have been in charge of the military


Februaey, 1923]


45


government. The customs have been collected and not only have the expenses of this work been paid for, but the national debt which amounted to approximately $30,0()0.000.00 in 1907 has been reduced by about half.*

At the request of the military government of the Dominican Republic the School of Tropical Medicine, Harvard University, sent a commission consisting of Drs. A. W. Sellards, W. L. Moss and G. H. Bigelow to Santo Domingo during the summer of 1920 to study yaws. The results of the observations made are herewitli presented in abstract.

Ci.ixicAL DESCRirxiox OF Yaws

As the disease is strictly limited to tropical and subtropical countries, it may be unfamiliar to many physicians who have not been in the tropics; lience a l)rief introductory description of yaws is given. f

Synonyms. — Framboesia tropica, yaws, buba, pian, etc.

Definition. — A tropical specific infectious and contagious disease caused by Treponema perteuue ©astellani and characterized by a framboesiform granulomatous eruption.

Dislrihittion. — West Indies, Central America, northern part of South America, Africa, Malay Peninsula, Siam, Ceylon, parts of China. It is said never to occur in the mountains and cold districts and rarely above an elevation of 800 feet.

Etiology. — The Treponema pertenue, first observed by Castellani in 1905, is now accepted as the etiological agent of the disease. There has been much discussion concerning the question of the identity of yaws and syphilis, and while some observers still maintain their unity, the evidence at hand seems sufficient to establish them as two separate diseases. Charlouis, as early as 1882, successfully inoculated yaws in a syphihtic patient, and syphilis in a patient infected with yaws. That yaws patients are not immune against syphilis is further indicated by the observations of Powell and Nichols and of others, who have described cases of syphilis supervemng on yaws.

Incubation Perioit. — Castellani gives the incubation period as from two to four weeks. Paulet, in 1848, inoculated 14 negroes with the secretions from framba?sic granulomata, and all developed yaws in from 10 to 20 days. In monkeys and other animals, experimentally inoculated with yaws, the incubation period varied from 16 to 92 days.

Symptomatology. — Tlie course of the disease, like tliat cif sypliilis, may be divided into a primary, secondary, and tertiary stage. Some authors add a fourth stage (paraframboesial affections), but it is questionable whether the observations on wliich this additional stage is postulated are reliable.


For thi> hi.stoiy of Santo Domingo the reader is referred to two fascinating books: "Santo Domingo — A Country with a Future," by Otto Schocnrich; and "Santo Domingo, Past and Present," by Hazzard; both unfortunately out of print. Free use has been made of these and of the Encyclopedia Bntannica in the brief historical note here given.

t This description is taken largely from the chapter on Framboesia Tropica, Manual of Tropical Diseases. Castellani and Chalmers, Third Edition, Wm, Wood & Co., 1920.


Primary Stage. — Incubation period two to four weeks, characterized by malaise, rheumatic pains, headache, sometimes irregular fever. The primary lesion (motlier yaw, madre buba) appears as a papule, which after a week becomes moist, and develops a yellow secretion. A crust may form, which on removal reveals, an ulcer witli a granulomatous base. The primary lesion may heal before, but usually persists until after the secondary eruption has appeared.

Secondary Stage. — The secondary stage begins in from one to three months after the primary lesion, and is characterized by a general eruption of granulomata over the body. It is preceded by malaise, headache, severe pains in the muscles, joints and bones. In some cases there may be fever of an intermittent type. The granulomata appear first as minute papules, which increase in size to an average diameter of about one centimeter. They may coalesce, become secondarily infected, and form large ulcerating areas. In the majority of cases — witliin three to six months in children, and six to twelve months in adults — the granulomata dry up, shrink, and disapjiear. In some cases the granulomatous eruption may continue for several years, new crops of nodules appearing from time to time in succession. The granulomata generally undergo involution witliin from two to four months, leaving behind, as a rule, some change in pigmentation. At times secondary ulceration occurs, leading to deeper scars, and occasionally the granulomata take the form of a circinate eruption.

The granulomata frequ-ently occur on the soles of the feet, less often on the palms of the hands, with a resulting condition closely resembling the syphilitic psoriasis palmaris and plantaris.

The alimentary, respiratory, excretory, and central nervous systems are rarely involved. The joints are frequently swollen and painful. Lesions of the mucosa are rare. Castellani and Chalmers have not observed alopecia, but state that various groups of lymphatic glands are found enlarged. There may be a moderate degree of ansemia of secondary type and an increase in the large mononuclears. The Wassermann reaction is positive in the great majority of recent cases, but is fairly often negative in old cases.

Tertiary Stage. — The disease often terminates with the secondary stage. Sometimes the secondary and tertiary stages merge, or there may be an interval of years between them.

The characteristic lesions of the tertiary stage are gummatous-like nodules and deep ulcerative processes, frequently resulting in great deformity. The bones are quite commonly implicated, but visceral lesions are rare or absent. Castellani and Chalmers think that Gangosa, an ulcerative condition of the palate, nose, and pharynx, is in reality a tertiary manifestation of yaws.

Histopatliology. — In the frandicrsic papules the surface epithelium is greatly thickened and numerous elongated downgrowths are seen. In patches the epithelial cells are swollen, vacuolated and degenerating. Circumscribed areas contain polymorphonuclear leucocytes. The layers near the corium are almost normal, but the corium itself is oedematous. There


46


[No. 372


is a diffuse cellular infiltration consisting of polj'morphonuclear leucocytes, large and small mononuclears, eosinophiles, plasma cells, mast cells, connective-tissue cells and some estravasated erythrocytes. Treponemata are present in the granulomata.

Treatment. — Brodin in 1910 tried antimony. " Castellani's "Yaws Mixture" contains tartar emetic, potassium iodide, sodium salicylate, and sodium bicarbonate. Salvarsan was first used by Nichols in the experimental disease in monkeys, and by Strong in the treatment of the disease in man. The cure of yaws with salvarsan or neo-salvarsan is one of the most striking examples of specific therapy that medicine presents.

The malady does not appear to be hereditary. The course of the disease is chronic, resulting in serious disability and much suffering in a majority of cases. The mortality from yaws is not high, and death, when it occurs, is usually due to secondary infections.

The Authors' Obsekvations In discussing the cases observed by us in Santo Domingo the following abbreviations are used for convenience of reference. If the reader will take a moment to familiarize himself with these, the understanding of what follows will be facilitated :

M. Madre buba, mother yaw, primary lesion. B'. Florid secondary eruption of granulomata. Eaj'ly secondary stage.

B. Sparse recurring secondary granulomata. Late secondary stage.

C. Clavus. Late lesions on the soles. Late secondary stage. P. Palmar lesions. Late lesions on tlie palms. Late secondary stage.

G. " Gomma." Tertiary stage.

H. History of yaws. No active lesions present. Quiescent.

PEIMAEY STAGE

We observed 64 eases in which a primary lesion was present. sometimes alone, oftener in combination with other manifestations of the disease. Briefly these cases may be presented in the following tabular form :


Diagnosis No. of cases

M 15

MB' 25

MB'C 13

MB 6

MBC 5


The extremes of age in this group of cases were 7 months and 60 years. The duration of the madre at the time of observation varied from one month to six years, and the size varied from 1 to 7 cm. with an average diameter of about 2.5 cm. Of the 15 cases in the above group which presented a madre buba as the only active lesion, five showed healed scars of secondary granulomata ; thus there were only 10 cases which


(rage age

6.6


Average durat in mos.

2.3


8.5


7.3


11.6


4.1


24.2


19.0


10.


175



had not progressed beyond the primary stage. In 54 cases, or 84 per cent, of our series, the madre persisted after the appearance of secondary lesions.

Location of Primary Lesion. — In 969 cases the location of the madre was determined either by liistory, observation of the lesion, or scar (in 635 cases, or 71 per cent, the scar was present). In 18 additional cases the patients stated that they had had no madre but that multiple granulomata had appeared simultaneously over the body. In two cases the patients claimed to have had two mad res simultaneously.


T.\BLE Showing Location of Madre

Cases

Lower extremities 803

Upper extremities 80

Head 39

Trunk 37

Genitalia 10

969


The primary lesion occurred on the right side of the body in 425 cases and on the left side in 468 cases. In the remaining cases, the site was noted as knee, ankle, etc., but the side of the body was not recorded.

These figures have been analyzed to determine if the site of the madre might throw any light on the mode of transmission of the disease. In this connection it is to be remembered that we are dealing with a bare-footed and bai'e-legged population, that even the adults arc scantily clothed, that the upper extremities are almost as much exposed as the lower, and that children of both sexes usually wear no clothing until they are about five years of age.

Sexual Transmission. — Sexual transmission as a common method may be excluded because of the fact that in only 1 per cent of our series was the primary lesion on the genitalia. Of these 10 individuals, 6 were males, 4 females, and 5 were under 8 years of age. Moreover, in 25 per cent of all the eases in our series the infection occurred before the fifth year of life.

Hereditary Transmission. — We saw no evidence of hereditary transmission in any case.

Insect Transmission. — We think that transmission by the bites of the common flying insects, such as flies, mosquitoes, etc., may be excluded on the basis of the distribution of the primary lesion ; in the great majority of cases, the madre occurred on the lower extremities, whereas the face and upper extremities are equally exposed to the bites of these insects.

The preponderance of the primai-y lesion on the lower extremities as compared with the trunk would likewise seem ta exclude transmission by the bites of such non-flying insects as lice, fleas, bed-bugs, and the like.

The fact that in such a large majority of cases (S3 per cent) the primary lesion occurred on the lower extremities led to a further analysis of these eases. The result is sho^vn in the


February, 1922]


47


following tabulation, and for comparison the figures for the upper extremities are given :

Cases Cases

Buttocks 13 Shouldeis 6

Thighs 30 Upper arms 4

Knees 55 Elbows 16

Legs 201 Forearms 17

Ankles 258 Wrists 8

Feet 240 Hands .' 25


Total


803


Total


It -will be noticed that the frequency with which the primary lesion is found on the various parts of the lower extremities increases strikingly as one approaches the ground. This suggests that if the disease is transmitted by the bites of insects, we should tliink of some non-flying insect which remains on or i near the ground, and does not normally prey on man or even 5 desire to domicile with him ; such as ants, ground spiders, and the like.

The possibility tluit trauma provided tlie poital of entry for the trei^onema has been suggested. The greater frequency of the primary lesion on the exposed parts of the body fits in with such an hypothesis. It seems probable also that in a bare-footed population the lower extremities would be the most frequently traumatized part of the body, and that the frequency of minor injuries would inci-ease as one progressed down the extremity. Examination of the preceding tal)le 1 shows that the frequency with which the madre occurs on the various parts of the lower extremities increases as one descends toward the foot.

Man, being a forward-going animal, is apt to get most of his knocks from in front; accordingly, we have analyzed the primary lesions occurring on the knees, legs and ankles with reference to their occurrence on the anterior, external, internal and posterior surfaces.

As far as our records showed this data, the results are set forth in the following table:

Anterior K.Ntei

Knees 18 S

Legs 107 35

Ankles 25 7£

In the case of the knees and legs it is shown that the anterior

\r surface leads by a large majority, the external surface comes

next, followed by the internal surface, while the posterior

surface finishes a poor last. This probably corresponds closely

to the exposure to minor traumata.

In regard to the ankle the circmnstances are different. Owing to its conformation the malleoli are about equally exposed to injury, while the anterior surface of the ankle is protected by the projecting foot which receives on its dorsum tlie injuries which would otherwise go to the ankle. This corresponds with the frequency of the madre on the various parts of the ankle, and with the fact that in the case of the foot, the primary lesion was found on the dorsum in a great majority of eases.


nternal


Poste


6


4


19


12


84


13


It appears probable from our observation, tlierefore, that trauma, frequently minor in character, usually determines the site of the primary lesion in yaws. This is entirely compatible with the view that the disease may be transmitted by direct contact, or that insects, flies, etc., may be vectors of the virus.

SECONDARY STAGE

Florid Secondary Stage. — In our series there were 144 patients who presented themselves during the florid secondary stage of the disease. In 38 of these the primary lesion was still present and in 37 the late lesions of the soles, known as clavus, had made their appeai'ance.

Briefly summarized these eases are presented in tlie following table :

Flohiu Secondary Stage


Diagnosis


No. of cases


Mg.


A vs. iluration


No. of cases with fresli Rraiiulomata < soles" palm


MB' .


. 25


8.5 yrs.


7.3 mos.


2 1


MB'C


. 13


11.6 "


4.1 "


7 1


B' ....


. 82


8.8 "


12.3 "


6


B'C ..


. 24


12.9 "


30.4 '


9 2


144 24 (16.67c) 4(2.8%)

Exclusive of tlie 38 still showing the primary lesion and previously analyzed, the youngest patient in this group was 1 year and the oldest 50 years.

While clavus may develop very early in the disease, witness the 13 MB'C cases with an average duration of 4.1 months, as a rule it does not develop until the disease has lasted more than a year. The average duration in the 82 patients who presented only secondary granulomata was 12.3 months, whereas the average duration of the cases which showed clavus in addition to the florid secondary eruption was 30.4 months. The greater frequency of granulomata on the soles as compared with the palms is indicated by their presence in the former situation in 16.6 per cent of this group as against 2.8 per cent in the latter situation.

From our observation and the histories we obtained, we are led to believe that, in the disease as it occurs in Santo Domingo, practically all of the patients, sooner or later, develop granulomata on the soles and become the victims of the painful condition wliich they designate as clavus.

The secondary eruption, as we saw it, corresponds closely to the text-book description as given by Castellani and Chahners. The individual granuloma does not difl'er materially in appearance from the primary lesion except tliat it usually does not become so large and seems less apt to undergo as deep ulceration. In our notes we described the earliest stage observed by us, as a small papule 1 to 2 mm. in, diameter, gray in color, and surrounded by a narrow zone of hyperemia. These papules increase rapidly in size to an average diameter of about 1 cm. and are usually elevated 0.5 to 0.75 cm. above the skin. The surface of the granuloma is covered with minute


48


[No. 372


bosses which give it a raspbern-like appearance from whicli the disease takes the name framba'sia (Figs. 4, 5, 6, and 8). In the early stage the granulomata are of a pearly gray color ; later they are apt to become covered by a thin yellow crust. After a few weeks or months retrogressive changes take place ; the lesions turn dark, often black in color, shrink in size, the surface becomes checkered, and after a time all that remains is a black crust, which on falling may leave no scar; more frequently pigmentary changes persist, which maj' consist of increased pigmentation, apigmentation or an apigmented area surrounded by a zone of increased pigmentation.

At no stage of the granulomata was vesiculation noted. They may go through their entire existence without ulceration, but more frequently they become secondarily infected, and consequently more or less deeply ulcerated. During the florid secondary eruption there may be hundreds of granulomata over the body, and no part of the cutaneous surface, from the croxvn of the head to the soles of the feet, is exempt from invasion. They show a special predilection for the muco-cutaneous borders — nostrils, mouth, genitalia, anus, and moist surfaces — axills, internatal region, between the upper thighs and popliteal spaces (Figs. 7 and 8). The mucous membranes are said to be rarely involved and we saw but one such case in our series, a single granuloma on the vermilion surface of the lower lip. We observed no ca.se of alopecia except as the result of iilcerative processes involving the scalp. One patient presented the typical circinate arrangement of the lesions on the face, which strikingly suggested the Indian war-paint appearimce (Fig. 12).

Usually there was no complaint of pain in consequence of granulomata on the body except in cases in which they were situated on the bearing surfaces of the feet, on the pahns of the hands or when extensively ulcerated.

Arthritis (Fig. 22) and dactylitis were fairly common, although they usually occurred later than the florid secondary stage of the disease. The knee-joints were involved most commonly, the elbows next in frequency. Usually a single joint was affected. The swelling was sometimes considerable and in one instance as much as 200 c. e. of shghtly turbid, straw-colored fluiil was aspirated from a knee-joint. This fluid was witlniut evident effect when injected into the peritoneal cavity of a guinea-pig. The dactylitis took the form of tlie usual spindle-shaped fingers, generally a single disit, sometimes several, being involved.

Lale Secondary Stage. — There were 190 cases in the series that were considered to be in the late secondary stage of the disease. The patients had passed through the florid secondary eruption as evidenced by the numerous scars of previous granulomata. Many of them had doubtless been entirely free from active lesions for variable lengths of time after which one or more recurrent granulomata had appeared. It is evident from the histories obtained that the disease may undergo an apparent spontaneous cure, but sooner or later granulomata reappear in a majority of cases and successive crops, consisting usually of only a few lesions, continue to make their appearance, with


free intervals between for many years. Briefly tabulated these cases may be presented as follows :


Late Secondary St.vje


niagnosii

MB .. MBC . B .... BC ... BCP .


. 6 . 5 . 60 .106 . 13


AvK.

242°yis. 10.0 ' 9.3 ••

16.5 " 20 5 '


19 mo.s. 172 " 29.6 " 5.4 VIS. 11.3


Total.. 190


No. of cases with

[rf-sh granulomata on

soles palms

2 1

1 1

16 1

69 1

10 4

98(51.6%) 8(42%)


Of the 190 patients in this group, 60 had as their only active manifestation of the disease a few secondary granulomata. 106 had grajiulomata plus elavus lesions and 13 had, in addition to the above, palmar lesions. Eleven cases which have already been analyzed with the madre cases are included here, as they appear to have been in a late secondary stage of the disease in spite of the persistence of the primary lesions.

The extremes of age in this group are 1 and 75 years; and while the former figure shows that a late secondary stage may be reached not only early in life but comparatively early in the disease, the average age in this group indicates that this is exceptional, and both the average age and average duration of the disease at the time of observation point to the chronicity of the secondary stage of the disease.

Clatus. — Perhaps the most curious and interesting effects of the disease are the late lesions which occur on the soles of the feet and it is to these that much of the pain and disability is due. We have adopted the term " Clavus " used by the natives, because it seems an appropriate one, although they use it to designate conditions of the soles long after anything suggesting a " Nail " has disappeared.

In a previous section attention has been called to the frequency with which secondary granulomata appear on the soles. Those occurring in this situation do not differ materially in appearance from granulomata seen elsewhere on the body (Fig. 13). They are perhaps more frequently secondarily infected, and are apt to ulcerate, probably as a result of the fact that the natives seldom protect them by any sort of dressing and continue to hobble about even when the feet appear to be in a shocking condition.

If the granuloma on the sole heals without ulceration, as it shrinks, it separates from the surrounding epidermis and becomes cireumvallate. The hard, dry black core must act like a foreign body beneath the foot and it is the " nail " which led to the use of the term clavus. Finally, the core drops out leaving the "nail hole," a circular opening 0.75 to 1 cm. in diameter with sharp-cut edges which descend vertically 2 or 3 cm. to a flat base.

Usually both soles are about equally involved, although rarely cases are encountered in which only one sole is affected (Fig. 15). The number of granulomata on each sole may vary from only a few to 20 or 30. Irregular erosion, the result of attrition of the epidermis intervening between the " nail holes " may account for the remarkable moth-eaten ai)])earauce


February, 1923]


49


of the soles which many of the old claviis cases present (Figs. 14, 17 and 18).

Under the designation " clavus " the natives include conditions difl'ering from those just described and which are not so readily explained as the result of pre-existing granulomata. Not infrequently the soles presented a condition which is aptly described by the term honey-combed. The entire sole, or, in some cases only the bearing surface, was filled with pits 0.5 cm. or less in diameter, conical in shape and extending to a depth of 2 or 3 mm. These pits are as closely and regularly set as the cells of a honey-comb, and no such number or distribution of fresh granulomata was observed as would suggest that each pit could represent the site of a previous tumor.

In otlrer cases the epidermis of the entire sole or a large part of it was greatly thickened, black, and dead-looking.

The natives applied the term clavus indiscriminately to any of the conditions just described. Pain was the characteristic common to all. Like the granulomata elsewhere on the body, the clavus lesions would clear up spontaneously in time, only to recur again and again throughout many years.

Occasionally a condition of the soles was seen to which the natives apply the name " Eajadura," which means a fissure or crack. The fissures, 2 to 3 cm. long and 2 to 3 mm. deep, are usually arranged vertically about the periphery of the heel. More rarely they occurred in the sole beneath the instep or ball of the foot, where they might attain a length of 6 or 8 cm., reaching to a considerable depth and, like tlio.se of the heel, cause much pain. We gained the impression that these fissures were also due to yaws, and for this reason have included in our analysis the relatively small number of cases encountered with clavus, although the term " Rajadura " is more descriptive.

Before proceeding to the analysis of the clavus cases it may be well to state that we have not included in this category ajiy cases which presented fresh granulomata as the only lesions of the soles. We have reserved the tenn clavus to designate only the late effects of the infection on the soles, and think that these should be regarded as belonging to the late secondary stage of the disease. It may be of interest to give here a description of the changes which took place in the clavus cases following the administration of neo-salvarsan leading to their cure. A statistical statement of the result of the treatment in all cases will be given in a later section of this paper, but the results obtained by treatment furnished corroborative evidence that these several different lesions were properly considered as a manifestation of yaws.

We were prepared to expect the improvement which resulted from the Tise of neo-salvarsan in the cases with fresh granulomata, whether they were situated on the soles or elsewhere on the body, but it seemed rather too much to expect that these old "moth-eaten," dilapidated soles would be rejuvenated by a few injections of any drug. The first few patients were given an injection more as a placebo on account of their importunity and the long distances that some of them had come, rather than with any expectation that they would be benefited. When these patients came back at the end of a week, however, their feet


not showing the least improvement in appearance, we received with surprise and incredulity the statement that they were cured of pain and when they adduced, in support of their claim, tiie statement that, whereas they had been obliged to come on horse the first time, they were able to return on foot, we were encouraged to try treatment on other cases. In the end clavus comprised the largest and in certain respects the most important group of cases that were presented for treatment.

A week after the first injection of neo-salvarsan there was, as a rule, no objective change in the feet, but almost without exception the patients affirmed that the pain was either greatly ameliorated or entirely gone. The injections were usually given at from seven to ten day intervals. About a week after the second injection, the old dead epidermis loosens up about the edges of the clavus holes, erosions and fissures, and begins to desquamate. This process goes forward rapidly until within a week or two after the second injection all the old epidermis has been shed, exposing healthy pink skin.

The followintr table summarizes the clavus cases :


Diagnosis


\o. of


AvB. Xge


.\vg. duration


Right


Sole affecte Left


1

Both


MB'C ....


.. 13


11.6 yrs.


4.1 mos.


8


11


6


MBC ....


.. 5


10. "


17.2 "


4


4


3


B'C


. . 24


12.9 "


30.4 "


21


23


20


BC


..106


16.5 "


5.4 yrs.


94


92


80


BCP


.. 13


20.5 "


11.3 "


11


13


11


C


..327


21.6 "


9. "


299


291


266


c+*


.. 41


29,4 "


12.9 "


33


35


27


cp


.. .50


32.7 "


1S.7 "


45


47


42


In 3 of the 579 cla\'us cases no note was made as to which sole was involved. It is ai>parent, however, that the right and the left sole are affected with equal frequency, and that in a uiajority of cases both soles are implicated (79 per cent in the above series).

Although we have seen clavus in an infant of one year, attention is called to the fact that the average age of the largest group in this series, the 327 patients who had clavus only, was 21.6 years, and that the average duration f of the disease in this group at the time they presented themselves for treatment was 9 years.

Palmar Lesions. — l\'rlia])s (•(irrespondiiig' to the clavus lesions, though not so definitely dependent upon granulomata, are the painful keratoses and fissures which, late in the disease, appear on the palms of the hands. There is sometimes more or less erosion of the epidermis and frequently an inability to open the hand fully and extend the fingers. This is apparently due to a certain amount of contraction of the hard, dry keratosed surface.


The group of 41 cases designated as C-f- had in addition to clavus some other condition which apparently was not dependent on the yaws infection. In a majority of cases the additional affection consisted of chronic leg ulcers.

t Wherever average duration is given in this paper it refers to the duration since the first appearance of the primary lesion and not to the duration of any particular stage, unless so specified.


50


[No. 372


The condition is usually painful, though less so than clavus, and interferes ^\-ith manual labor. It is a much rarer lesion than clavus, and appears to develop in older individuals and later in the disease, though it seems to be equally amenable to treatment.

In our series this condition was met with 68 times, but only five of these patients had palmar lesions (exclusive of scars) as the only effect present. These 68 cases are briefly summarized in the accompanying table :

No. of Averas** Average

Diagnosis cases age duration

BCP 13 20.5 yrs. 11.3 yrs.

CP 50 32.7 " 18.7 "

B 5 25. " 14. "

It is rather striking that of the cases presenting a single manifestation of yaws, clavus forms the largest group, 327 cases, while those showing palmar lesions (other than fresh granulomata) constitute the smallest group — five cases. It is also striking in the above simimary of the palmar cases, that those associated with clavus as the only other lesion form the largest number — 50 out of 68 cases.

It is interesting that the average duration of the disease in the CP group, 18.7 years, is the longest of any group of the entire series, not excepting those in the tertiary stage of the disease.

Studded Lesions. — In a number of cases we observed nodular skin lesions which we believe to be a manifestation of yaws (Fig. 20). The earliest stage of these nodules was either not observed or else was not recognized as the beginning of lesions which in a later stage of their develojiment became quite familiar to us. Apparently, these lesions may occur on any part of the body, although we never observed them on the head or face. The forearms and legs were the commonest sites in the cases which we observed. The thighs and trunk were frequently implicated and lesions starting on the dorsum of the foot and extending on to the plantar surface were noted.

A typical picture of the condition as observed by us may be described as follows: The individual lesion consists of a moderately hard skin nodule, 1 cm. in diameter, elevated 3 to 4 mm.-, not painful, unaccompanied by itching and without striking pigmentary changes until after regression, when increase of pigment may mark its former site-. These nodules are thickly studded and regularly set over an area 8 to 10 cm. in diameter. The size of the area involved increases 'by an advancing margin consisting of an almost unbroken }ow of nodules. As this peripheral advance occurs, healing takes place in the center of the area. If the nodules do not ulcerate this retrogression is accompanied by desquamation of the epithelium. The nodules gradually flatten out and finally disappear, leaving no trace, or more often a circular area of increa.sed pigmentation.

In no case were the nodules observed to pass tlirough a vesicular stage, but they frequently underwent ulcerative changes varying from very superficial ulceration involving only the individual nodules to extensive and deep ulceration which sometimes became confluent over large areas of the body and


extended to the subcutaneous tissues. The degree and character of the resultant scarring probably depends upon the depth to which the ulceration had extended. Sometimes there remains only an increase of pigmentation, as mentioned above ; sometimes the skin over the entire area involved wa.s left thin and crinkly and in patterns like those which form on the surface of hot chocolate which has been thoroughly boiled and allowed to stand awhile. Again, where the ulceration has extended to a greater depth there may result complete leucoderma and sometimes painful keloid (Fig. 21).

We have called these lesions " studded " for the lack of a more descriptive term. The area involved, when small, is usually circular, but as it increases in size it is apt to become elliptical or more often it approaches a rectangular shape but with rounded corners. If situated on an extremity, the long diameter of the area coincides with the axis of the limb. Seldom does it completely encircle an extremity even when large areas are involved.

We regret that we had no opportiuiity of studying these lesions histologically. They do not in the least resemble the primary and secondary granulomata. We do not know if they should be considered a late secondary manifestation of the disease or if they belong to the tertiary stage.

We have notes on 37 cases, the youngest patient exhibiting these lesions being 5 years of age and the oldest 80 years. The average age in this group was 28.8 years, which comes next in length to the gomma group (29.3 years). The average duration of the disease (yaws) in this group was 13.5 years with the extremes of duration 5 months and 39 years. This average duration is exceeded only by the P, G, and CP groups.

Our belief that these skin lesions are a manifestation of yaws is based on the fact that they were observed only in patients who gave a liistory of yaws, or who had other recognized yaws lesions, and on the observation that they apparently responded specifically to neo-salvarsan therapy.

We are inclined to regard them as a late secondary manifestation of tlie disease.

Tertiary Stage

The impression gained from a fairly close study of over a thousand cases leaves no doubt in our minds that yaws and sj'philis are separate diseases. Exclusive of the evidence contained in the literature of inoculation experiments, the occurrence of yaws and syphilis in the same patient, the difference in the response of the two diseases to mercury, the possibility of reinoculation in yaws, the localization of the disease to the tropics and below certain altitudes, the grounds afforded by our ovni experience on which we base our opinion are as follows : Syphilis is universally recognized as a venereal disease, the primary lesion is, in the vast majority of cases, located on the genitalia and even in the cases not contracted through sexual intercourse, in which the primary lesion is extra-genital, the course of the disease is in no way modified by the method of inoculation or the unusual seat of the chancre. Yaws, as we have already pointed out, can be excluded from the category of venereal diseases by the fact that in a series of approximately


February, 1922]


51


a thousand cases the primary lesion was located on the genitalia in only 1 per cent and infection took place in 35 per cent before the fifth year of life. Moreover, the primary lesion in yaws differs distinctly in appearance from chancre, even when the latter is extragenitally located, the madre buba, as a rule, being larger, the surface rougher ; moreover, in no case was there the hardness sometimes present and usually considered characteristic of the syphilitic lesion.

In no case did we observe any evidence or obtain any history of hereditary transmission of the disease.

The duration of the madie buba and the high percentage of cases in which it persists after the onset of the secondary stage of the disease (84 per cent in our group of 64 madre cases) is contrary to what is observed in syphilis.

Wlien we come to consider the secondary stages of the two diseases we find more striking contrasts and more especially do we note the remarkable pleomorphism of the secondary lesions of syphilis and the faithful monotony of the secondary granulomata of yaws. The versatile Spirocheta pallida may occasionally produce a counterfeit yaws but never did we observe a yaws patient in the secondary stage of the disease who failed to show either typical granulomata or scars which we felt sure were the marks of typical granulomata. Even the secondary infections and ulcerations which may result do not disguise the nature of the lesions.

Although Castellani and Chalmers say that " occasionally, peeling, whitish patches may be seen on the pahns of the hands and soles of the feet closely resembling the syphilitic psoriasis palmaris and plantaris " we have never observed in syphilis anything resembling the clavus and palmar lesions which we have described as occurring in yaws. On the other hand, none of the yaws cases which we observed presented such characteristic secondary manifestations of syphilis as the macular skin rash, mucous patches and alopecia.

In the tertiary stage of the disease the absence of visceral involvement especially of the heart, blood vessels, liver and kidneys is in striking contrast with the frequency of their involvement in syphilis. Moreover, in a series of over a thousand cases in which the average duration for the entire series was 20.3 years and 79 of which were in the tertiary stage, none showed any evidence of central nervous system involvement. We do not wish to attach undue significance to the absence of central nervous system lesions in this series of cases because the relative immunity of the colored race to such involvement in syphilis is well known.

Finally, the marked difference in the response of yaws and syphilis to neo-salvarsan is a not ujiimportant point in favor of the duality of the two diseases. We have seen the lesions of a patient in the florid secondary stage of the disease melt away completely after a single injection of 0.6 gm. of neo-salvarsan. Indeed the cure of yaws by this agent is the most dramatic therapeutic performance that we have ever witnessed.

But although a decision between syphilis and yaws in the primary and secondary stages was arrived at with little difiiculty, there were cases presenting tertiary lesion in which we found it impossible to make the differential diagnosis (Figs.


23, 24, and 29). There were patients with periosteal thickenings of the long bones ; especially of the tibia, radius and ulna. There were those with typical spindle-shaped swellings of one or more fingers, and others with the bridge of the nose destroyed and -with perforation of the hard and soft palate (the condition known as gangosa) . There were cases in which old ulcerative processes had led to loss of substance of the cranial and other bones. Some of the patients who had an eye that had ulcerated out, and some in whom the nose was almost completely destroyed or fingers or toes had been lost, suggested the ravage of leprosy. Whether these results were due to syphilis or yaws we are unable to state positively. The patients themselves applied the term " Gomma " to all tertiary lesions and stoutly maintained that they were due to the latter disease " yaws."

There were other patients exhibiting tertiary manifestations which bore no resemblance to syphilis as we see it in the United States to-day. These included extensive skin ulcerations, great enlargement and deformity of the hands and especially of the legs and feet.

As previously mentioned, pain in the joints and often an effusion was a frequent complaint in the secondary stage. Many of the tertiary cases showed arthritic enlargement and partial or complete fixation.

That those cases, objectively indistinguishable from syphilis, were actually yaws, we must, of course, leave unsettled. In favor of the view that they were yaws are the following facts :

The patients themselves, who are familiar with both diseases, maintained that the condition from which they were suffering was yaws. Practically all of this group denied liaving had syphilis (this denial deserves some consideration, because little or no shame attaches to venereal infection among the natives) . There were 79 individuals in the " Gomma " group and of these 67 were able to give the location of the primary lesions. Corroborative evidence of the correctness of their statements as to the location of the primary lesion was furnished by a scar in 34 cases. In only two of the 67 cases was the location of tlie primary sore at all a usual one for a chancre, the upper lip in one case and perineum in the other.

Summary of the Gomma Cases

No. of Average age Duration in years

Diagnosis cases in years Extremes Average Extremes

G 67 29.3 3-85 16.3 7/12-59

MG n

BG 3

BCG 2 ^ 252 7-60 9.7 1-40

CG 5

CPG 1 J

The youngest patient in the entire gomma group was 3 years, the oldest 85 years. The average age for the entire group, as well as the duration of the disease at the time of observation, was greater, as would be expected, than in any other group with the exception of the cases with palmar lesions. The extremes both of age and duration are strikingly far apart.

It is of interest that in one case the primary lesion persisted after tertiary manifestations had made their appearance.


52


[No. 373


That the disease may progress to the tertiary stage in a comparatively short time is shown by the fact that in 10, or 7.9 per cent, of the gomma cases the disea.se had existed 2 years or less at the time of observation.

Latent Yaws

We have inehided in our series 127 individuals who had no active lesions of yaws at the time they were observed but who gave a history of having had the disease and who, in most cases, exhibited confirmatory evidence in the way of scars. Brief histories were taken and examinations made of all these cases, inasmuch as they furnished certain statistical data of some interest. Moreover, being impressed with the chronicity of the disease, the frequent recurrence of active lesions after longer or shorter periods of latency, with the doubt in our minds that spontaneous cure occurred in any considerable proportion of the cases, and at the earnest solicitation of these individuals many of whom would say " I had clavus last month and I will have it again next month " or " I have a breaking out of the buba every year," we made a practice of giving one or more injections of neo-salvarsan to patients in whose cases the history and examination satisfied us that they probably had liad yaws.

The 137 cases which come under this lieading may be tabulated as follows :


Diagnosis

H

H-f

H ulceration


21.2 22.9 27.6


' Extremei

1-80 2-62 10-80


Duration in years Average K.\trenies

8.8 yrs. 2/12-50

10.2 " 6/12-45

10.6 " 3/12-59


Of the cases in this group those designated H were without active lesions of any disease, but gave a satisfactory history of having had yaws. Under H-f- we have grouped those individuals who, in addition to giving a history of having had yaws, had symptoms or signs of some other disease such as various skin eruptions, pains in various parts of the body, extensive scarring and other manifestations. One man gave a history of chancre and had a scar on the prepuce. The H ulceration group comprise 45 eases which in addition to a history of yaws showed active ulceration on some part of the body, usiiaUy old leg ulcers, which we could not definitely identify as yaws, and in most cases believed to be due to some other condition.

It seems not unlikely that this group of 137 individuals must have included many sufferers from latent yaws who, had they been left untreated, sooner or later would have again developed active lesions and not only suffered the disability arising from the disease themselves, but would also have become active agents in the spread of the disease. The statistics of this group seem to lend weight to this view. The average age in this group (33.8 yrs.) is lower than the average age of various other groups which showed active lesions, e. g., groups MB, C-|-, CP, P, G, and G-I-. Moreover, the average duration in this group (9.7 j'ears) is shorter than that in several groups showing active lesions, e. g., C-f, CP, P and G.


Indeed in 33, or 36 per cent, of these 137 cases the primary lesion had developed within 2 years prior to the time they applied for treatment. It seems highly probable that some of th^se 33 patients may have been in a free interval between the primary and secondary stages of the disease and that the majority of them were in a latent period following the early secondary eruption.

With a very limited time in which to work among a given population and with the object of accomplishing as much as possible toward eradicating the disease from the locality in which we were stationed, we think the policy of treating patients who gave a history of having had the disease, even though without active lesions at the time, is fully justified.

Gexeral Remaeks The epUrocldear ghinds were noted in 886 cases and were found to be palpably enlarged in 519, or 58.5 per cent; they varied in size from a grain of wheat to 1 or 3 cm. in diameter. Sometimes two or three glands. 1 cm. in diameter, were found in the epitrochlear region.

The femoral glands were noted in 131 cases and found enlarged in 100 per cent of the cases noted. In a majority of cases the femoral glands formed a visible ovoid swelling in Scarpa's triangle frequently attaining a width of 3 or 4 cm. and a length of 5 or 6 cm. It seems probable that the enlargement of this group of glands may have been due to the frequent pyogenic infections on the feet and legs that occur in a barefoot population.

The spleen was noted in 216 cases and found enlarged in 58, or 36.8 per cent. Malaria is common among the natives and may account for the large percentage of cases with splenic tumor.

The Wa.ssermann test was made in 91 cases and found strongly positive in 78 cases (85.7 per cent), moderately positive in 4 cases, weakly positive in 1 case and negative in 8 ca.ses. The 8 eases with negative Wassermann reactions were distributed with reference to diagnosis as follows :

2 cases diagnosed B

1 " '^ c+

1 " " SL

1 " " H

1 " " H-f

1 " " G

1 " " G+

The Wassermann test was repeated in a iew case^ after one or two treatments, the interval between the two tests being not over a few weeks. In no case did the result of the second test differ from that of the first. If it had been possible to perform the test at a longer interval after treatment, the results might have been different.

Thus it will be seen that in the small number of cases in which we have data on the Was.sermann reaction the negative reactions all occurred in the late secondary stage, the tertiary stage, or in patients who had no active lesions but who gave a history of yaws.


Febkuary, 1922]


53


Sequen^ce and Duration of the Various Stages of Yaws III the discussion of this entire series of cases we have tried to take up the various distinctive manifestations of the disease in the order of their development. In so chronic a disease as yaws it was obviously impossible, in the limited time at our disposal, to observe a single case from the appearance of the madre buba, tlu'ough the various stages, to the final deformity produced by the gummatous lesions. Moreover, it is apparent from the preceding sections that there is much overlapping of the various stages, many patients presenting two or even three different manifestations of the disease at the same time. In one case the madre buba persisted after the tertiary lesions had developed. Indeed, not being able to observe the sequence of events, we were often ignorant as to what it had been. We believe, however, that a statistical study of the data collected enables us to unravel this tangle. Accordingly, we have tabulated those cases that presented only a single manifestation of the disease, arranging them according to the average age of each group. The table also shows the extremes of age as well as the average and extremes of duration for each group.

No. of Aire Duration

Diagnosis cases Averatre Kxtremes .U-erage Extremes

M 15 6.6 yis. 9/12-12 yrs. 2,3 mos. 1/12-7/12 yrs.

B' 82 8.8 • li-50 ' 12.3 " 1/12-7 "

B 60 9.3 •• 1-42 29.6 " 4/12-17 "

C 327 21.6 " 3-68 ' 9 yr.s. 4/12-58 "

H 127 23.8 " 1-80 " 9.7 " 2/12-59 "

P 5 25 " 15-45 ' 14 " 10-19 "

SL 37 28.8 " 5-80 " 13.5 " 5/12-39 "

G 67 293 " 3-85 " 16.3 " 7/12-59 "


720


This series comprises 780 cases and the number included in a majority of tlie groups is large enough to give averages that are probably fairly reliable. It seems to us that this arrangement according to average ages probably indicates the sequence in which the various lesions develop. Confirmatory evidence in support of the correctness of this view is furnished by the fact that the average duration of the disease, at the time of observation, in the various groups, with one exception (Studded Lesions) follows the same orderly progression as do the ages. The fact that the extremes of age and duration in each group may vary widely does not, in our opinion, invalidate the deduction which we have just made.

We have attempted to use the data contained in the above table to answer certain other questions. It will be noted that the average ages and average durations are given as of the date of observation, and in the case of duration this does not represent the actual average duration of the disease or any particular stage of it. To illustrate : A patient in the primary stage of the disease may hiive given a history of having had the madre buba 2 months at the time he applied for treatment; it is quite possible that this lesion would, without interference, have persisted for another 2 months. Thus in that particular case the duration of the primary lesion would have been 4 months. If our reasoning is correct, the actual average duration of the madre buba may be estimated by determining


the average duration of the lesions at the time of observation in a sufficiently large number of uuselected cases under natural conditions and multiplying this average by two. Thus, in our series of madre cases, the average duration at the time of observation was 2.3 months, and if 15 cases were a sufficiently large number for generalization, we could say that the actual average duration of the madre stage of yaws is 4.6 months.

Furthermore, if one stage supervened promptly on the termination of the preceding stage, we could in turn calculate the actual average duration of each of the manifestations in the above series. For example, we would arrive at the average duration of the B' lesions by multiplying 12.3 months by 2 and subtracting the average duration of the madre stage. Thus we would find 20 months as the average duration of the florid secondary stage of the disease. The difficulty about applying such simple mathematical calculations to a disease like yaws is that there may be an interval free from active lesions and symptoms between the various stages of the disease, on the one hand, and, on the other hand, a considerable overlapping of the stages. This difficulty could have been overcome if it had been possible for us to obtain accurate histories of the date of onset of each stage of the disease. This we did not attempt, owing to the ignorance of a majority of tlie patients and the limited time at out disposal.

Although we obtained a history of tlie primary lesion persisting for 6 years in one case and saw patients with a florid secondary eruption', in one case 3 years, in another 7 years and in still another case 19 years after the onset of the disease, if we may accept the history given by these patients, it seems probable that the average duration of the florid secondary eruption is less than 2 years and that the late secondary lesions, especially clavus, may recur throughout a long period which, while frequently much longer, averages about 16 years and that in some cases patients may live to extreme old age, 85 years in one case in our series, with tertiary lesions.

Result of Treatment

Neo-salvarsan was used in the treatment of the cases here reported. The drug was dissolved in freshly distilled water in the proportion of 0.1 gm. to 2 c. c. and injected within 30 to 45 minutes after being put in solution. The intravenous method was used in all cases except for young children with V ins difficult of access, these patients receiving the injections intra-muscularly in the buttock. The dose varied from 0.075 gr. for an infant under 1 year of age to 0.6 gm. for a fully developed adult. Intermediate doses were given in proportion to age and body weight. While reactions were frequent, including chill, fever, headache and general malaise, we observed no permanent ill efl'ects and in none of the patients who received the intramuscular injections did we observe abscess formation which reached the surface, althougli some of them showed a painful brawny swelling.

It should be remembered that we were working under field conditions with no facilities for hospitalizing patients even over night, that many patients came two, three, and even four days journey on horse or on foot, over difficult trails, beaten


54


[No. 372


upon by tropical sun and often drenched by tropical rains, with the ground for a bed and the sky for shelter. Oftentimes within an hour after receiving an injection these patients started on the return journey to their distant homes again facing the hardships of the trail. The fact that no patient declined to return and receive further injections, when advised to do so, indicates as clearly as any statistics we might give either that the natives did not regard the reactions of a very serious nature or else that they considered the benefits to be derived as by far outweighing the discomforts of the reactions.

From a statistical standpoint the results of treatment in our series is of little value owing to the limited time over which the cases were observed and the fact that many patients received but a single treatment and were not seen again. The latter circumstance is easily explained. In the interior of Santo Domingo communication is largely by word of mouth and news spreads chiefly by means of the more or less chance wanderings of the natives. Wlien we went up into the interior and set up our camp it was five days before the first patient, an old woman with " gomma," presented herself for treatment. Two days later three children with a florid secondary eruption were brought to us. During the next few days patients ventured in in small numbers, but when the news of the results obtained in the early cases treated began to spread, patients came in rapidly increasing numbers. By the end of the first month we were getting an average of about 25 to 30 new cases a day, and during tlie last two weeks of our stay the daily number of new and old cases ranged between 100 to 300. In spite of the fact that we sent out notices well in advance of our departure to stop new patients from coming in, the daily number increa.sed almost up to the last day of our stay. Their appeal for help was so piteous and their condition so distressing that we could not refuse them a single injection, although we knew that we should never see them again. This accounts for the large number of cases in which we have no note as to the result of treatment.

It has been claimed that 90 per cent of yaws cases can be cured by a single injection of salvarsan. Our experience was not so favorable, though, if we had had a longer time in which to observe tlie results, we could have allowed a longer interval between injections and might have found it necessary to repeat the treatment in a smaller percentage of the cases. Moreover, the number of patients requiring more than one injection to effect a cure might have been fewer if 0.9 gr. of neo-salvarsau instead of 0.6 gm. had been adopted as the adult dose.

We have designated our results as cured, practically cured, much improved, improved, and unimproved. No patient was recorded as cured unless all the lesions (scars excepted) had entirely disappeared. If there remained at our last opportunity of observation otily so much as a few black crusts almost ready to fall, where a week or two before there had been an abundant crop of fresh granulomata, we designated such cases as " practically cured " although we were convinced that could we have seen these patients a week later, without further treatment, they would have fallen into the cured list. The remaining designations, much improved, improved, and


unimproved, are sufficiently clear without further comment, except to say that due conservatism was used in applying them and that, if error was made, it was probably on the side of underrating the actual improvement.

As to the ultimate results obtained we have no observations and even if the present condition of these patients could be ascertained, sufficient time has not elapsed to determine the permanence of the cures. We believe, however, that Ehrlich's ideal of " steriUzatio magna " is more nearly obtained with salvarsan or neo-salvarsan in yaws than in the treatment of any other disease.

There are obvious difficulties in giving a statistical statement of the results of treatment which can be correctly interpreted.


TABLE SHOWING


RESULT


OF


TREATMENT


IN


570 CASES



1 K


Results


Cured


PraotioaUy cured


Much improved


Improved


Unimproved



Number of doses


Number of doses


Number of doses


Number of doses


Number of doses


1 a


1 "^ Ma §,


1


2


3


4


1


2


31


4


1


2


3


4


1


2


3


4


1


2


3


4


M

MB' ... MB'C. MB.... MBC...

B'

B'C...

B

BC .... BCP...

C

C+....

CP

P

SL

G

G+....


15 25 13 6 5 82

..

60 106 13 327 41 60 6 37 67 12

sm


i 10

2

3

3 16

4 24 26

7 143 13 16

S 17 24

4 318


11 15 11

3

2

66 20 36 80

6 184 28 35

2 20 43

8 670


2 2

13 2 4

1

29


2 3

1 11 4 9 10 3 5 1

2

51


1

2

1

5

1 1

1 12


1


5 4 2

14 J3


2

1 3

6 5 3 6

9

1

36




6 2

1

8 3 11 36 3 27 7 5 1 3

lis


1

3

1 3 7

16 3

7

2 4 4 51


5

1 13


2 2


1 1

5

3

7

55 4

10 1 4 8

99


1

1

3

10 2 1

\

1

21


5 6



1

1

41 6 9

3 15 2

T8


••

10


1 1




93 83 179 126 89


As previously stated, many of the cases which were designated as " practically cured " might have fallen into the " cured " eolimm without further treatment, if there had been an opportunity for examing the patients after the lapse of another week. Moreover, the percentage of cures after one injection might have been materially increased, if we had allowed a longer interval of time in which to determine the result of the first injection.

The interval between injections was one week in the majority of cases; exceptionally it was as short as five days and in a moderate number of cases it was ten days or two weeks. The final result of treatment, as far as we had opportunity to observe it, was usually made one week after the last injection.

The above table gives the results of treatment in the various groups as noted at the last observation on each case and shows the number of injections each patient had received


THE JOHNS HOPKINS HOSPITAL BULLETIN, FEBRUARY, 1922



PLATE II


Fig. 2



ng.3


Priinaiy Stage. Madrc Buba or Mother Yaw.


THE JOHNS HOPKINS HOSPITAL BULLETIN, FEBRUARY, 1922


PLATE III


^







Fig. 7


ng.8


Stage. Figs. 4, 5, 6 and S, Florid Secondary Eruption. Note Swel ling of the Femoral Lyniiih-Glands in Fig. o and the Predilection for the

Muco-Cutaneous Borders and Moist Surfaces in Figs. 7 and S.


THE JOHNS HOPKINS HOSPITAL BULLETIN, FEBRUARY, 1922




PLATE IV


FiglO




Tig 11 Fig 12

Secondary Stage. Figs. 9 and 10 show Granulomata of Unu.sual Size. Fig. 12 shows Circinate Arrangement of Lesions on Face, Giving the Indian War Paint Appearance.


THE JOHNS HOPKINS HOSPITAL BULLETIN, FEBRUARY, 1922



ng 13


F1914


F13.15



Fi^ 16



Figl?


Fi^lB


Late Secondary Stage. Figs. 13, 14, 15, 17 ami IS Clavu.s. Note the Fresh Granuloiuata on the Soles ^n Fig. 13. the Unilateral Invol^ement_ in Fig. 15, ami the Marked Erosion in the Remaining Cases. Fig. 16 sliows Marked Thickening of the Skin and Siibmitaneoiis Tissues of the Feet, Probably a Rare, Late Manifestation of Yaws.


THE JOHNS HOPKINS HOSPITAL BULLETIN. FEBRUARY, 1922



ri^i9




Fig. 19.— A '


Fig.?i


rig22


Yaws " Family. Fig. 20, " Stiulded Lcsion.s." Fig. 21, Leucoderma Following " IStuddcd Lesions. Fig. 22, Arthritis, Late

Secondary Stage.


THE JOHNS HOPKINS HOSPITAL BULLETIN, FEBRUARY, 1922


PLATE VII



\


ri^23




i


v"


r



"^


'


s»«»




M



".

,%


>.'r'iii

.- ^


^nr


MkL


_i-il


i


hqZb




H



fig. 2 b

Tertiary Stage. " Gomma." Figs. 23 and 24. Indistinguishable Clinically from Syphilis.


THE JOHNS HOPKINS HOSPITAL BULLETIN, FEBRUARY, 1922



Fig 28



liH 29

Tertiary Stage. Fig. 28.— Bears Some Resemblance to Leprosy. Fig. 29.— Indistinguishable Clinically from Syphilis.


February, 1922]


55


at the time of this note. The 127 patients who gave a history of having had yaws but who had no active lesions of the disease at the ti. :e they presented themselves for treatment are not included in this table, as we had no means of judging of the results of treatment in these cases. We have also excluded from the table 31 miscellaneous cases of yaws in which tlie results of treatment were difficult to determine owing to complicating diseases or for other reasons. Exclusive of these two groups there are 888 cases and in 570 of these we have notes as to the result of treatment.

Briefly summarized, irrespective of the number of injections each patient received, this table shows the following results :

Cases Approx.

Cured 93 16.32

Practically cured 83 14.56

Much improved 179 31.41

Improved 126 22.10

Unimproved 89 15.61

Total 570 100

There were 362 cases in the above series in which the final result noted was after a single injection of neo-salvarsan. Briefly summarized they are shown in the following table :

Results After a Single Injection of Neo-Salvaesan

Cases Per cent

Cured 29 8.01

Practically cured 43 11.88

Much improved 113 3151

Improved 99 27.34

Unimproved 78 2154

Total 362 99.98

Perhaps one gets a better idea of the efficacy of treatment in yaws by considering the cases which showed no improvement following the administration of neo-salvarsan. In the above series of 570 caises only one patient (a gomma case) showed no improvement after three injections and only ten patients (3 gomma and 7 clavus cases) showed no improvement after two injections. This is certainly in marked contrast to the 113 cases which showed marked improvement after a single injection.

We have further analyzed our figures to see if they will show what stage of the disease is most readily amenable to treatment. For this purpose it seems probable that more reliable information will be obtained by combining the cured and


practically cured groups and considering only those cases in which the result noted was obtained after a single injection. They are summarized in the following table:

Cured or practicaUy cured Total No. ot .\verage No. of cases after one injection

Oiagnosis cases ageinyears Results noted Number Percent

M 15 6.6 11 7 63.63

MB' ... 25 8.5 15 6 40.00

MB'C .. 13 11.6 11 2 18.18

MB ... . 6 24.2 3

MBC .. 5 10.0 2

B' 82 S.8 66 27 40.91

B'C .... 24 12.9 20 7 35.00

B 60 9.3 36 5 13.88

BC ....106 16.5 80 7 8.75

BCP ... 13 20.5 6

C 327 21.6 184 8 4.34

C+ .... 41 29.4 28 1 3.57

CP .... 50 327 35

P 5 25.0 2

SL 37 28.8 20 2 10.00

G 67 29.3 43

G+ .... 12 252 8

The number of cases comprising some of the groups in the above table is too small to be of value for statistical purposes, but taken as a whole the figures seem to show what we would expect, i. e., the earlier the stage, the more readily does it respond to treatment.

Acknowledgments

We desire to express our appreciation of the courtesy and co-operation shown us during our stay in Santo Domingo by Eear Admiral Thomas Snowden, U. S. N., Military Governor, Commander Eeynolds Hayden, M. C, TJ. S. N., Secretary of the Department of Sanitation and Beneficence, Lieutenant Commander Ralph M. Warfield, C. E. C, IT. S. N., Secretary of the Department of Development, Public Works, etc., and to Mr. W. A. Elders, Administrator-General of the SantiagoSanchez Railway.

We are deeply indebted to Dr. Stone and Mr. Hermann who acted as interpreters for us and whose intimate knowledge of the island and its inhabitants was of the greatest service.

To Mr. H. H. Raymond, President of the Clyde Steam Ship Company, we wish to make grateful acknowledgment for extending to the members of the commission and their equipment free transportation from New York to Santo Domingo and return.


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56


[No. 378


STUDIES ON A CASE OF CHROMIC ACID NEPHRITIS


By Ralph H. Major

(Fram The Henry Ford Hospital, Detroit, Michicjan)


A great deal of light has been shed upon the obscure points of kidney patliology by the newer studies on experimental nephritis. These observations have not only clarified many of our ideas about diseased kidney function, but have also contributed much to a clearer understanding of the complex and highly specialized functions of the kidneys in health.

In the production of experimental nephritis, chemical substances have been employed almost exclusively; arsenic, cantharidin, uranium nitrate, potassium bichromate, the tartrates and mercuric chloride being most frequently used. As the result of these studies we know that in a general way arsenic and cantharidin are glomerular poisons, while uraniimi, potassium bichromate, and the tartrates and mercury seem to have an especial affinity for the tubules. The more recent work, particularly that of MacjSTider," has shown that. although a hard and fast line cannot be drawn, yet we are still justified on the basis of outspoken affinity in speaking of glomerular poisons and of tubular poisons.

The study of cases of poisoning from these chemicals that come into the hospital for treatment is important because of their relation to experimental nephritis. Corrosive sublimate has furnished the largest number of cases in this group. Lewis and Eivers ' have published extensive chemical studies on a case of bichloride poisoning and have pointed out that, while the therapeutics of this condition have merited much attention in the literature, studies on the metabolism of these cases have been very meager and superficial. The number of other ]niblished cases of chemical nephroses is extremely small.

The following studies on a case of chromic acid poisoning are presented as a contribution to the subject of kidney pathology and particularly for comparison with the numerous observations in the literature on experimental chromate nephritis. This patient, suffering from an inoperable carcinoma of the face which had been treated by the application of chromic acid crystals, lived for four weeks after the -development of the nephritis. During this period extensive studies of his blood and urine chemistry were carried out, marked variations from the normal were encountered and certain interesting changes brought about by therapeutic measures were noted. These observations have a certain interest as a study of the acidosis accompanying chromate nephritis and as a commentary on theories of renal secretion, particularly the " modern theory " of Cushny.^

Chromic acid was used as a cauterant as long ago as 184,5 when Alexander IJre " described several cases of hemorrhoids treated with this agent. Marshall" in 1857 reported a scries of cases of venereal warts in which the local application of an aqueous solution of chromic acid produced excellent results. Many similar reports appear in the literature during the succeeding years, although Gubler* in 1871 stated that "the


absorption of chromic acid is not free from danger, as patients have been poisoned by a too extensive application to the surfaces of their bodies." .J. William ^Vhite" in 1889 reported the case of a young womaji who died 27 hours after the local application of an aqueous solution of chromic acid to an extensive crop of venereal warts.

Industrial poisoning among workers in chrome plants from chromic acid has long been recognized. Pye," in 1885, reported such cases and described the perforation of the nasal septum long known among workers as " chrome holes." He stated that the possession of such " chrome holes " is an object of pride to the workers, who often carry in their pockets a bent wire which they pass through the hole for the amusement of their friends.

Gergens," in 1877, studied the toxic effect of chromic acid on rabbits and noted that it produced an acute nephritis. Kabierske,' in 1880, studied the pathological changes produced in the kidney by this chemical and Posner" continued and amplified these studies. Both Kabierske and Posner described in detail the exquisite tubular nephritis also. Kossa ' observed that potassium chromate produced a hemorrhagic nephritis, but was interested particularly in the accompanying glycosuria which he described as " chromic acid diabetes." Viron " noted the frequent occurrence of both glycosuria and albuminuria in animals poisoned with chromate, but did not carry out any extensive pathological studies. Among later investigators who used the chromates for the purpose of studying experimental nephritis are Schlayer and Hedinger,"" Heinike and Meyerstein,° Ophiils," Hellin and Spiro," Pearce Hill and Eisenbrey." Weber," Euschaupt," Austin and Eisenbrey ' and MacXider." These observers noted its particular though not exclusive affinity for the renal epithelium, and MacNider states that in the earliest stage there is a vascular injury followed rapidly by a tubular involvement which soon dominates the picture.

The patient upon whom these observations were made was a man of 59 and was suffering from a very extensive carcinoma of the left cheek which had been present for two years. This lesion, when first seen, involved almost the entire cheek, extending from the inner canthus of the left eye down upon the left half of the nose and thence down and over the cheek. The patient's mother had died of chronic nephritis, one brother had died of acute nephritis and another brother still living was suffering from chronic nephritis. The patient's urine was examined for the first time on December 8, 1920, and was normal in every respect, as was also a specimen examined on December 11, 1920. On December 11 the carcinoma was curetted and crystals of chromic acid applied until an eschar had formed. The urine, when examined on the following day, showed 4 gm. of albumin per liter. The amount of albumin


February, 1922]


57


in the urine increased and three days later there were 7.3 gm. per liter, and hyaline and granular casts, red blood cells and numerous degenerated epithelial cells made their appearance.

On December 14, a little more than 48 hours following tlie application of the chromic acid crystals, the patient had an almost total suppression of urine and the blood urea was 60 mg. per 100 c. c. From this time on he waged an unsuccessful fight which terminated with his death on Jan- , uary 9, his blood urea on the day of his death reaching 340 mg. per 100 c. c. Many marked changes in his blood chemistry and in the excretion of fluids and solids took place. An intravenous phenolsulphonephthalein test was carried out on six occasions. On December 17, December 18, December 23 and on January 8 none was excreted, on December 27 and 31 the urine showed a faint unreadable trace. One gram of potassium iodide taken by mouth on January 30 was excreted in 24 hours. The McLean index which was determined every day varied from 0.01 to 0.47, the lowest values being at the onset of the nephritis and again towards the end.

A very marked feature of this case was the comparative wellbeing of the patient for more than two weeks following the appearance of the nephritis. During this period of 18 days he felt comparatively well except for some loss of appetite and occasional headaches, in spite of the fact that his kidneys were very severely damaged, his excretion of solids markedly diminished, and his blood urea soaring up around values ten times that of the normal high average. He was very impatient of the therapeutic measures, which consisted of forced fluid by mouth, saline purges, sodium bicarbonate administered by moutli, and rectal saline irrigations.

On December 28 his pulse became somewhat irregular and on December 31, 19 days after the onset of the nephritis, vomiting appeared which became increasingly frequent and was especially severe the la.st five days. His urinary output remained at a high level but sank very low the last five days, apparently because of the markedly lowered fluid intake. The patient never showed the slightest symptoms of uremia, no visible edema developed and the blood pressure varied from 145 to 110 systolic and 90 to 75 diastolic. He was given glucose intravenously, caffeine subcutaneously but gradually became weaker and weaker, the cardiac arrhythmia became more marked and he died on January 10, 30 days after the application of the chromic acid crystals.

At autopsy only the kidneys were removed. They measured "l"3x 5 X 4.5 cm., the right kidney weighed 176 gm., the left 197 gm.; both were mottled in appearance, the cortex was swollen and the glomeruli were very indistinct. There was no free fluid in the abdominal cavity and no general anasarca.

Microscopically tlie kidneys showed extensive destruction of the tubular epithelium with debris in the lumen and marked swelling of many intact epithelial cells, the changes being especially marked in tlie convoluted tubules. There were a few mitotic figures seen in these cells, although the evidences of regeneration were not striking. Similar observations in experimental chromate nephritis were made by Kahierske (I. c.) and by many later investigators. Tlie glomeruli were


for the most part normal in appearance, although in some sections a few fibrosed glomeruli were seen. There were numerous areas of small-round-cell infiltration and much fibrosis in many places between the tubules. Because of the patient's age, it is doubtful Just how much of the glomerular and interstitial fibrosis was due to the chromate. In general, the picture was that of a pure tubular nephritis.

A great deal of care was exercised in obtaining specimens and there is every reason to believe that no gross errors occurred that would materially affect the calculations. As the chemical studies were quite extensive, it is simpler to discuss each group separately.

METHODS

The non-proteid nitrogen, creatinin, creatin, uric acid and sugar determinations in the blood were carried out by the method of Folin and Wu. The blood urea was estimated by the method of Van Slyke and Cullen, blood amino acids by the method of Van Slyke, blood chlorides by the method of McLean, Van Slyke and Donleavy, blood calcium by the method of Halverson and Bergheim, phosphates by the method of Bloor.

The urine chlorides were determined by the method of Volhard and Arnold, the total nitrogen by the method of Folin and Farmer; creatinin, creatin and uric acid by the methods of Folin, Benedict and Myers and of Folin, and the urea by the method of Van Slyke and Cullen.

1. Fluid Exchange

The observations on the fluid intake and output in this patient were quite complete and cover the entire of his stay in the hospital. The patient was urged at the onset of the nephritis to drink large amounts of water, which he did.until a few days before death. No initial polyuria was observed such as has been described in thq vascular reactions due to glomerular poisons. On the second day following the development of the nephritis, the urinary output was markedly reduced, falling to 10 c. c. Following the drinking of large amounts of water, the urinary output the following day again rose to 310 c. c, and there was a constant increase until the si.xth day after the partial suppression. The total amount of urine excreted was 1360 c. c. During the period of lowered urinary excretion, the urine contained large amounts of albumin with numerous epithelial cells and epithelial ca.sts — evidence of extensive tubular destruction. This suggests that the swelling of the tubular epithelium and choking of the tubules with debris may have caused the reduced urinary output, as observed by MacNider in experimental nephritis.

The urinary secretion remained uniformly high until four (lays before the death of the patient when it fell sharply reaching 280 c. c. the day before the patient died, this reduction corresponding in time to the period of continual vomiting with a marked dinrinution of fluid intake. The urinary record did not represent the entire fluid output, since the patient was purged constantly and considerable amounts of fluid were passed in this way. The specific gravity of the urine which


[Xo. :rrZ


was 1.025 the day the nephritis appeared, afterward varied from 1.011 to 1.019, the average specific gravity, as determined by adding all the observations together and using their number as a divisor, being 1.014. The reaction of the urine was acid before any therapy was instituted, but following the administration of sodium bicarbonate became alkaline, becoming again acid whenever this therapy was discontinued.

2. T0T.\L XlTRDGEX AXD ChLOHIDE EsCRETIOX

The total nitrogen, non-proteid nitrogen and chloride excretion fell very sharply following the onset of nephritis reaching the low level of 1.17 gm., .96 gm. and .59 gm., respectively. This marked depression persisted for four days and tlien was followed by a slow and gradual rise, until ten days after this depression the patient excreted 13.5 gm. of total nitrogen,


_"



n


V


Cl-RVE 1.

13.4 gm. of nonproteid nitrogen and 1.62 gm. of chlorides in 24 liours. Similar curves of nitrogen excretion were observed by Pearce, Hill and Eisenbrey, by Green, and by Austin and Eisenbrey in experimental chromate nephritis. If this curve of nitrogen and chloride is super-imposed on a curve of the fluid output, it will be seen that the two follow each other closely. The nitrogen output is reduced, the chloride output is markedly reduced, yet their fluctuations correspond closely to those of the urinary secretion.

3. Urea

The urea nitrogen excretion was constantly low. The first determination made after the onset of nephritis showed a total 24 output of 6.33 gm. On December 15. the daV following the marked suppression of urine, it fell to 0.81 gm. and on the following day it was only 1.2 gm. It later rose slowly, reaching the highest level on December 29 when it was 9.49 gm. During the last five days of the patient's illness it again showed a de


cided depression, falling to 1.62 gm. on the day before. the patient's death. In comparing these figures with those of t>\e total nitrogen and non-proteid nitrogen excretion, it is seen that, while the.se two are markedly diminished, yet their depression is not relatively so great as that of the urea excretion.

The blood urea, on the other hand, was high at the onset — 65.38 gm. on December 14, and afterwards showed a constant and gradual rise, reaching the high value of 340 mg. per 100 e. c. the day before death. For two weeks from December 2 to January 3, during the period when the patient, as previously mentioned, felt comparatively well, the blood urea varied from 168 to 227 mg.

These urea values are interesting in the light of Cushny's well-kno'wn, modern theory. Cushuy states that " the modern view requires that the urea, Uke the other constituents of the urine, pass out by the capsule." He cites, in proof of this view, that the excretion of urea ceases at the same time as the secretion of water by the kidney. In this patient, however, the secretion of water, after an initial drop, rose rapidly and continued at a high level while the urea output remained very low and the blood urea continued to increase markedly.

Urej, curves similar to that in this case have been frequently noted in cases of chronic dift'use nephritis showing at autopsy both glomerular and tubular lesions. Such urea values, as noted in our case of tubular nephritis, raise the question whether the older view, that urea is excreted mainly by the tubules, does not explain the picture better than the modern theory.

Further evidence in favor of this older view is found in the work of Oliver." This observer, working with xanthydrol, which produces a characteristic crystalhne product with urea, found evidence of urea excretion in both the glomeruli and tubules. The marked diminution of urea excretion in our case of chromium nephritis would indicate that a part of the urea may have escaped through the intact glomeruli, while still another fraction may have been secreted by such portions of the tubular e]iithelium as were not destroyed.

4. Ceeatixix and Ceeatik • The 24 hour excretion of creatinin fell to 0.23 gm. two days after the onset of nephritis, but following this rose gradually to normal and hovered about 1 gm. for two weeks. The la.st few days it was iigain depressed, falling to 0.3 gm. the day before death.

The blood creatinin on the other hand was +.56 gm. on December 14 and rose rapidly until the fifth day after the onset of the nephritis when it reached 14.8 mg. per 100 c. c. From this time to the end it showed a gradual rise, reaching 17 mg. the day before death. Here again the patient although comparatively free from symptoms for two weeks, showed blood creatinin values varying from 13.68 mg. to 15.68 mg. per 100 f. c, demonstrating the point emphasized particularly by Myers and Lough" that values above 5 mg. indicate an early fatal termination and also that a progressive increase in blood creatinin is a sign of unfavorable prognostic import.


February, 1922]


59


The blood creatin was increased to 12.48 mg. per 100 c. c. on the day following the partial suppression of urine and on the next day fell sharply to 5 mg. It remained about this



latter level until a few days before death when it rose abruptly, reaching 18.72 mg. per 100 c. c. on the day before death. The excretion of creatin varied from to .17 mg.

5. Uric Acid The blood uric acid values were constantly high, varying from 4 mg. per 100 c. c. on December 14, the second day of the nephritis, to 9.15 mg. on January 5. There was not the



Curve 3.


same striking increase in blood uric acid as there was in the blood urea, although the amounts were increased, the average reading being 6 mg. per 100 c. c. The uric acid excretion was consistently low varying from 6 nig. to 30 mg. in 24 hours.


This patient was on a very low purine diet and the determinations show a very definite uric acid retention, which does not seem in complete harmony with the theory of Cushny which postulates a glomerular excretion of uric acid.

6. Chlorides The plasma chlorides in this case of chromic acid nephritis show a consistently low level varying from 378 mg. to 558 mg. per 100 c. c. following the development of the kidney lesion. The total chlorides in the urine were even more markedly lowered, varying from .1 gm. to 2.76 gm. in 24 hours. No marked fluctuations were observed, although the general tendency was a gradual lowering in the amount excreted as the lirocess continued. The low values were doubtless due in part to the low chloride content of the diet, and the absence of any increase in the plasma chlorides in the presence of an extensive tubular nephritis would seem to be evidence in favor of the excretion of tliese substances by the glomeruli.

7. Sugar

The urine of this patient frequently showed traces of sugar which apparently bore no relation to amount of blood sugar present. The urine during the first 12 days showed traces of sugar on three occasions, although the blood sugar was not higher than 159 mg. per 100 c. c. The last four days of the patient's illness the blood sugar varied from 204 mg. to 229 mg. but with no glycosuria. Intravenous injections of glucose given on three occasions produced no marked change in the blood sugar curve.

Kossa {I. c.) in experimental chromate nephritis noted glycosuria in the absence of a hyperglycemia and explained this phenomenon by the assumption of an increased glomerular permeability. Cushny has attacked this theory of increased glomerular permeability used so long in phloridzin glycosuria, and has pointed out that a failure on the part of the tubular epithelium to re-absorb the excess of glucose excreted by the glomerulus explains the phenomenon equally well or better. In chromic acid nephritis with extensive tubular necrosis, such a failure of reabsorption is presumably present.

8. Carbon Dioxide, Acetone, Phosphates and Amino Acids

Evidence of acidosis in this patient was present soon after the onset of nephritis, the carbon dioxide tension in the plasma on December 15 falling to 37 per cent. Tlie patient was immediately given sodium bicarbonate by mouth with the result that there was a sharp rise to 64 per cent the following day. As the result of such alkali therapy a normal carbon dioxide teni^ion was maintained until the onset of vomiting when the tension fell to 30 per cent, and did not later rise above 37 per cent.

Acetone was present in the urine the last ten days, although it was absent in the early stages in spite of other evidence of acidosis. Determinations of inorganic phosphates in the blood were made on four occasions, sbowing 35 mg. per 100 c. c.


60


[No. 372


^ « i '5 S I



o 2 £ -J

S 6 < s


<5


THE JOHNS HOPKINS HOSPITAL BULLETIN, FEBRUARY, 1922


PLATE IX



Fri. 1.- .Mirnji>liL.lu';rul'h of \\\y kHlni.\- sliowing desquamation of the tubules, interstitial fibrosis and areas of small, round cell infiltration. (Hematoxylin and eosin. Bausch and Lomb obj. 4 oc. X 10.)




7 / *,r


f . ,



Fici. 2. — Micrui'liotujiraiih of tla kidney .sliowing extensive tubular desquamation with an intact glomerulus. (Hematoxylin and eosin. Bausch and Lomb obj. 4 oc. X 10.)


February, 1922]


61


on December 29 aiid 50 mg. per 100 c. e. on January 8, two days before death. The phosphate excretion in the urine was consistently low, falling to 0.1 gm. the day before death. These findings indicate an early phosDhate acidosis complicated later by an acidosis of acetone body origin. Determinations of the blood amino acids were made on four occasions and showed high values. 12.7 mg. per 100 c. c. on December 30, 1920, 33 mg. on January 7, 1921J 24 mg. on January 8 and 31.3 mg. on January 9.

CONCLUSIONS

In this case of chromic acid nephritis the kidney lesion present was that of a pure tubular nephritis. No edema was noted clinically and no anasarca or ascites was present at autopsy. No symptoms of uremia were present, and the patient, during the greater part of his illness, felt comparatively well.

The urine output following a temporary depression was high, but the urine itself was of low specific gravity and the excretion of nitrogen, chlorides, phosphate, creatinin, uric acid and urea was markedly diminished. Glycosuria appeared from time to time but it bore no apparent relationship to the amounts of blood sugar present.

A study of the blood chemistry showed very liigh values for urea, inorganic phosphates, amino acids and creatinin and values higher than normal for uric acid. Determinations of the carbon dioxide content of the blood plasma showed definite evidence of acidosis which responded promptly to alkali therapy.

In conclusion it is a very great pleasure to acknowledge the assistance of Dr. R. L. Haden, under whose direction the chemical studies were carried out.

BIBLIOGRAPHY

1. Austin, J. H., and Eisenbrej-, A. R.: E.xperimental Acute Nephritis: The Elimination of Nitrogen and Chlorides as compared with that of Phenolsulphonephthalein. J. Exper. Med., 1911, XIV, 366.

2. Cushny, A. R.: The Secretion of the Urine. London, 1917.

3. Gergens, E.: Beobachtungen ueber die toxische Wirkung der Chromsaure. Arch. f. exper. Path. u. Pharmakol, 1877, VI, 148.

4. Gubler: Quoted by White (l. c).


5. Heinike, A., and Meyerstein, W. : Experimentelle Untersuchungen ueber den Hydrops bei Nierenkrankheiten. Deutsches Arch. f. klm. Med., 1907, XC, 101.

6. Hellin, Dionys, and Spiro, Karl : Ueber Diurese. Arch, f . exper. Path. u. Pharmakol., 1897, XXXVIII, 368.

7. Kabierske. Eugen: Die Chromniere. Inaug. Dissertation, Breslau, 1880.

8. Kossa, Julius: Ueber Chromsiiure-Diabetes. .\rch. f. d. ges. Physiol., 1902, LXXXVIII, 627.

9. Lewis, D. Sclater, and Rivers, T. M.: Chemical Studies on a Case of Bichloride Poisoning. John Hopkins Hosp. Bull., 1916, XXVII, 193.

10. MacNider, William deB: A Study of the Renal Epithelium in \'arious Tj'pes of Acute Experimental Nephritis. J. Med. Research, 1912, XXVI, 79.

11. MacNider, Wra. deB.: A Study of the Renal Epithelium in Various Types of Acute Experimental Nephritis and of the Relation which exists between the Epithelial Change and the total Output of Urine. J. Med. Research, 1912, XXVI, 79.

12. Marshall: The Application of a Solution of Chromic Acid as a new Stimulant Escharotic in Warts and other Growths. Lancet, 1857, 1,88.

13. Myers, Victor C, and Lough, Walter G.; The Creatinin of the Blood in Nephritis, Its Diagnostic Value. .\rch. Int. Med., 1915, XVI, 536.

14. Oliver, Jean: Mechanism of Urea Excretion. J. Exper. Med., 1921, XXXIII, 177.

15. Ophtils, W.: Some Interesting Points in regard to Experimental Nephritis. J. Med. Research, 1908, XVIII, 497.

16. Pearce, R. M., Hill, M. C, Eisenbrey, A. R.: Experimental Acute Nephritis: The Vascular Reactions and the Ehmination of Nitrogen. J. Exper. Med,, 1910, XII, 196.

17. Posner, Carl: Studien ueber pathologische Exsudatbildungen. Virchows Arch. f. Path. Anat,, 1880, LXXIX, 311.

18. Pye, Walter: On the Local Lesions Caused by the Alkaline Salts of Chromic Acid. Ann. Sur,, 1885, I, 303.

19. Ruschaupt, W, : Beitriige zur Diurese. Arch. f. d. ges. Physiol., 1902, XCI, 595.

20. Schlayer and Hedinger : Experimentelle Studien ueber Toxische Nephritis. Deutsches .\rch. f. klin. Med., 1907, XC, I.

21. Ure, Alexander (Quoted by Ernest Hart) : Lancet, 1857, I, 543.

22. Viron, L.: Contribution a I'etude physiologique et toxicologique de quelques preparations chromees. These de Paris, 1885,

23. Weber, S.: Experimentelle Untersuchungen zur Physiologie und Pathologie der Niereniunktionen. Arch, f . exper. Path. u. Pharmakol., 1906, LIV, I.


ADAPTATION OF BACTERIA TO GROWTH ON HUMAN MUCOUS MEMBRANES WITH SPECIAL REFERENCE TO THE THROAT

FLORA OF INFANTS

By Arthur L. Bloomfield {From the Biological Division of the Medical Clinic, The Johns Hopkins University and Hospital)


In studying the problem of respiratory infection it soon became apparent that certain fundamental data in regard to growth and colonization of bacteria in the upper air passages must be assembled before real progress could be made. It has seemed advisable, therefore, to digress from the original practical problem to more purely biological questions of growth adaptation of bacteria on the respiratory mucous membranes.


The recent trend of bacteriological study clearly indicates that the conditions allowing and promoting the growth of microorganisms are vastly more complex than the early investigators in this domain were led to believe. The newer studies on the importance of hydrogen-ion concentration, and of growth accessory substances in particular, have opened roads which will undoubtedly lead into very extensive and prolific fields of


62


[No. 372


research. However, these considerations ajjply more especially to growth of bacteria on artificial nutritive media, and the facts at hand help but little in elucidating the conditions of growth of micro-organisms in the body. It seems that factors more elusive than those of acid-base equilibrium and such matters govern the actual conditions of human parasitism. To illustrate — while the pneumococcus is found in the mouths of approximately 50 per cent of normal people, we find that the pH of the mouth secretions is almost always greater than that which would allow the initiation of growtli of the pneumococcus in the test-tube.' Furthermore, certain organisms such as B. coli and Friedlander's bacilli, whose growth requirements in viiro are simple, do not colonize on the mucous membranes of the upper air passages, whereas more fastidious organisms such as streptococci are invariably present. It is apparent, tlierefore, that one cannot directly apply test-tube criteria in explaining growth on human mucous membranes.

The immediate question under consideration, then, concerns the factors which allow growth of certain organisms in the upper air passages and prevent that of others. In a previous study it was pointed out that the bacteria found in the tliroat considered from this standpoint fall into several distinct groups.' There is in the adult (with aerobic methods) a constant habitual flora consisting of non-hemolytic streptococci. Gram-negative cocci, and diphtheroids. These organisms are found widely disseminated through the mouth cavity — on tongue, tonsils, and pharynx. They occur in normal and in sick people, in those with foci of infection, and in those whose tonsils have been removed. All the evidence indicates that they grow free on the normal mucous surfaces. In distinction to this group we find another comprising for the most part potentially pathogenic organisms. These are usually localized in some definite area of infection such as the tonsil, adenoid, or a sinus, or they may occur temporarily arid sporadically on the free surfaces of the mucous membranes — evidently transients which have not actually colonized. What then are the conditions which create such a sharp and fundamental difference between these two groups of organisms? An analysis of the known facts in regard to test-tube growth requirements of the two groups does not answer this question. For example, hemolytic streptococci are no more fastidious than many of the green-producing strains, and yet the former are not members of the normal habitual flora, whereas the latter are constantly present. It seems to us necessary to fall back on a theory of adaptive parasitism, -ndthout attempting to define the exact chemical conditions involved. In other words, is it possible that the non-hemolytic streptococci gradually — perhaps during a period of thousands of years — became adapted to growth on human mucous membranes, while other bacteria show a total absence of such ada]itation or only partial and abortive degrees ?

It seemed that some information about this question might be gleaned from a study of the development of the throat flora in infants. Such individuals would furnish a most desirable experimental material for the following reasons: First, one would be dealing with a virgin soil not previously the habitat


of any bacteria. Secondly, no local foci of infection such as diseased tonsils or sinuses are present to obscure the bacteriological picture. Thirdly, the teeth with their associated collections of food particles in which organisms of various sorts may breed have not yet appeared. If it should turn out that any particular varieties of organisms appeared early, regularly, and in large nimibers, one might reasonably assume that such organisms possessed a high degree of inherent adajitation to growth in these regions. The present paper concerns itself, therefore, with a study of the throat flora of infants.

Literature

No attempt will be made to review the extensive literature on the bacteriology of the mouth. Our work concerns itself with defining the position of certain well-known organisms rather than with enumerating all bacteria which may be recovered from the upper air passages under all conditions. Brailovsky-Lounkevitch " has reviewed the literature on the bacteriology of the mouth in infants. She, as well as other observers, has found no organisms during the first few hours of life but notes their rapid appearance after the twelfth hour. Beyond this important observation, however, we find little but a general catalogue of organisms without infonnation which would indicate their significance.

Methods Tlirough the courtesy of the staff of the Obstetrical Department it was possible to examine infants shortly after birth and at more or less regular intervals thereafter. A swab wag passed over the palate and pharynx and plated on rabbit blood agar and oleate Iiemoglobin agar according to the method described elsewhere.' Care was taken to secure a good spread of colonies and careful qualitative and quantitative observations were made of the various bacteria present. A note was made of the relation of the culture to the time of nui'sing.

Results

The results are presented in Table I. The first fact of importance is that the cultures made very shortly after birth — under 12 liour^, and before the baby had nursed — were sterile. This was so in seven ca.ses (I, V, IX, XI, XII, XIV, XIX). In Case XVII a culture made three hours after birth and before nursing yielded four colonies of staphylococcus albus and two colonies of a diphtheroid. From the twelfth hour on (at which time nursing began) organisms were uniformly present in large numbers. In Case VII a culture made at 12 hours, but before nursing, showed a few white staphylococci and several hundred green streptococci. The above findings confirm those of previous observers.

A few words may now be said about each of the organisms encountered in later cultures.

1. Staphylococci (albi). — White staphylococci were present in over half of the cultures but were not a constant finding. They varied in number in various cultures from a few to innumerable colonies. 5Iany types, both hemolytic and non


February, 192-2]


G3


TABLE I Kesults of Throat Cultdres from Infants^



Aug. 6— eel. 11 hrs. (has not nursed)


.\ug. 8— at. 3 days


1 Aug. 15- irf. 10 days



Aug. n-at. 12 days


Case I— K


Xo trrowtli


Staph, albus Tetragenus Strept. (green) Strept. (grey)


20 a few


Staph, albus Tetragenus Strept. (green) Strept. (grey) Staph, aureus


a few many

50


Stajih. albus x

Strept. (green) x Strept. (grey) x



.•Vug. 8-(ct. 14 hrs. (has nursed)


Aug. 9— at. 2 days


Aug. la— at. 8 days


Aug. 17— «t. 10 days


Case II— E


Tetragenus " 1 Gram+ diphtheroid 1


Tetragenus

Gram-neg. cocti Strept. (green) Strept. (grey)


many ma ny a few


Tetragenus

Strept. (green) Strept. (grey) Staph, albus


50


Tetragenus many

Strept. (green) many Strept. (grey) x Stapli. albus few



Aug. 8— ret. 16 hrs. (has nursed)


Aug. 9— at. 2da.v8


Aug. 10-iE/. 10 days


Aug. 15— at. 8 days (culture while nursing)


Case 111— K...


Staph, albus 16


Spreaders


Tetragenus sev. Ii Strept. (green) Strept. (grey)


Lindred many


Staph, albus sev. hundred Tetragenus few Strept. (green) x Strept. (grey) x Staph, aureus 5



Aug. 9-(rt. 14 hrs. (has nursed)


Aug. 10— at. 2 days


Aug. lo- at. 7 days


Aug. 19— at. 11 days


Case IV— Fr...


Stapli. albus 200 .Strcpt. (green) a few Utrrpt. (grey) many


Staph, albus Strept. (green) Strept. (grey) Tetragenus


many

X


Strept. (green) Strept. (grey) Tetragenus.

a few


Staph, albus many Strept. (green) x Strept. (grey) many Tetragenus many Staph, aureus few



Aug. 2S-«B(. 16 hrs. (has nursed)


Aug. 24-<t(. 11 days


Aug. 26-0B(. 4 days



Case V—


Staph, albus iOO Strept. (grey) 50


Stapli. albus


120


Staph, albus Strept. (grey) Strept. (green)


few many





Gram+ coccus Tetragenus


many


Tetragenus Staph, aureus Gram+ bacillus


many

a few

6




Aug. 1— (C(. 7 hrs. (has not nursed) Aug. 2—<f.I. .'SO hours



Aug. 5-at. 3 days


Aug. 8-(E(. 6 days


Case VI— 8....


Xo growth


Staph, albus Tetragenus Strept. (green )

Gram-neg. bacillus


300 a few nmny


Staph, albus sev. hundred Tetragenus a few

Strept. (green) oo


staph, albus sev. hundred Tetragenus a few Strept. (green) sev. hundred Strept. (grey) "

Staph, aureus 12



Aug. '2— at. 13 hrs. (has not nursed)


Aug. 3 at. 31 hours


Aug. 5-(c(. 3 days


.Kug. S—at. 6 days


Case Vll— .T. . .


Staph, albus few Strept. (green) sev. hundred


Staph, albus Strept. (green) Strept. (grey) (iram-neg. cocci


few

00

few


Staph, albus Strept. (grey)


few


Staph, albus co

Strept. (grey) few



Aug. 9— at. 22 hrs. (has nursed) Aug. 5— a(. 3 days


Aug. 9— at. 7 days



Aug. li-at. 10 days


Case VIII— Sn.


Staph, albus -x: Strept. (gresn) many Strept. (grey) many


Strepl. (green)

Tetvageniis Gram+ diphtheroid


few


Strept. (green) Tetragenus Gram-neg. Ijacillus


X

numy


Staph, albus many Strept. (green) x Strept. (grey) few Tetragenus many



July 19- at. 6 hrs. (has not nursed)


July 20— at. 31 hours


July 22-15*. 3 days



July 29— «(. 10 days


Case IX— Ce...


Xo growth I Staph, albus

Strept. (green)


50


Strept. (green) Tetragenus


X

many


Staph, albus few Strept. (green) x



1 Lactis aerogcnes


4




Staph, aureus few


00 = innume

rable. ^The numbers i


ndicate number of colonics.






64


[No. 372


TABLE I— Continued



July 22-at. 20 hrs. (has just nursed)


July 23-at. 2 days

July 29— at. 8 days



Case X— H....


Strept. (green) =o Tetragenus 50 Gram-neg. cocci 50 Diphtheroid few


Strept. (green) co Tetragenus many

Staph, albus 5 Staph, aureus 1


Strept. (green) co Tetragenus few

Strept. (grey) few Staph, albus many




Jul.v 23- (cf. IJ hrs. (has not nursed)



Aug 2-ict. lO.days



Case XI— Hi...


Xo growth



Staph, albus sev. hundred Tetragenus few Strept. (grey) many Strept. (green) few




July 14-(pf. Ihr. (has not nursed)


July 15— (r«. 1 day


July 19-(rl, 5 days


July 2»-ffil. 9 days


CaseXII— Ch..


Xo growth


Staph, aureus 1 Tetragenus sev. hundred Strept. (grey) many


Staph, aureus 6 Tetragenus many Strept. (grey) many Stapli. albus co Diphtheroids few


Strept. (grey) Staph, albus Diphtheroids Strept. (green)


few many



Aug. 20-(t(. 23 hrs. (has nursed)


Aug. 2a-<e(. 3 days | Aug. 2S-(Bt. 9 days



Case XI 1 1— K..


Staph, albus 10 (iram-neg. cocci 20 Gram-neg bacillvs 50 Strept. (g recti) ra


Staph, albus 30 Gram-neg cocci oo

Strept. (green) co Diphtheroids few


Staph, albus few

Strept. (green) oo Tetragenus oo




Aug. 2S-at. 1 hr. (has not nursed)


Aug. 24-<t(. 1 day


Aug. 26— fft. 3 days


.\ug. 2S-eel. 5 days


CaseXIV— W..


Xo growtll


Staph, albus few Gram-neg. cocci many Strept. (green) many


Gram-neg. cocci many Strept. (green) co Strept. (grey) co Tetragenus x Staph, aureus 10


Staph, albus Gram-neg. cocci Strept. (green) Strept. (grey) Tetragenus


few few

few



Aug. n-<Et. 23 hrs. (culture while nursing)


Aug. 18— one. 2 days


Aug. 20-at. 4 days


Aug. 25— cf. 9 days


Case XV— N...


Strept. (grey) co Strept. (green) » Tetragenus oo B. lactis aerogenes 50


Strept. (grey) co Strept. (green) few Tetragenus co


Strept. (grey) many Strept. (green) many Tetragenus co

Staph, albus 200


Strept. (grey) Strept. (green) Tetragenus

Staph, albus


1 50



Aug. 14— <f (. 23 hrs. (has nursed)


Aug. 22-at. S days


Aug. 2i—(Et. 5 days


Aug. 29-(tl. 9 days


Case XVI— A.C.


Staph, albus 10 Gram-ueg. cocci few Tetragenus sev. hundred Strept. (grey) co Strept. (green) « 


Staph, albus . oo

Tetragenus many Strept. (green) co Strept. (grey) many Diphtheroids few


Staph, albus many

Tetragenus oo Strept. (green) oo Strejit. (grey) oo

Staph, aureus 50


Staph, albus

Tetragenus Strept. (green) Strept. (grey)

Staph, aureus


few

X

few



Aug. lo— est. 3 hrs. (has not nursed)


Aug. 16— <^^ 1 day


Aug. 18— et. 3 days


.\ug. 23— a(. 8 days


CaseXVII- Ca.


Staph, albus 4 Diphtheroid 2


Tetragenus oc Strept. (green) oo Strept. (grey) co


Tetragenus co Strept. (green) numy Strept. (grey) co Staph, aureus 6


Staph, albus

Tetragenus Strept. (green) Strept. (grey) Staph, aureus


10 many



July 19— (j(. 24 hrs. (has nursed) July 22 - ttt. 4 days


Aug. 1—at. 14 days


.Kxjg.a-at. 16 days


Aug. lO-at. 26days


Case XVIII— Fr


Tetragenus 300 Strept. (green) co

Diphtheroids oo Beta hemol. strept. 100


Strept. (green) many

Diplitheroids few Beta hemol. strept. 10 Staph, albus oo Gram-neg. coccus co


Staph, albus 300 Gram-neg. coccus oo


Tetragenus Staph, albus


many


Tetragenus sev Strept. (green) Strept. (grey)

Staph, albus


huiid. 100


February, 1922]


65


hemolytic, were encountered, the type often differing in successive cultures from the same infant. The organisms corresponded with the varieties found on the skin (see below). Their logical source is clearly the mother's skin and the milk, and this supposition is supported by the much lower incidence of white staphylococci in adults. There seemed to be no evidence of a real carrier state, but the presence of these bacteria indicates multiple transient infestations derived from nursing at four-hour intervals.

2. Staphylococci (aiirei). — These organisms were found in a few cultures — in no case as constant inhabitants. Their significance was clearly that of transients derived from the mother's skin and milk.

3. Micrococcxis Tetragenus. — Organisms of this group were found in a large percentage of the cultures but by no means constantly. Two main types were encountered (1) large uniformly Gram-positive forms in clumps and groups of four, and (2) smaller forms, many of which failed to retain the Gram stain. The colony formation also varied markedly. These organisms are common skin inhabitants, which pretty clearly explains their source.

4. Diphtheroids. — In marked contrast with what occurs in adults, diphtheroids were found only in occasional cultures and then in small numbers. Their significance was clearly that of transients, and their ubiquity on the skin surfaces and in the mouths of adults offers a ready explanation of their source. There was, however, no tendency to extensive or permanent gro\\i;h on the mucous membranes of infants.

5. Gram-negative cocci. — These organisms are found practically constantly and in large numbers in the mouths of adults. In sharp contrast to this observation was their incidence in infants. We found them in only four cases of the series (II, VII, X, XIII).

6. Streptococci. — This group was of particular interest. As is seen in the table, streptococci appeared early, usually within 24 hours, and in nearly every case were constantly present in large numbers. Two main types were encountered, first, small green-producing colonies consisting usually of elongated cocci in long chains, which exhibited many variations in form and much variability to Gram staining, and secondly, small grey colonies of intensely Gram-positive forms in short chains. No obvious source for these organisms in the infants surroundings was found save the mouths of the attendant adults.

7. Other Organisms. — A variety of other organismsm were encountered in single instances — clearly transients of no significance — a Gram-negative bacillus (Cases VI, and VIII), B. lactix aerogenes (Cases IX and XV). In Case XVIII beta hemolytic streptococci were obtained in small numbers on the first two cultures. They were clearly transients and did not colonize or produce disease, but their source was obscure. Unfortunately, no examination of the mother's milk or breasts was made.

In summary, then, the infant's mouth, sterile at birth, rapidly becomes the site of profuse bacterial growth. The flora is relatively simple compared to that of adults consisting


essentially of ( 1 ) a group of organisms constantly introduced during the process of nursing and corresponding pretty clearly with that of the skin of adults — Staphylococcus albus, M. tetragenus. Staphylococcus aureus, etc., (2) a small group of variable transients, and (3) non-hemolytic streptococci.

With a view to confirming the source of some of the above organisms a series of cultures wa.s made from the skin and from the breast nipples of a group of adults associated with the infants. A swab dipped in sterile salt solution was rubbed over an area about one inch in diameter and plated on the same kinds of media used for the throat cultures. The results summarized in Tables II and III indicate the source of certain of the orgairisms found in the infants' mouths, such as albus, aureus, tetragenus. etc.

TABLE II

Cultures from Skin op Arm of Adults M. — 50 colonies Staph, albus (non-hemolytic). 4 colonies Staph, albus (hemolytic). 6 colonies M. tetragenus.

A few colonies of a coarse rod-like Gram-neg. bacillus. E. — 50 colonies Staph, albus. 1 colony Staph, aureus.

Many colonies Gram-neg. coccus. C. — 1 colony Staph, albua (non-hemolytic).

2 colonies Staph, albus (hemolytic).

2 colonies Staph, aureus.

Many colonies spore-bearing Gram-neg. bacillus.

Many colonies M. tetragenus. G. — Many colonies M. tetragenus.

A few colonies Gram-neg. cocci.

A few colonies Staph, albus.

2 colonies Staph, aureus. S. — 00 colonies Staph, albus (hemolytic).

A few colonies Staph, albus (non-hemolytic).

Many colonies M. tetragenus.

A few Gram-pos. diphtheroids. J. — Many colonies Staph, albus.

A few M. tetragenus. D. — .K few colonies Staph, albus.

A few colonies M. tetragenus.

A few colonies Gram-pos. diphtheroids.

A few colonies Gram-neg. diphtheroids. A. — 1 colony Staph, albus. E. — 12 colonies .Staph, albus.

1 colony coarse Gram-neg. bacillus. C. — 12 colonies Staph, albus. D. — Spreaders.

TABLE III Cultures from Nipples of L.^ctating Women Case 1 — 00 colonies Staphylococci (several kinds).

Many colonies M. tetragenus.

Many colonies Diphtheroids.

A few colonies spore-bearing bacilli. Case 2 — 00 colonies Staphj-lococci (white).

Many colonies Gram-positive spore-bearer.

Many colonies M. tetragenus.

A few colonies spreaders. Case 3 — Many colonies M. tetragenus.

Many colonies Staph, albus.

Many colonies Gram-positive bacillus.


66


[No. 3:2


TABLE III— Continued

Case 4 — 00 colonies Staph, albus.

Many colonies M. tetragenus.

A few colonies Staph, aureus.

00 colonies Gram-positive diphtheroids. Case 5 — A few colonies Staph, albus.

Many colonies Gram-negative coccus.

Many colonies Yeast.

Many colonies long chain — grey streptococcus. Case 6 — 00 colonies Staph, albus.

Many colonies M. tetragenus,

A few colonies Staph, aureus.

Many colonies Diphtheroids

Spreaders.

D1SCUS.SION Above we have outlined the bacteriological facts elicited in the present study. A few words may now be devoted to the general questions raised at the beginning of the paper. It was hoped that further information might be obtained about the mucous membranes of the upper air passages as sites for bacterial growtli and the nature of their relation to the organisms found. It may be recalled that in healthy adults we deal with an apparently habitual constant and completely adapted flora, with transients, and with foreign organisms associated with acute disease or chronic foci of infection. The question of another group of partly adapted organisms has been raised in another place" and evidence of their occurrence collected. In the present work it is of interest to note the absence of such organisms as influenza bacilli, pneumococci, and hemolytic


streptococci which are relatively frequent in adults. Without direct contact observations no definite conclusions can be drawn, but the absence of these organisms rai.ses the possibility of an inherent lack of complete adaptation to free growth in the throat, and suggests that special conditions are necessary for their colonization.

Of major importance, however, from the ])rosent point of view is the practically constant finding of non-hemolytic streptococci in large numbers beginning within a few hours after birth. One seems forced to conclude that an inherent adaptation to free growth on the mucous membranes of the upper air passages exists in the case of this group of bacteria. We state the matter in this way at the present time, for as yet no exact information is available as to actual chemical or biological factors which allow the colonization of this group rather than that of hemolytic streptococci or a host of other organisms. These observations do, however, seem to give a hint that subtle biological adaptations are of importance in explaining the presence of bacteria under certain conditions, rather than the cruder and more specific chemical facts of bacterial gro^^^h demonstrable in the test-tube.

BIBLIOGRAPHY

1. Bloomfield, A. L.: Bull. Johns Hopkins Hosp., 1920, XXXI, 118.

2. Idem: Bull. Johns Hopkins Ho.sp., 1921. XXXII, 1.

3. Brailovsky-Lounkevitch : Ann. de I'lnstitut Pasteur, 1915, XXIX, 379.

4. Bloomfield, A. L.: Journal Am. Med. Assn.. 1921, LXXVII, 187.

5. Idem: Bull. Johns Hopkins Hosp., 1921, XXXII, 387.


DERMOID CYSTS OF THE OVARY

A REPORT OF FOUR CASES

By K.VRL H. Maetzloff (From the Department oj Gynecology oj The Johns Hopkins Hospital and University)


1. A small dermoid cyst accidentally discovered by needling an enlarged ovary.

2. A large dermoid of the left ovary; a small dermoid cyst of the right ovary.

3. A dermoid cy.st in the wall of a large multilocular ovarian cystadenoma.

4. A spinal-cell carcinoma developing in a dermoid cyst of the ovary.

These four cases of dermoid cyst of the ovary are presented not because such growths are rare, but because the pathological specimens came to our laboratory in rapid sequence and each case presented something of interest either clinically or pathologically. The case histories and pathological findings in brief are as follows :

Case 1. — G. G., a white female, unmarried, 24 years old, was operated on by Dr. Cullen at the Church Home and Infirmary, November 15. 1920. The patient had had several attacks of pain localized in the right iliac and the inferior portion of the right lumbar quadrants. Some of these attacks had been associated with nausea, vomiting and a chill. The historj- was otherwise irrelevant. On account of the


iliac pain the patient herself asked for a careful examination of the ovary.

The physical examination was essentially negative except for definite abdominal tenderness and muscle spasm on deep palpation o\er McBurney's point. A pelvic examination was not made.

At operation the appendix was found to be involved in a wellmarked chronic inflammatoiy process. It was removed. The right tube and ovary were next brought to view through the (gridiron) incision. The tube appeared to be normal. The ovary, however, was about half as large again as normal, and presented several large follicular cysts. Three of these were punctured with a straight intestinal needle, and all yielded a clear, colorless fluid. The ovary, however, being still somewhat too large, the needle was passed deeply into its median half where no definite cyst was apparent, and immediately there escaped a verj' small drop of an oily fluid, which against the white surface of the ovary and on account of the good illumination appeared as a small, shimmering globule. The diagnosis being evident, an elliptical incision was made over the surface of the ovary and a dermoid cyst about 3 cm. in diameter was at once encountered. This was shelled out intact, about two-thirds of the ovary being left. The left ovary was normal.

Pathological Examination (Gyn. Path. No. 26372).— The spicunen consists of a small tumor mass measuring 3.5x3x3 cm. (Fig. 1).


February, 1922]


67


About one-half of this mass (marked "dermoid cyst") has a regular, pale gray surface with a few fine shreds of pale gray tissue attached. On palpation this portion is of semi-fluid consistence and possibly is slightly fluctuant. The other part, which represents the portion of the ovarj- included in the elliptical incision, appears elevated, pale red, smooth, glistening and translucent. On section a thin-walled cyst is seen filled with a graj', granular, greasy material which is soft and contains a few strands of hair. In one portion of the wall is an elevated, thickened area from which a tuft of hair projects. The cyst wall is as thin as tissue paper except over the portion where some ovarian tissue is attached; here it is 3 mm. thick and has several smooth-walled, cystic areas in its substance.

Microscopic examination shows the picture of an ordinary simple dermoid cyst lined with stratified epithelium several layers thick. In the wall of the cyst are numerous hair follicles and sebaceous glands. In its cavity are pink-staining detritus and some strands of hair. The cystic areas to one side are thin-walled structures lined with a single layer of flat or low ciiboidal epithelium. These are enlarged Graafian follicles.

Comment. — The point of particular interest in this case is the discovery of a dermoid cyst embedded in the substance of an ovary so as to be entirely surrounded by ovarian tissue. Careful scrutiny of the fluid obtained by paracentesis led to the discovery of a dermoid cyst which would otherwise have been overlooked, and the patient wotild very probably, at some later date, have been compelled to submit to another abdominal operation. The value of paracentesis ovarii as a diagnostic procedure is well brought out in this case and can equally well be applied to other selected cases of small ovarian enlargement when it is desirable to be particularly conservative in preserving ovarian tissue and when the spilling of some of the cyst contents will not lead to peritoneal implantations as sometimes happens in ovarian papillary cystadenomata.

C.4SE 2.— M. M. (Gyn. No. 26756), a white, married woman, 2-para, 39 years old, was admitted to The Johns Hopkins Hospital April 6, 1921, complaining of pain in the lower part of the abdomen. There was nothing of importance in the family or personal history. The menstrual history was normal.

Present Illness. — For about a year the patient had complained of a dull pain in the left iliac quadrant and gradually had become conscious of a mass in this situation which seemed to be enlarging, so that finally it gave rise to a sense of fullness whene^er she sat down.

The physical examination was essentially negative except for the presence of a- Ann, rounded, smooth mass which was movable and apparently about 15 cm. in diameter. This mass was situated in the left iliac fossa and extended upward to a point mid-way between the umbilicus and the s>-mphysis pubis in a position in front of and to the side of the uterus, which appeared to be es.sentially normal. The right o\-ary was in its usual position and seemed to be about twice the normal size.

At operation. .A|iril 9. 1921, the left ovary was represented by a large tumor mass lying in the left ihac fossa and twisted on its pedicle through an arc of 90° (Fig. 2). The left tube and ovar>- were rpmoved in the usual manner. The right ovary also contained a yellowish cyst, through the wall of which some hair could be seen. This cyst was resected, leaving in .ntu about what would correspond to one-third of a normal ovary. The usual prophylactic appendectomy was performed and the uterus was left in good position by a modified Coffey suspension.

Patholoi/icnl Examination (Gyn. Path. Xo. 26756).— The resected portion of the right ovar>- measures 4.5x3 x 2 cm. It is .smooth, pale gray and glistening except for one area where the surface has some small vesicle-like areas which are yellow, glistening and translucent. The tumor is doughy in consistence. On section (Fig. 2: insert) a


cystic structure is seen filled with a sebaceous material in which there is some hair. The hair grows in a tuft from the inner aspect of the roughened yellow area noted on the external surface. The lateral portion of the mass, where it has come in contact with the normal ovarian substance, has several small, yellow, cystic areas.

The tumor mass on the left measures 12 x 10 x 9 cm. and has attached to it a normal Fallopian tube. The tumor is smooth, glistening, reddish, and a fine tracery of blood vessels is seen over its surface. It is doughy in consistence and on section presents the appearance or an ordinary dermoid c\st with \-er>' little hair.

Sections of the tumor removed from the right ovary show a thin wall, the inner aspect of which is lined with a stratified squamous epithelium, without papilla, beneath which are numerous hair follicles. The yellow cystic areas seen in the gross are small cavities lined with stratified epithelium and here one also sees numerous multinuclear giant cells scattered in the stroma forming the cyst wall.*

Sections from the large cyst on the left show a similar inner epidermal lining, without papillae but with an occasional hair follicle and sebaceous gland. Numerous epidermal inclusions are seen, some of these having the appearance of undifferentiated sweat glands. Comment. — This patient passed through an uncomplicated convalescence and when last heard of had been menstruating normally at 26-day intervals. The case affords a good illustration of the feasibility of conserving the ovarian function when some normal tissue is present, thereby avoiding the danger and inconvenience of a premature operative menopause. The dermoid cysts are interesting in that they replaced about five-sixths of what should have been normal ovarian stroma; nevertheless, the patient has had and is still having her normal catamenia.

Case 3.— L. T., a white woman, 35 years old, married, 3-para, was admitted to the Church Home and Infirmary March 27, 1921, complaining of an abdominal tumor and indigestion. The family and l)ersonal history have no bearing upon the case; the menstrual hi,story had been normal.

Present Illness. — One year before admission the patient first noticed that she had an abdominal tumor which had gradually become larger.

The physical examinUtion was essentially negative except for the abdominal findings. A large, s\-mmetrical, rounded, and freely movable tumor mass was felt in the hvpogastrium extending from the peh-is up to the umbilicus. This tumor could not be differentiated from the uterus on pelvic examination and we thought we were dealing with a freely movable myomatous uterus.

Operation. — The patient was operated on by Dr. Cullen on March 28, 1921. A small quantity of free straw-colored fluid was found in (he pelvis. The right o\-ary was represented by a large ovarian cyst, which with its Fallopian tube was removed. Convalescence was uneventful and the patient is now well and free of all s>-mptoms.

Pathological Examination (Gyn. Path. No. 26733). — The specimen consists of a large blue-domed mass to which is attached a Fallopian tube. The latter measures 10 x 1 cm. It is flattened out and stretched over the tumor mass. Its surface is covered by a smooth and glistening serosa, its lumen is patent and it.s fimbriae are delicate. A small appendix \esiculosa is present.

The tumor mass measures 20 x 17 x 10 cm. Its sm-face has a bluish sheen, is smooth, glistening, and slightly irregular in outline. Palpation elicits definite fluctuation. On section a large multilocular cystic structure (Fig. 3) is seen. It was filled with a thick, tenacious.


Dr, Cullen has frequently drawn attention to these giant cells and he feels that they are the precursors of the squamous epithelium. He goes further and states that " wherever an alveolar structure is noted in an ovaiy and where these alveolar spaces are more or less lined with giant cells, one can be ab.solutely certain that a dermoid cvst exists in that ovarj-."


[No. 372


glistening, glairy, translucent, mucinous material. One area in the cyst opposite the tube has a rather honey-combed, bubble-like appearance and here the contents are particularly viscous. On the inferior portion of the posterior wall of the cyst is seen an encapsulated mass different from the remainder of the cyst and measuring on its cut surface 1.7x1.5 cm. (Fig. 3; insert). It is easily recognized as a dermoid cyst containing some yellowish-gray, greasy material and a few strands of hair which do not appear to grow as a tuft from any one situation.

Sections from various portions of the cyst wall show a scanty stroma with numerous capillaries and occasional areas of round and polymorphonuclear cell infiltration. The inner aspect of the cyst wall is lined with a single layer of columnar epithelium which only in a few places is thrown up into a low fold. The individual cells are goblet-shaped and have a pink -staining cytoplasm with a pale central zone and a well-defined cell outhne. The nuclei are at the base of the cells, are oval or round and in some cells are crescentic in shape. The cells rest on a poorly defined membrana propria. The smaller cystic areas mentioned in the gross description contain a moderate quantity of a homogenous, pink-staining material in which are large vacuoles. In short, it is a picture of a tj-pical multilocular cystadenoma. Sections of the dermoid cyst taken through its wall where it borders the cavity of the multilocular ovarian cyst (Fig. 4) show the dermoid lined with several layers of cuboidal epithelium. In its wall are hair follicles as well as sweat and sebaceous glands. In the cavity of the dermoid is some detritus in which can be identified some strands of hair. The dermoid is separated from the cavity of the adjoining cyst by a narrow strand of pink-staining fibrous stroma which in turn is lined with a single layer of columnar epithelium similar to and continuous with the previously de.scribed epithelium lining the remainder of the multilocular cyst. No recognizable ovarian stroma is seen.

Comment. — This is the first time in the history of this hospital, as far as the records of the gynecological and patliological departments show, that a dermoid cyst has been seen in association with, and included in, the wall of a multilocular pseudomucinous cystadenoma of the ovary. Although pseudomucinous cysts of the ovary are not particularly rare and dermoid dysts of the ovary are even less uncommon, nevertheless, this particular pathological association is very unusual and the literature on it is quite meagre.

Case 4. — E. R., a white woman, married, 37 years old, 3-para, was admitted to the Church Home and Infirmary complaining of a " lump in her stomach" and was operated on by Dr. Cullen, March 1, 1921. There is nothing of importance in the family or personal history. Her menstrual history is apparently normal.

Symptoms of the present illness were first noted in December, 1920, when she began to have discomfort in the hypogastrium, and a sense of pressure, most noticeable near the rectum, which was aggravated by exercise and by the sitting posture. Lying down gave marked relief. The patient had felt a tumor mass in her abdomen about two months before entering the hospital and she was certain that the tumor had definitely, though slowly, increased in size.

The physical examination was essentially negative except for the abdominal findings. The hver edge was felt just below the costal margin, being sharp, of about normal consistence and not tender. The lower half of the abdomen was quite tender, especially in the left iliac quadrant. Here a firm, tender, mass was felt extending from the left iliac fossa upwards to within 3 cm. of the level of the umbilicus. This same mass coidd be felt behind the cervix and was only moderately movable on bi-manual manipulation.

At operation a quantity of oily fluid containing white flakes and gi-umous-like material was found free in the peritoneal cavity. The site of the left ovary was occupied by a niptured cyst which extended out into the left side of the pelvis and lay upon the rectum behind, its anterior and medial surfaces being adherent to the posterior


aspect of the uterus. This tumor mass with its attached Fallopian tube was removed as completely as possible, but a portion was left attached to the left side of the cervix. The peritoneal cavity was drained with two pelvic and two abdominal cigarette drains.

The patient had an intermittent pyrexia up to 101° F. for two days after the operation. At times she was jaundiced, bile was found in her urine, and she had constant rectal pain up to the time of her discharge from the hospital, March 27, 1921. She failed rapidly and died at her home June 21, 1921.

Pathological Examination (Gyn. Path. No. 26643). — The specimen consists of a Fallopian tube attached to a tumor mass (Fig. 5). The Fallopian tube, 7x0.5 cm., is pale gray, smooth, and ghstening; its fimbrise are delicate. The tumor mass measures 10 x 8 x 7 cm. It occupies the position of an ovary in relation to the tube to which it is attached by a meso-ovarium which has a normal appearance. Its outhne is rather iiTegular and it has several distinct nodular areas. The portion of the tumor adjoining the proximal part of the tube is pale graj-, smooth, glistening, and fluctuant, while the part inferior to the distal segment of the tube is pale gray, and finn, with opaque yellow nodular areas. The most dependent of these nodular areas,

a, has a roughened sm-face which shows some loss of substance, just at the point where the tumor had been adherent to and blended with the cervix. On section the tumor is seen to be a unilocular cyst with a dirty brown liquid conteiit in which are seen small, flattened yellow masses and large quantities of hair. Its walls measure from 1.5 mm. to 3.5 cm. in thickness; they are smooth and somewhat irregular, the irregularities corresponding to the nodular areas seen externally. At its thickest part, a, the external surface is rough and shows some loss of substance, while its internal aspect has several tufts of hair arising from it. The cut surfaces of this area are yellow, opaque, and granular, with pale gray, glistening and translucent columnar areas, which appear to be invaginations or ingrowths from the external surface. The other areas of nodular thickening show quite smooth surfaces both externally and on section.

In sectio7is taken through various portions of a the cyst is lined with a stratified epithelium, eight to ten cells thick. These cells are round and oval, their nuclei take a fairly deep blue color with the hematoxylin and eosin stain and most of them have well-defined nucleoli. Each nucleus is surrounded by a small quantity of eosinstaining cytoplasm. The cells are irregular in size, shape, and staining reaction, large mononuclear forms and mitotic figures being common. There is no papillary arrangement of the epithelium, no stratum gcrminativum as such is obseiTed, nor are any hair follicles, sebaceous or sweat glands seen. As one follows the epithelium along from the thinner portion over into the thicker part of the cyst wall, a marked metaplasia is noted and the cells invade the cyst wall in long strands and solid alveoli (Fig. 6). Much central necrosis is present in the alveoli and many bizarre degeneration forms are seen. Numerous epithelial pearls, giant cells and mitotic figures also occiir. The tumor in places occupies the entire cyst wall and penetrates it at one point over a fairly broad area. The predominant type of tumor cell is round or polyhedral, with a nucleus which has a finely granular chromatin distribution with a well-defined, dark-blue-staining nucleolus and is surrounded by a moderate quantity of cytoplasm. This cytoplasm takes a faint eosin stain or no stain at all and is surrounded by a well-defined pink cell membrane.

Sections through b (Fig. 5) show a picture similar to that seen in the sections taken from a, with the exception that no epithelial pearls are observed and the neoplastic process, while involving the entire thickness of b, does not penetrate the serosa. The epithelium lining the inner surface of the cyst about the periphery of this nodule (Fig. 7) is stratified, three to four cells thick; it shows no evidence of malignant metaplasia, being identically the same as the epithelium from other non-malignant portions of the cyst wall.

Sections through a (Fig. 5) show a picture similar to those from

b, there being no microscopic continuity between the two neoplastic processes.


THE JOHNS HOPKINS HOSPITAL BULLETIN, FEBRUARY, 1922


Outline of ovary ^^ Dermoid cyst



Fic. 1.— Gyn. Path. Xo. 20372.


al po..,0,, ' ';^.^ ,. Ka,,.-I,d dermoia povtio,



Resected Dei-moid



FiK. 2 (;\n I'lili \'i. 26756. Case 2, showing the dermoid with its twisted pedicle on the kit aud I he Muall quantity of normal ovarian tissue left on the right side after the resection.




Fig. 3. — G.yn. Path. No. 26733. Case 3, a multilocular pseudomucinous cystadenoma of the ovary containing a dermoid cyst.


Fig. 4,— Case 3 (Gyn. Path. No. 26733). From the dermoid cyst where it adjoins the cystadenoma. The columnar epithelium lining the cystadenoma is seen at the superior margm of the picture and the wall of the adjacent dermoid at the inferior margin.


THE JOHNS HOPKINS HOSPITAL BULLETIN, FEBRUARY, 1922


I





Fh;. .5. — Gyn. Path. Xo. 26643. Case 4. Spinal cell carcinoma in a dermoid cyst of the ovaiy.


Fig. 6.— Case 4 (Gyn. Path. No. 26643). Section from Fig. 5 (a) showing spinal cell carcinoma with epithelial pearl at (a).


•'JC**'




•.':'i<<r'fe'ifc'4



^.^•^^^^TLv ' ^c^^r .



Fig. 7.— Case 4 (Gyn. Path. Xo. 26643). Section from nodule (b). (Fig. 5.) At the upper margin is the epidermal lining of the cyst, being here only a few cells thick and showing no evidence of malignant change. Two low power fields to the right of this area the epithelium assumes the character of that seen in Fig. 8 and invades. At the most inferior part of the photomicrograph is seen a small clump of cancer cells. This pictm-e is taken from the periphery of (b) to show the benign appearance of the epithelium which completely surrounds these carcinomatous nodules.



Fig. 8.— Case 4 (Gyn. Path. Xo. 26643). From the cyst wal adjacent to the meso-ovarium showing the thickness of the epithelium without invasion. These cells under high power show numerous mitotic figures and marked irregularity in size, shape, and staining reaction. Mitotic figure at (a).


February, 1922]


69


The sections of the Fallopian tube and the meso-ovarium present nothing noteworthy. However, the adjacent wall of the cyst is lined with stratified epithelium, eight to twenty-five cells thick, which do not invade but are irregular in size, shape and staining reaction and show frequent mitotic figures (Fig. 8).

Comment. — It is interesting to note that while sections through a (Fig. 5) show many epithelial pearls, they are not seen in any of the others. The latter, however, show more welldefined cells and much less evidence of degeneration than at a. The carcinomatous change in various isolated portions of the cyst with intervening areas of non-malignant epidermal cells is striking and raises the question whether these small malignant nodules represent lymphatic metastases or whether they indicate separate foci of independent malignant change. The latter does not seem at all improbahle when one considers the apparent malignant metaplasia without invasion noted in the epidermal cells (Fig. 8) lining the cyst in the portion adjacent to the meso-ovarium.

A simple dermoid cyst of the ovary, without evidence of a more complex teratomatous structure, undergoing malignant change, is in our experience very rare. Out of a total of some


200 patients who have had dermoid cysts in one or both ovaries (confirmed by examination of microsections) we have had only one other authentic case of carcinoma developing in an ovarian dermoid cyst. This occurred in 1899 in a white woman, 46 years old, who was lost track of. She was operated on, her convalescence proved unsatisfactory and she was not expected to live more than a few months after she left the hospital. It is reasonable to assume that she died as the result of the carcinoma. Sections showed a well-defined spinal cell carcinoma occurring in a dermoid cyst. The incidence of carcinomatous change occurring in a dermoid cyst of the ovary in this clinic is one per cent and our mortality from this disease is, we feel safe in saying, one hundred per cent.

I wish to express my indebtedness to Mr. Max Brodel and Ills pupil, Miss A. K. Lovett, for their excellent illustrations.

ADDENDUM

The value of puncture of the ovaiy as a practical diagnostic procedure was again demonstrated by Dr. Cullen in January, 1922, in the case of a young woman, C. H. I., No. 27512, whose history, operative findings, and treatment are almost a replica of Case 1 in this article.


TITLES OF PAPERS APPEARING DURING THE YEAR 1921, ELSEWHERE THAN IN THE BULLETIN, BY THE PRESENT AND FORMER MEMBERS OF THE JOHNS HOPKINS HOSPITAL AND MEDICAL SCHOOL STAFF


PREPARED IN THE LIBRARY


Atchley, D. W., Palmer, W. W., and Loeb, R. F.

Studies on the regulation of osmotic pressure. I. The effect of increasing concentrations of gelatin on the conductivity of sodium chloride solution.— J. Gen. Physiol., Bait., 1921, iii, 801-806.

AUBR, J., Eind WiTHERBEE, W. D.

Studies on decreasing the reaction of normal skin to destructive doses of X-rays by pharmacological means and on the mechanism involved. — J. Exper. M., Bait., 1921, xxxiii, 791 813.

Baeb, W. S.

Past, present and future of orthopedic surgery. — South. M. J., Birm.. Ala., 1921, xiv, 42-48.

Baek, W. S., Brackett, E. G., and Rugh, J. T.

Report of the Commission appointed to investigate the results of ankylosing operations of the spine. — J. Orthop. Surg., Host., 1921, iii. 507-514.

Baetjer, F. H., and Waters, C. A.

Injuries and diseases of the bones and joints: their differenti.il diagnosis by means of the roentgen rays. — N. Y., 1921, P. B. Heeber. 349 p., 4°.

Baird, J. H.

The examination of the eye in neuro-surgical patients. — Viyginia M. Month., Richmond, 1920-21, xlvii, 532-536.

Bardeen, C. R.

The relation of ossification to physiological development. — ,J. Jtadiol., Omaha, Neb., 1921. il, 18.

The von Pirquet standard of normal body weight as compared with other standards (Correspondence). — .J. Am. If. Ass., Chicago. 1921, Ixxvii, 1988-1990.

Barker, L. F.

Multiglandular syndromes. Survey of literature from Sept. 1, 1920. to March 1, 1921. — In: Nelson Loose-Leaf Med., Lond. & N. i'., 1921. iii, 238-239.

The importance of psychiatry in general medical practice. — In: A Psychiatric Milestone. Bloomingdale Hospital, 1821-1921. N. Y , 1921, 55-75. [Privately printed.]

Diagnostic criteria in epidemic encephalitis and encephalomyelitis. — Arch. Neurol, d Psychiat., Chicago, 1921, vi. 173-196. The value of drugs in internal medicine. — J. Am. M. Ass., Chicago, 1921, Ixxvii, llSl-ll.'iS.

The classical endocrine syndromes. — N. York M. J., ictc.l, 1921, cxiii, 353-363.

Neutrophilic myelocytes in the cerebro spinal fluid of n patient suffering from myeloid leukemia and their significance for the diagnosis of niyeloleukemic infiltration of the leptomeninges. — ^auth M. J., Birmingham. Ala., 1921, xiv. 437-442.


On the care of patients manifesting high blood pressure. — Yirgima M. Month., Richmond, 1921. n. s., xlviii. 1-7. Also: South. M. d 8., Charlotte, N. C, Ixxxiii, 134-141.

Group diagnosis and group therapy. — Illinois M. J., 19_1, xxsix, 1-J. Also: J. Iowa State U. Soc, 1921. xi, 113-120. BATJMG.4ETEN, W., FiscHEL, W., and SOPER, H. W.

Hospital diets. — J. Missouri M. Ass., St. Louis. 1921. xvlii, 263.

B.\YNE-JoNES, S., and Wilson, D. W.

Specific immunological reactions of Bence-Jones proteins. — Proc. Soc. Exper. Biol. & Med., N. Y., 1920-1921, xviii, 220-222.

Beall, F. C.

The diagnosis of cancer of the colon. — Texas State J. M., Fort Worth, 1921-22, xvii, 343-348.

Beall, F. C, and Jagoda, S.

Injection of the bile ducts with barium. — J. Am. M. Ass., Chicago, 1921. Ixxvi. 1483-1484.

Beall, K. H.

Epidemic encephalomeningomveloneuritis ("Sleeping sickness"). — Texas State J. M., Fort Worth. 1921-22, xvii, 157-161.

Bernheim, B. M.

Whole blood transfusion and citrated blood transfusion ; possible differentiation of cases. — J. Am. M. Ass., Chicago, 1921, Ixxvii, 275279.

Berry, J. M.

I'\irther observations on the presence of roentgenological shadows associated with bursitis. — Albany M. Ann., 1921, xlii, 54-59. The differential diagnosis between tuberculosis of the hip and osteochondritis deformans juvenilis (Perthes) or osteochondral trophopathy of the hipjoint (Legg). — Albany U. Ann... 1921, xlii, 90-93. Perthes' disease. — All>any M. .Ann., 1921. xlii, 94-99. An unusual exostosis of the tibia. — Albany M. Ann., 1921, xlii, 130131. —

Reports from the Roentgen-rav Department of the Albany Hospital. — Albany M. Ann., 1921, xlii, 347-355.

BiDGOOD, C. Y.

A study of methods of procedure in resection of the oesophagus. — Ann. Surg.. Phlla., 1921, Ixxiv. 546-556.

Blackfan, K. D.

Biologic therap.\' : III. The use of antimeningococcus serum in the

treatment of epidemic meningitis. — J. Am. M. Ass., Chicago, 1921,

Ixxvl, 36-37. Blackfan, K. D., and Little, K.

A clinical and radiographic study of the thymus in infants. — .im. J.

Dis. Child.. Chicago. 1921. xxii, 459-470. Also: Tr. Am. Pcdiat. Soc,

1921, xxxiii.


[No. 37-2


Blanton, S.

The medical significance of tile disorilers of speccli. — /. Am. M. Ass.,

Chicago, 1921, Ixxvii, 373-377.

Speech defects in school children. — Moit. liny.. X. V., l'.)i;i. v, 820 827.

Block, E. B.

The etiologlc relation of wcirms to epilepsy. — /. Med .Lis. Georgia, Atlanta, 1021. x. 30U.

Bloodgood, J. C.

Cancer of the t^mgiie a preventable disease (Correspondence). —

J. Am. M. Ass., Chicago. 1SI2]. Ixxvii. 220.

Cancer of the tongue: a preventable disease. — ./. .Im. M. Ass..

Chicago, 1921, Ixxvii, 13S2-87.

Early diagnosis of cancer. — 'Wisconsin M. J., Milwaukee. 1921-22, xx.

235-236.

The patholog.v of chronic ejEtic mastitis of the female breast ; with

special consideration of the blue-domed c.vst. — Irr/i. Siirii., Chicago,

1921, iii, 445-.-i42.

Bloomfield, a. L.

The serial duantitative method of culture in the study of respiratory

disease. — J. Am. M. Ass., Chicago, 1921, Ixxvii. 187-192.

The mecbimism of the bacillus carrier state with special reference to

the Friedliin.ler b icillus.— J»i. Rei: Tiibere., Bait., 1920-21, iv, 847 85.1.

Blumee, G.

Gonococcus infection.— /«.- Oxford Med.. 1921, v, 39-70.

Report of a ease of extensive cavernous angioma of the head, face and

neck, with attacks of fever and somnolence. — Boston II. d- S. J., 1921,

Ixxxv, 58-60.

Some remarks on " cases and observations by the Medical Society

of New Haven County." — Bull. Hoc. Med. Hist.. Chicago, 1921. ii.


205


218.


(iroup practice (Correspondence). — Ixxvii. 2081.

President's address. Connecticut Connect. State Med. Soc, 1921, 69-77


'. Am.

State


M. As


Chicago, 1921. Society. — Proc.


Boggs, T. R., and Pincoffs, M. C.

Diseases of the peritoneum. — ;«; Oxford Me.l.. 1921. iii. 517-581.

Bowcock, H. M., and P.wllin. J. E.

Recent improvements in dietetic treatment of ilialieles mellit J. Med. Ass. Georgia, Atlanta. 1921, x, 076.

Boyd, M. L.

Nephritis am xiv. 36.8-:i71.

DU Bh.4Y, E. S. Svcculai- iim ruiiture into


-.Vniith. M. -J.. Bin


The


. sm of the descending tl into the' lower lobe of the left lum; ort <if a case and remarks on ruplui ni.— -l»i. ./. M. Sc, Phila., 1921, <


Ixi. 41


aorta, with direct left pleural cavity, lernoptysis in aortic 7 418.


of the thyrcuil lobes — Endocrin


Brooks, B., and Allison, N.

Bone atrophy; an experimental and clinical study of the changes in bone which result from non-use. — Surg.. Oiinec. & Oist., Chicago, 1921, xxxiii, 250-260.

Brown, N. W.

Determination of rehitive activity olofiu. Glendale. Cal.. 1021, v. 29-32

Brown, T. R.

Carcinoma and other tumors of the gastrointestinal tract. — In:

Nelson Loose- Leaf Med., Lond & N. Y.. 1921, v. 295-299.

Tuberculosis of the gastrointestinal tract. — In: Nelson Loose-Leaf

Med., Lond. & N. Y., 1921, v. 301-308.

Ulcerations of the gastro-intestinal tract (Not including peptic

ulcer).— / II.; Nelson Loose-Leaf Med.. Lond. & N. Y.. 1921, v, 309-310.

R51e of diet in etiology and treatment of migraine and other types

of headache. — J. Avi. M. As.-i., Chicago, 1921. Ixxvii, 1396-1400.

The absence of pancreatic secretions in sprue and the employment

of pancreatic extract in the treatment of this disease. — Am. J. M. So.,

Phila., 1921, clxi, 501-507,

A note on the administration of pancreatic extract in the treatment

of sprue. — J. Trap. M. [etc.]. l>ond.. 1021, xxiv, 90-92.

Bunting, C. H.

Diseases of the lymph

1920, to March 1. 1921. 1921, iii, 3G8. The leukocytic jiicture clxii, 1-9.

Bunting. C. H.. and Huston, J.

Fate of the lymphocyte. — J. Ej-per. M.. Halt.. 1921,

Burrows, M. T.

The causes of i:lisease. — J. Missou

384-392.

The reserve energy of actively growing embryonic tisi

Exper. Biol. <f Med., N. Y.. 1920-21. xviii, 133-136.

BuRWELL, C. S., and Jones, C. M.

The removal of bile and blood from the urine in iierforming the

phenolsulphouephthalein test of renal function. — .1. .tin ]/ Ass, Chicago, 1021, Ixxvii, 462-463.

Burwell, C. S., and White, P, D.

The clinical significance of changes in the form of the electrocardio gram.— J/rri. Clin. N. Am., Phila., 1921, iv, 1839-1861.

Bukwell, C. S., White, P. D., and Marvin, H. M.

The action of quiuidine sulphate in heart disease, to abolish the circus movement of auricular flutter and fibrillation. — Boston M. tC S. J., 1921, ixxxv, 647 650.


influenza. — .im. J. M. Sc.. Phila., 1921,


Ass., St. Louis. 1021, xviii, Pioc. Soc.


il I Editorial). — ./. .li,


Chicago, 1921,


from the writings of William Osier. 2. ed.. d I'ress ; Boston. Houghton, .MiSlin & Co., 356


Camac, C. N. B,

The live wire mir; Ixxvii, 129-130.

Cam.^C, C. N. B., cump. Counsels and ideals, 1021, London. Oxfo p. 8-.

Campbell, C. M.

I'ersonal factors in disease. — In: Nelson Loose-Leaf Med., Lond. &

-N. Y., 1921, vii, 145-153.

History of insanity during the past century, with special reference to

the .McLean 1 lospital — /Jostoii If. d S. J., 1921. clxxxv. 538-544.

Tile use nf tlroi.'s in ueurologj' and psychiatry. — ./. .4m. M. Ass.,

( liicaj.-". 1021. l.\xvii. 1228-1230.

Mental hygiene in industry. — Ment. Hyg., N. Y.. 1921. v, 468-478.

The education of the emotions. — Proc. Head Mistresses Assoc, Bost.,

Mass., April, 1921.

CARTER, D. W., Jr.

Irregularities of the heart beat. — Dallas M. !.. 1921. viii. 13.

The role of the pituitary body in the etiology and therapy of diabetes

insipidus. — Texas State J. M., Fort Worth, 1921, xvi, 491-495.

C.\SH, J. R.

On the develoimient of the lymphatics of the stomaih in the embryo pig. — Contrih. Embryol. No. 57, i Carnegie Inst.), Wash., 1921, xiii, 1-15.

C^SH, J. R., and Young, H. H.

A case of pseudohermaphrodismus masculinus showing hypospadias, greatly enlarged utricle, abdominal testis and absence of seminal vesicles. — ./. Vrol. Bait., 1921, v, 405-430.

C.4ULK, J. R.

Factors which influence results and mortality rate in kidney surgery. Analytic study of two hundred and sixty-three operations. — J. Am. M. Ass.." Chicago," 1021. ixxvif. 843-848.

A new m-tliod of leiuoving the median bar type of prostatic obstruction. — ./. Mi.isoiiri M. A.VS., St. Louis. 1921. xviii. 191. Renal tuberculosis. — ./. Urol.. Bait, 1921, vl. 97-113. Presentation of a cautery punch for the removal of minor obstructions at the vesical neck, with new method of anesthesia. — South. M. J., Birmingham, Ala., 1921, xiv, 816-810.

Chesney, a. M.

An immunologic study of bacillus influeuzip. — /. Inleet. Dis., Chicago, 1921. xxix, 132-140.

Churchman, J. W.

Sterilization of dosed cavities by la violet. Description of technic. — /. An 24-26.

Further studies on the behavior of bacteria toward gentian violet. — J. Exper. M.. Bait.. 1921. xxxiii. 569-581.

The cause of the parallelism between the gram reaction and the gentian violet reaction. — Pioc. Soc. Exper. Biol d- Med., N. Y., 1920-21. xviii, 17-18.

The isolation of gentian positive individuals from a suspension of a gentian negative organism (B. coli). — Proc. Soc. Exper. Biol, d Med., N. Y.. 1920-21, xviii. 19.

Relation of the gentian violet reaction to dilution of implanted suspension. — Proc. Soc. Exper. Biol, d Med., N. Y'.. 1920-21, xviii. 20. The effect of repeated re-inoculations of gentian violet agar with genti.in i".sitiv.' uruanisms. — Proc. Soc. Exper. Biol, d Med., N. T.,

The sill riive .1(1 inn of gentian violet in relation to chemotherapy. — Proc. Sue. Lxv>r. ISiol. d Med., N. Y., 1920-21. xviii, 21-22.

Chubchm.\n, J. W.. and Kahn, M. C.

Communal activity of bacteria. — ./. Exper. M.. Hall.. 1921. xxxiii, 583-591. Alio: Proc. Soc. Exper. Biol, d .Med.. N. Y., 1920-21, xviii.


Clarke, T. W.

•Colic" in the nursing infant. — : xxi, 138.

Clough, M. C. and Clough, P. W. A study of the reactions following .If. .;.. Birmingham. Ala., 1921, xiv

Cobb, S.

Some neurologii iv. 1,'!63-1S70.


VorA- State ./. .1/.. N. Y., 1921.


aspects of chorea. — Med. Clin.


an elephant. — Im. ./.


Cobb. S., and Coleman, C, C.

The course of recovery following trauma Surg., Chicago. 1921, iii, 132-139.

Cobb, S., Forbes, A,, and C.wtell, M.

An electrocardiogram and an electromyugr Physiol., Bait.. 1921, Iv, 385-389.

Cobb. S., and P.\rmenter, D. C.

Headache. — J. Indiist. ling.. N'. Y. & Bost.. 1021. iii. 173-178.

Cole, R.

Acute lobar pneumonia. (Survey of literature from Sept. 1, 1020. to March 1, 1021,) In: Nelson Loose-I>eaf Med.. Phila. & Lond.. 1921, i.


Antipneumococcus serum

112.

Prevention and specific

Lond.. 1921. xxix, 58.

Colston, J. A. C.

The value of pyelography — J. Vrol., Bait.. 1921. v


—J. Am. M. Ass., Chicago. 1921. Ixxvi. Illtreatment of pneiunonia. — ,1. State Med.,


iiplasms id" the kidne


February, 1933]


71


Cornell, W . B.

Group mental hygiene. — Am. J. Insan., Bait, 1920-21, Ixxvii. 335-342. The organization of state institutions for feeble-minded in the United States. — Cnnaa. J. Mcnt. Hyg., Montreal. 1921, iii, 65-70.

Corner, G. W.

Abnormalities of the mammalian embryo cccurring before implantation. — Contrib. Emhryol., No. 60 (Carnegie Inst. i. Wash., 1921, xiii. 61-66.

Cyclic changes in the ovaries and uterus of the sow. and their relation to the mechanism of implantation. — Contrih. Emhryol.^ No. 64

(Carnegie Inst.), Wash.. 1921, xiii, 117-146.

A review of some recent work on the mammalian reproductive cycle. —

J. Mammalogy. Bait.. 1921, ii, 227-231.

A case of true lateral hermaphroditism in a pig with functional ovary. — J. Vrol., Bait., 1921, v. 481-485.

The ovarian cycle of swine. — Science, N. Y. & Lancaster, 1921, liii. 420-421.

COWDRY, E. V.

The Office of Imperial Physicians, Peking. — J. Am. M. Ass., Chicago, 1921, Ixxvii. 307-310.

The reticular material of developing blood cells. — J. Exper. M., Bait., 1921, xxxiii. 1-12.

Crawford, A. C, and George, J. M.

The testes and certain vaso-motor reactions of the penis. — J. UroL, Bait., 1921, V, 89-ll.S.

CCLLBN, E. K.

Diverticulum of the first portion of the duodenum. — .Irch. Surg., Chicago. 1921. ii. 542-564.

Tuberculous ulcer of the anterior vaginal wall with resection of ulcer. — Surg., Gyncc, rf- Ohst., Chicago, 1921, xxxiii, 76-78.

CULLEN, T. S.

Three cases of subperitoneal, pedunculated adenomyoma. — .irch. Surg.,

Chicago. 1921. ii, 443-454.

The weak spot in American surgery. — Stirg., Gyncc. <£ Obst., Chicago,

1921. xxxiii. 67-72.

Early squamous-cell carcinoma of the cervix, accidentally discovered

when the body of the uterus was being curetted for hiemorrhage

cau.sed by hyperplasia of the endometrium and by a small submucous

myoma. — Surg., Gyncc. rf Obst., Chicago, 1921, xxxiii, 137-144.

CUSHING, H.

The personality of a hospitnl. 538.

Disorders of the pituitary gland; retrospective and prophetic M. Ass., Chicago, 1921. Ixxvi, 1721-1726.

Further concerning the acoustic neuromas. — Laryngoscope, St. Louis,

1921, xxxi, 209-228.

The special field of neurological surgery after another Interval. —

Illinois M. J., Oak Park, 1921, xxxix, 133-141 ; 185-195. Also:

Wisconsin M. ./., Milwaukee. 1920-21. xix. 501-520. Alio: Ohio .If. J.,

Columbus, 1921, xvii. 293-302; 373-380. Also: J. Jotca State If. Soc,

Clinton, 1921, xi, 337-342 ; 385-394 ; 426-430.

Dandy, W. E. ,„„,

The treatment of brain tumors. — J. Am. M. Ass., Chicago. 1921, Ixxvii. 1853-1859. , . . ^.

The diagnosis and treatment of hydrocephalus due to occlusions of the foramina of Magendie and Luschka. — Surg. Oynec. «f Ohst.. Chicago, 1921. xxxii. 112-124. „ „ , ^v ..

An operation for the removal of pineal tumors. — Surg., Grjnec. d OOst., Chicago. 1921. xxxiii. 113-119.

D.wiDsoN, E. C, and Dieuaide, F. R.

Terminal cardiac arrhythmias. Report of three cases. — Arch. Int. ilcd., Chicago. 1921. xxviii, 663-677.

Davis, D. M., and Swartz, E. O.

Action of niercurochr.ime -220 on the gonococcus. — .7. Anu M. Ass..

Chicago, 1921, Ixxvi, 844-846.

The action on the gonococcus of various drugs commonly useil in the

prophylaxis and treatment of gonorrhea. — .7. Vrol., Bait., 1921. v,

235-253.

Davis, E. G.

Urinary antisepsis ; a study of antiseptic propertie;

excretion of 204 aniline dyes. — Am. J. il. Sc, Phila..

267.

The significance of hematuria. Report of 46 cases. — J. Iowa State M.

Soc, Clinton. 1921. xi, 315.

The Young-Stone operation for urethrorectal fistula. Report of three

cases. — Surg., Oynec. i(- Ohst.. Chicago, 1921, xxxii. 225-231.

Davis, E. G., and Beck, G. H.

Urinary antisepsis. The secretion of antiseptic urine by man following the oral administration of proflavine and acriflaviue. I'reliminarv report. — J. Urol., Bait. 1921. v, 215-233.

Davison, W. C.

Biologic therapy, xiii. 1921, lxx%-i, 242-243.


-Boston il. <f S. J., 1921, clxxxv, 529Am.


Pertussis vaccine.


Davison, W. C, and Josephs, H. W.

The serotherapy of bacillary dysentery in children. — .7. Am. M. Ass., Chicago, 1921, l-xxvii, 1863-1.864.

Davison, W. C, and Rosenthal, L. V.

a bacteriological study of the fecal flora of infants and children (the lack of association of nutritional disorders with a so-called " putrefactive " intestinal flora). — Am. J. Dis. Child., Chicago, 1921. xxii. 284-298.

Denzer, B. S.

The size of the infantile palate. — Am. J. Dix. Child., Chicago, 1921. xxii. 471-47C.


Denzer, B. S., and Anderson, A. F.

Absorption of fluid injected into the peritoneal cavity. — .l»i. J. Dis. Child., Chicago, 1921, xxi, 565.

Dick, Gladys H., and Dick, G. F.

Experimental inoculations in scarlet fever. — J. Am. M. Ass., Chicago, 1921, Ixxvii, 782-785.

Dickson, E. C.

Botulismus. — In: Oxford Med., 1921. v, 231-257.

Pathology of botulism. (Correspondence.) — J. Am. M. Ass., Chicago,

1921, Ixxvii. 483-484.

Dickson, E. C, and Shbvky, R.

Botulism ; a study of the action of the toxin of B. botulinus upon the living tissues. — Proc. ,'ioc. Exper. Biol, d Med., N. Y., 1920-21, xviii, 313.

DiEU.AIDE, F. R.

The determination and significance of the electrical axis of the human heart. — .irch. Int. Med., Chicago, 1921, xxvii, 558-570.

Dunham, Ethel C.

Peritonitis as a complication of scarlet fever. — -4»i. J. Dis. Child.. Chicago, 1921, xxii, 307-309.

Dunton, W. R.

The psychosis of a famous portraitist. — Maryland Psychiat. Q., Bait., 1921, X, 50-54.

The passing of the Henry B. Faville School. — Maryland Psychiat. Q., Bait., 1921, X, 77-78.

Eb.augh, F. G., and Hoskins, R. G.

A case of dystrophia adiposogenitalis.1921, V, 21-28.


-Endocrinology. Glendale, Cal.,


Elmendorf, D. F.

Vincent's angina.


-.Mil. Surgeon. Wash.. 1921. xlix. 287-291.


Phila. & Lend.. 1921, J. B. Lippincott Co.,


Emerson, C. P. Clinical diagno 726 p. 8°.

The problem of the so-called primnrv ansemias from the internist's standpoint. — Chicago M. Recorder. 1921. xliii, 125-136. Teaching of general medicine. — J. Am. M. Ass., Chicago, 1921, Ixxvi, 869.

The acute element in the chronic nephropathies. — J. Am. M. Ass., Chicago, 1921, Ixxvii. 745-749.

Emmons, A. B., 2d.

Health in mercnntile establishments. 111. Common sanitary defects in stores. — J. Indust. Hyg.. N. Y. & Bost.. 1921. iii. 29-30.

Emmons, A. B., 2d., and Goldthwait, J. E.

A work chair. — J. Indu.it. Hyg., X. Y. & Bost. 1921, iii. 154-1.=SS.

Erlanger, J. Blood vol 207.


ind its regulatii


-Physiol, Rcc, Bait.. 1921, i, 177


duodenal ulcer. — Penii. M. J.


ERL.4NGEB, J., and White, H. L.

The effect on the composition of the blood of maintaining an increased blood volume by the intravenous injection of a hypertonic solution of gum acacia and glucose in normal asphyxiateci and shocked dogs. — Am. J. Phmiol., Bait.. 1920-21, liv, 1-29.

Estes, W. L., Jr.

Early diagnosis of perforated gastric 1920-21. xxiv, 307 309.

Eyster, J. A. E., and Meek, W. J.

Reactions to hemorrhage. — Am. J. Physiol., Bait.. 1921. Ivi. 1-15. The origin and conduction of the heart beat. — Physiol. Per., Bait., 1921, i, 1-43.

Fabyan, M., Tyzzer. E. E., and Foot, N. C.

Further observations on " blackhead " in turkeys. — J. Infect. Dis., Chicago, 1921, xxix. 26S-286.

Felton, L. D.

A colorimetric method for determining the hydrogen-ion concentration of small amounts of fluid.— 7. Biol. Chem., Bait., 1921, xlvi, 299-305.

Finney, J. M. T.

A personal appreciation of Sir William Osier. — J Chicago. 1921. Ixxvii. 2013-2019.

Acute intestinal obstruction. — Surg., Gyncc. <f Ohst. xxxii, 402-408.

Finney, J. M. T., and Friedenwald, J.

Pylorospasm in adults: its medical and surgical treatment. M. Sc, Phila., 1921, clxii, 469-481.

Finney, W. P., Jr.

Malignant timiors Surgeon, Wash., 1).


jlm. M. Ass., Chicago, 1921,


1917-1919.— Jfi(.


Fleurnoy, H.

Tin asile d'ali^n(^s an .Tnpon. Commuuic. au Congr^s des M4declns Ali^nistes de France. Strasbourg, Aoflt, 1921.

Flexner, S.

Biologic therapy. General considerations regarding serum and vaccine therapy.— 17. Am. M. Ass., Chicago, 1921. Ixxvi. 33-34. Biologic therapy. iv. Serum treatment of bacillary dysentery. — J. Am. M. Ass., Chicago. 1921. Ixxvi. 108-109.

Lethargic I encephalitis. History, pathologic and clinical features, and epidemiology in brief. — Stud. Rockefeller Inst. M. Research, N. Y., 1921, xxxvi, 119-131.


72


[No. 372


Flexner, S., and Amoss, H. L.

Experiments on the nasal route of infection in poliomyelitis. — Stud. Rockefeller Inst. U. Research, N. Y., 1921, xxxvl, 45-56.

Foley, F. E. B.

Clinical uses of salt solution in conditions of increased intracranial tension. — Surg., Oynee. d Ohst., Chicago, 1921. xxxiil, 126-136.

Fowler, H. A.

Tuberculosis of the kidney complicated bv impacted pelvic calculus. — J. Urol., Bait., 1921, v, 345-351.

Frontz, W. a.

A clinical and pathological study of contracted bladder. — J. Urol., Bait, 1921, V. 491-.^ill.

The more common causes of chronic urinary obstruction in male children. Tr. South. Med. Assoc, 1921, xv. Abstr. Jn: J. Am. M. .Iss , Chicago, 1921, Ixxvii, 1996.

Gaenslen, F. J.

The diagnosis and treatment of chronic lesions of the hip-joint. —

Minnesota Med., Saint Paul, 1921, iv, 630-635.

Sling suspension method of exercise in infantile paralysis. — Surg.,

Gynec. rf Ohst., Chicago, 1921. xxxii. 274-276.

Sacro-iliac joint arthrodesis by bone-splitting method. — Wisconsin

M. J., Milwaukee, 1921, xx, 20-22.

Gaenslen, F. J., and Schneider, C. C.

Treatment of tuberculosis of the ankle iu tlie adult. — J. -4m. M. Ass., Chicago. 1921, Ixxvii, 116.8-1171.

Gaenslen, F. J., and Thalheimer, W. M.

Congenital syphilitic epiphysitis in adolescence.Bost., 1921, ill, 8-17.

Gaither, E. H.

Interpretation of digestive symptomatology. Relative to change

in svmptoms and extrinsic factors. — J. Am. M. .-iss., Chicago, 1921,

Ixxvii, 1407-1410.

Chronic appendicitis. — South. M. J., Birmingham. .\la., 1921, xiv, 190 199.

Geraghtt, J. T.

Biologic therapy : II. Use of vaccines a

urethritis and its complications. — J. .4»i.

Ixxvi. 35.

Some general considerations regarding prostatectomy.-— .?o»(7i. M. J.,

Birmingham, Ala., 1921, xiv, 48-51.

Geraghty, J. T., Short, J. T., and Schanz, R. F.

Multiple renal calculi, unilateral and bilateral ; some observations. — T. Am. M. Ass.. Chicago, 1921. Ixxvii, 901-904.

GiFFIN, H. Z.

Present status of splenectomy as a therapeutic measure. — Minnesota Med., St. Paul. 1921. Iv, 132-13S.

GiFFiN, H. Z., and Szlapka, T. L.

The treatment of pernicious anemia by splenectomy ; second report. — J. Am. M. Ass., Chicago. 1921, Ixxvi. 290-295.

GiLMAN, P. K.

Amebic abscess of liver. — Calif. State J. M., San Fran., 1921. xix, 239. Acute suppurative thyroiditis. — Calif. State J. M., San Fran., 1921, xix. 294. Branchial cysts and fistulas. — J. Am. M. Ass., Chicago, 1921, Ixxvii,


~J. Orthop. Surg.,


20-28.

Cvsts and flstul.'e of the thyroglossal duct. Chicago, 1921, xxxii, 141-149.


-Surg., Gynec. & Ohst.,


ind treatment of cardiac disorders. — Tr. M. Ass.


VON Glahn, W. C., and Herman, N. B.

Carcinoma of the supra-ampullary portion of the duodenum.M. So., Phila., 1921. clxi, 111-119.

Goddard, C. H.

The recognition Alahatna, 1921.

Goetsch, E.

The diagnosis and treatment of hyperthyroidism. — .Y. To) 7; M. J.,

[etc.]. 1921. cxiii, 378-383.

Hyperthyroidism in pregnancy. — Ti. Y. State J. M., N. Y., 1921, xxl.

317-324.

Goodpasture, E. W.

Myocardial necrosis in hyperthyroidism. — J. Am. M. Ass , Chicago, 1921. Ixxvi. 1545-1551.

The infliience of thyroid products on the production of myocardial necrosis. — J. Eiper. M., Bait., 1921, xxxiv, 407-423.

Goodpasture, E. W., and Talbot, F. B.

Concerning the nature of "protozoan-like" cells in certain lesions of infancy. — Am: J. /)«. Child., Chicago, 1921, xxi, 415-425.

Greenhill, J. p.

.\ histological study of fetus and implantation site in a case of missed abortion. — .4«t. J. Ohst. tf Ounce, St. Louis. 1921. 11. 188-194, Tuberculous salpingitis. A clinical study of 200 cases. — Johns Hop kins Hasp. Rep., Bait, 1921, xxi, 97-156.

GUNDRUM, F. F.

Focal infection. — Calif. State J. M., San Fran., 1921. xix, 296.

"' ' exophthalmic goitre. — Calif. State J. M., San Fran.,

Guthrie, C. G.

A simple and accurate metabolism spirometer. Spirometer measurement of oxygen consumption bv the rebreathing method. — .Arch. Int. Med., Chicago, 1921, xxviii. 687-702.


GwYN, N. B.

The epidemiology and diagnosis of encephalitis lethargica. — Canad. U. Ass. J., Toronto, 1921, xi, 169-173.

H.\LSTED, W. S.

Ablation d'un an^vrysme de la premiere portion de I'art^re sousclavi^re gauche. — Lyon chirurg., 1921, xviii, 1-6.

Hanson, H.

Sanitary conditions in Peru. — Am. J. Pub. Health, N. Y.. 1921, xi, 13-10.

Harrop, G. a., Krogh, a., and von Liebermann, P.

studies on the physiology of capillaries. 11. The reactions to local stimuli of the blood-vessels In the skin and web of the frog. — J. J'hysiol, Cambridge, 1921, Iv, 412422.

Hastreiter, R. F.

Nitrous oxide oxygen, analgesia and anesthesia in obstetrics. — South. Calif. Fract., Los Angeles. 1921, xxxvl, 17-18.

Hazen, H. H.

The diagnosis of syphilis. — Am. J. Syph., St. Louis, 1921. v, 472-475. Public health activities in venereal disease control. — Am. J. Syph., St. Louis. 1921. V, 674-076.

Roentgen-ray treatment of cutaneous cancer. — J. Am. if. .4s«., Chicago, 1921, Ixxvi, 1222-1227.

Henderson, D. K.

War psychoses, the infective, exhaustive group. — Glasgow M. .J., 1921, xcvi, 321-336.

Hennington, C. W.

Abdominal incision. — X.


York State J. M., N. Y., 1921. xxi, 81 83.


Herman, N. B., and von Glahn, W. C.

Carcinoma of the supra-ampullnry portion of the duodenum. — .4m. -J. M. Sc, Phila.. 1921. clxi, 111-119.

Heuer, G. J.

Surgery of the thorax. — Jn: Surgery, its Principles & Practice. (Keen) Phila. & Loud., 1921, viii, 332-414.

Higgins, W. H.

Unusual relapse in typhoid fever. — Virginia M. Month., Richmond, 1921-22, xlviii, 347-348.

HiNMAN, F,

Multiple renal and ureteral stones in with results of ureteronephrectomy. — , Ixxvi. 237-238.

HiNMAN, F., and Gibson, T. E.

Squamous cell carcinoma of the bladder: a study of heterotopic epidermization. with a review of the literature and report of cases. — J. Urol, Bait., 1921, vi. 1-50.

HiRSCHPELDER, A. D.

Saligenin. a new non-toxic local anesthetic and its mercury derivative, a new antiseptic. — Miiincsota Med., St Paul, 1921, iv, 399-402.

Ejrschfelder, a. D., Hart, M. C, and Kucera, F. J.

Mercurv saligenin. a new antiseptic. — Proc. Soc. Eiper. Biol, rf Med., N. Y.. 1920-21, xviii. 77-79.

Hirschfelder, D., Hart, M. C., and Kucera, F. J.

Further studies on saligenin : its mercury derivative and allied compounds. ( Proceedings.) — J. Pharmacol, d Expcr. Therap., Bait., 1921. xvii. 325-326.

Hirschfelder, D., and Quigley, J. P.

The relation of substitution in the carbinol group to the pharmacological action of some phenyl carbinols (Proceedings). — ./. Pharmacol, d Eiper. Therap., Bait.. 1921, xvii, 326-327.

Hitzrot, J. M.

Fractures of the head and the neck of the radius. — .4m. J. Surg., X. Y.. 1921, XXXV. 100-109.

Hitzrot, J. M., and Murray, C. R.

The factors that influence the prognosis in fractures at the base of the radius. — .Am. J. Surg., N. Y., 1921, xxxv, 17-29.

Hitzrot, J. M., and Weeden, W. M.

The treatment of acute suppurative pleurisy. — .Ann. Surg., Phila., 1921. Ixxiii, 531-544.

Hohman, L. B.

Epidemic encephalitis (lethargic encephalitis) its, psychotic manifestations, with a report of twenty-three cases. — .Arch. Neurol, d Psjichiat., Chicago. 1921, vl. 295-333. .Also: [Abstr.] Maryland Psychiat. Q.. Bait. 1921, X, 73 77.

Holmes, J. B.

Recent progress in anatomy, physiology and pathology of childhood. — Am. J. Dis. Child., Chicago, 1921, xxil, 61 103.

Holmes, W. R.

The illness ', 1921. X, 628.


nd death of Napoleon.


Georgia, Atlanta


HosKiNS. R. G.

Some current trends in endocrinology. — .J. Am. i 1921. Ixxvii. 1459-1462.

The reaction to epinephrin administered by rectum. Ej-per. Therap., Bait.. 1921, xviii, 207-211.

HosKiNS, R. G., and Ebaugh, F. G.

A case of dystrophia adiposogenitalis.1921, V, 21-28.


.Ass., Chicago, '. Pharmacol, i


-Endocrinology, Glendnle, Cal.,


February, 1932]


73


Howard, C. P.

Obesity. — In: Oxford Med.. 1921. iv, 105-213.

Hemochromatosis. — In: Oxford Med., 1921. iv. 215-222.

Oclironosis — In: Oxford Med.. 1921, iv, 223-22S.

Riclsets. — In: Oxford Med., 1921, iv, 229-252.

Scun-y. — In: Oxford Med.. 1921, iv, 253-272.

Trentment of pneumonia with special reference to the use of serum. —

Canad. M. Ass. J., Toronto. 1921, xi, 709-713.

Howard, C. P., and Gibson, R. B.

A case of allsaptonuria with a study of its metabolism. — Arch. Int. Med., Chicago. 1921, xxviii, 632-637.

Howell, W. H.

Dr. Meltzer's influence on American physiology. — Proc. Soc. Exper.

Biol, d Med., N. Y.. 1920-21. xviii. 25-36. Memorial Number for S. J.

Meltzer.

Samuel James Meltzer. — Science, N. Y. & Lan(?aster, 1921, liii. 99-106.

Howland, J.

Prolonged intolerance to carbohydrates. (Proceedings.^ — Arch. Pediat.,

N. Y., 1921, xxxvlii, 393-396.

Measles.— y?i.- Oxford Med., 1921, v, 499-517.

Scarlet fever. — In: Oxford med., 1921. v. 519-546.

Rubella. — In: Oxford med., 1921, v, 547-551.

Howland, J., and Kramer, B.

Calcium and phosphorus in the serum in relation to riclicts. — Am. -1 . Dig. Child., Chicago, 1921, xxii, 105-119.

Howland, J., Kramer, B., and Tisdall, F. F.

Observations on infantile tetany. — Am. J. Dis. Child., Chicago, 1921, xsii, 431-437.

The clinical significance of calcium concentration in the serum of children and possible errors in its determination. — .Im. J. Dis. Child., Chic.igo. 1921. xxii, 560-564.

Howland, J., Tisd.^ll, F. F., and Kramer, B.

The concentration of sodium and potassium as compared with that of calcium and magnesium in the serum of patients with active infantile tetany. — Proc. Soc. Ed-per. Biol. & Med., N. Y.. 1921. xviii. 252-253.

HuGHSoN, W., and Weed, L. H.

The sliull as a closed box. (Proceedings.) Anat. Record, I'hila.. 1921, xxi, 8.S.

HUNNER, G. L.

Intractable blaSder symptoms due to ureteritis. — Tr. Am. Urol. Ass.,

1920. Bait., 1921, xii, 193-219.

Hunt, R.

Some factors relating to the toxic action of arsphenamin. — J. Am. U. Ass., Chicago, 1921, IxxVi. 854-S59.

HUKD, H. M.

The earlv days of the Training School for Nurses at the Johns Hopkins Hospital. — Johns Hopkins Nurses Alumnae Mag., Bait., 1921, XX. 63-71.

HURWITZ, S. H.

Some aspects of pernicious anemia and its treatment. — Calif. State J. M., Sin Fran.. 1921, six, 275.

Ingraham, C. B.

Imprcsssions gained from the use of radium during the past year. —

Colorado Med.. Denver. 1921. xviii. 72-75.

Vaginal cysts.^^/. Am. M. Ass., Chicago. 1921. Ixxvii. 14S7-14S9.

Jeidell, Helmina.

Active immunization against diphtheria. — Northwest Med., Seattle.

1921. XX. 89-92.

Josephs, H. W., and Davison, W. C.

The serotherapy of bacillary dysentery in children. — J. Am. if. Ass., Chicago, 1921. Ixxvii, 1863-1S64.

Keidel, a.

The treatment of neurosyphilis. — South. M. .7. , Birmingham, Ala., 1921. xiv, 595-601.

Keidel, A., and Moore, J. E.

Internal hydrocephalus in a syphilitic, probably due to Intraspinal treatment. — .im, J, M, Sc. Phila., 1921. elxii, 209-215. Stomatitis and aplastic anemia due to neoarsphenamin. — Arch, Dermatol, d Si/ph., Chicago. 1921. iv. 169-176.

Dermatitis and allied reactions following the arsenical treatment of syphilis. — .Arch. Int. Med., Chicago, 1921, xxvii. 716-747. Comparative results of colloidal mastic and colloidal gold tests. — Arch, Neurol, <C Psychiat,. Chicago. 1921. vi. 163-172. Studies in asymptomatic neurosyphilis. I. A tentative classification of early asymptomatic neurosyphilis. — Arch, Neurol, d Psychiat., Chicago. 1921. vi. 286-291.

Studies in familial npurnsyphilis. 1. Conjugal neurosyphilis. — J. Am, M, Ass,, Chicago. 1921. Ixxvii. 1-7.

Kelly, H. k,

Lafayette Houghton Bunnell. M. D.. discoverer of the Yosemlte. — Ann.' Med. Hist.. N, Y.. 1921. ill. 179-193.

Operation for renal calculi. — -V. Tor7.- Jlf. J,, Fete.]. 1921. cxiii. 1-3. Drainage in pelvic abdominal surger.v. — N. York M, J, [etc.], 1921, cxiv, .391-394.

The treatment with radium of cancer of the bladder. (Correspondence.) — Surg.. G:/nec. d Ohst.. Chicago. 1921. xxxii. 187. A diary of Robert Battev. M. D. — Thcrap. Gas.. Detroit. 1921, xlv, 612-62(5. .Also: J. Med. Ass. Georgia. Atlanta, 1921. x. 693-695. Uterine polyps. — Therap. Gas., Detroit, 1921, xlv, 761-767.

Kelly, H. A., and Frickb, R. E.

The use of pessaries. — Therap, Gaz,, Detroit, 1921, xlv, 5-9.


I's wealth. — ^Voman Citizen, N. Y-. A\igust,


M. Ass., Chicago, 1921.


-Arch. Int. Med,, Chicago, 1921,


Kbtron, L. W.

A note on the treatment of larva migrans. — Arch. Dermatol, d Syphil,, Chicago, 1921. iv, 368-369.

Kempp, E. J.

A minimum course in psychopathology for medical students. — Y. York M. J. [etc.], 1921, cxiii, 309-313.

Kempf, Helen C.

Child health, the natii 1921.

Kline, B. S.

Spirochetal pulmonary gangrene.Ixxvii, 1S74-1S77.

Kline, B. S., and Fishberg, M.

Spirochetal pulmonary gangrene.xxvii. 61-70.

Knox. J. H. M., Jr.

The health center, what it may mean to a community. (Proceedings.) — Arch. Pediat.. N. Y.. 1921. xxxviii. 451. The claim of the child. — Bambino. Columbus. Ohio. 1921. 10-11. Erysipelas in childhood. — South. M. /., Birmingham. Ala., 1921, xiv, 387 393.

Kramer, B., and Howland, J.

Calcium and phosphorus in the scrum in relation to ricliets. — .Im. J. Dis. Child., Chicago. 1921. xxii, 105-119.

Kramer, B., and Tisdall, F. F.

A clinical method for the quantitative determination of potassium in small amounts of serum. — J. Biol, Chem,, Bait.. 1921. xlvi, 339-349. Simple method for the direct quantitative determination of sodium in small amounts of serum. — J. Biol, Chem., Bait., 1921. xlvi, 467473.

A simple technique for the determination of calcium and magnesium in small amounts of serum. — J, Biol, Chem,, Bait, 1921. xlvii, 475481.

Methods for the direct quantitative determination of sodium, potassium, calcium, and magnesium in urine and stools. — J, Biol, Chem,, Bait, 1921, xlviii, 1-12.

The direct quantitative determination of sodium, potassium, calcium, and magnesium in small amounts of blood. — J, Biol. Chem,, Bait., 1921, xlviii, 223-232.

Kramer, B., Tisdall, F. F., and Howland, J.

Observations on infantile tetany. — Am, J, Dis, Child,, Chicago. 1921,

xxii, 431-437.

The clinical significance of calcium concentration in the serum of

children and possible errors in its determination. — Am. J, Dis. Child.,

Chicago, 1921, xxii. 560-564.

The concentration of sodium and pot.nssium as compared with that

of calcium and magnesium in the serum of patients with active

infantile tetany. — Proc, Soc, Exper. Biol, d Med,, N. Y.. 1921, xviii,

252-253.

Kratjse, a. K.

Tuberculosis : infection, patholo.gy. etiology and bacteriology. — In:

Nelson Loose-Leaf Med., Lond. & X. Y., [1921], i. 309-336.

Some problems of medical education in tuberculosis. — Am. Rev.

Tuhcrc, Bait., 1921-22. v. 755-768.

The tuberculosis problem. Some thoughts on its solution. — Am. Rei:

Tuberc, Bait.. 1921-22. v, 769-763.

Essays on tuberculosis, xx. Some phases of resistance. Part Iv.

Immunity. (Continued.) Immunization with living virulent bacilli:

Trudeau's work. — J, Outdoor Life, N. Y., 1921, xviii. 31-34; 48-49.

Essays on tuberculosis, xx. Some phases of resistance. Part It.

Immunity. (Continued.) Cattle immunization. — J, Outdoor Life,

N. Y.. 1921. xviii. 63-67. „ ^ ,

Essays on tuberculosis, xxi. Some phases of resistance. Part Iv.

(Immunity, concluded.) The nature of resistance: its determining

factors. — J. Outdoor Life, N. Y.. 1921. xviii. 247-252.

Essays on tuberculosis, xxii. Reinfection. — J, Outdoor Life, N. 1..

1921, xviii, 2~" """


The elementary pathology of tubcr1921. xviii. 305-312: 327. The pathology of tuberculosis: '. Outdoor Life, N. Y., 1921,


xvlil.


Essays on tuberculosis, xxiii.

culosis. — J, Outdoor Life, N. Y.

Essays on tuberculosis, xxiv,

spread throughout the body.—

34*^-347 35.8-359

Course of' tubercle bacillus from sputum to the child. — N, T. State J.

M.. N. Y., 1921. xxi, 83.

Enseignement de la tuberculose aux ftudiants. Mission AmSricaine

de preservation contre la Tuberculose (Fondatlon Rockefeller), Paris,

1921.

Tuberculin : its nature and its effects on tuberculous animals ; Its

.iction. Bureau of Correspondence and information. Texas State

Tuberculosis Sanatorium. 1920. Pamphlet No. 22. [1921.] 23 p. 8".

Leonard. V. JT.

A building designed to meet the requirements of group practice. — Mod. Hasp., St. Louis. 1921, xvii. 131 132.

Levy, R. L.

Advanced heart failure. General s.vmptoms. — In: Nelson Loose-Leaf Med.. 1921, iv, 285-300.

Restoration of the normal cardiac mechanism in auricular fibrillation by quinidin. Preliminary report. — -J, Am, M. Ass., Chicago, 1921, Ixxvi, 1289-1293.

Leyt, R. L., and Cohn, A. E.

Experimental studies of the pharmacology of quinidin. — Proc. Soc, Exper, Biol, d Med., N. Y.. 1920-21. xviii, 283-284.

Lewis, D. S., and Moff.att, C. F.

Standardization of certain preparations of digitalis. — Canad. M. Ass. J., Toronto. 1921, xi, 755-758.


74


[No. 372


Lewis, W. H., and Webster, L. T.

Migration of lymphocytes in plasma cultures of human lymph nodes. —

J. Exper. M., Bait. 1921. xxxiii. 261-269.

(Jiant cells in cultures from human lymph nodes. — J, Exper. M., Bait.,

1921. xxxiii. 349-360,

Wandering cells, endothelial cells, and fibroblasts in cultures from

human lymph nodes.—/. Exper. M., Bait.. 1921, xxxiv. 397-406.

LiNTHICUM, F. H.

Ozena and its relation to tuberculosis. — -4m. J. il. Sc, Phila.. 1921, clxii. 216-226.

LiSSER, H.

Does the pituitary .secretion iniiuence the development of the prostate? — -Y. York M. J., [etc.]. 1921. cxiii, 391-393.

Little, H. M.

The least common multiple in obstetrics. — .Iw, J. Obst. A Crynec, St. Louis. 1921, ii, 67-76.

LOWSLEY, 0. S.

The role of the prostate and seminal vesicles in arthritis. With a discussion of surgical and nonsurgical treatment. — N. York M. J., [etc.], 1921. cxiii, 641-646. -4(so.- Tr. Am. Urol. Ass., 1920, Halt., 1921. xii, lis.

A perineal operation for removal of stone in the lower end of the male ureter. — Surg., Oynec. d Obst., Chicago, 1921, xxxii, 300-306.

Lyman, D. R.

The limitations and possibilities in the federal care of tuberculous exservice patients. — Xat. Tuberc. Ass., Tr., N. Y., 1921, xvii.

M.icCallum. W, G.

The pathology of the pneumonia in the United States army camps durin;; the winter of 1917-18. — Johns Hopkins Hasp. Rep., Bait., 1921, XX. 1 147.

r;ithul(i;;ical anatomy of pneumonia associated with influenza, — Johns Hopkins Hasp. Rep., Bait., 1921, xx, 149-249,

MacCallum, W. G., and Moody, L. M.

Alastrim in Jamaica. — Am. J. Hyg., Bait,. 1921. i 388-409.

McCann, W. S.

Tho effect of the ingestion of foodstuffs on the respiratory exchange

in pulmonary tuberculosis. — Arch. Int. Med., Chicago, 1921, xxviii,

847-S5S.

Limitations of metabolism determinations in diagnosis. — Med. Clin. ^.

Am., Phila., 1921, iv, 1483.

McCrae, T.

Arthritis deformans. — In: Oxford Med., 1921, iv, 367-403. Pain in lower back. — Med. Clin. N. Am., Phila., 1921, iv, 973.

Macht, D. I.

On the absorption of local anesthetics through the genito-urinary organs. — J. Pharmacol. iC- Exper. Therap., Bait., 1920-21, xvi, 435-448, Pharmacological examination of cinnamein, benzvl succinate and benzyl nitrite. — Proc. Soc. Exper. Biol. & Med., N. Y., 1920-21, xviii, 177-179.

Macht, D. I., and Bloom, W.

Kxperimentnl inquiry into the cerebral and neuromuscular manifestatiiuis of digitalis. — .irch. Int. Med., Chicago. 1921, xxviii, 678-686. rhysiolugical and pharmacological studies of the prostate gland. III. ICffect of prostatectomy on the behavior of albino rats.— »/. Urol., Bait., 1921, y, 29-41,

A pharmacodynamic analysis of cocain action of the cerebrum. — Proc. Sop, Exper. Biol. <£ Med., N. Y., 1920-21, xviii, 81-82, Comparative study of ethanol. caffeine and nicotine on behavior of albino rats. — Proe. Soe. Exper. Biol. <f Med., N. Y",, 1920-21, xviii, 99-100,

The effect of prostatectomy on the behavior and learning of albino rats. — Proc. Soc. Exper. Biol. <{• Med.. N. Y.. 1920-21, xviii, 100-101. Comparative study of ethanol. caffeine and nicotine on the development of frogs' larvse, — Proc. Soc. Exper. Biol, d Med., N, Y,, 1920-21, xviii. 241-242.

Macht, D. I., and Ting, G. C.

Response to drugs of excised bronchi from nonual and diseased animals. — J. Pharmacol, d Exper. Therap., Bait., 1921, xviii, 111119.

Experimental inquiry into sedative properties of some aromatic drugs and fumes. — J. Pharmacol, d Exper. Therap., Bait,. 1921, xviii, 361. "' ' " -J. Pharmacol, d Exper.


Major, R. H.

The treatment of empyema Phila,, 1921, clxii, 397-406.


ith gentian violet,


J. M. Sc,


M.4RRI0TT, W. McK,

Ahnnrinal met.-ibolism in infancy and its relationship to symptomatology,— nnt, ./. Child. Dis., Lond,. 1921, xviii, 129-13.').

Marshall, H. W. ,

Scoliosis,— Bos/oii M. d S. J.. 1921, clxxxiv, 31-40. When to open knee-joints, — Boston M. d S. J., 1921. clxxxiv, 291-297, A case of myositis ossificans traumatica developing from torn coracoclavicular ligament without other bony fracture, — Boston M. d S. J. 1921. clxxxiv. 380-384. A questionnaire on sacro-iliac Joint lesions. — Boston M. d S. ./,, 1921.


-/. Ai.


M. Ax


Mason, C, C and Shohl, A. T.

Pood at the front lines, — Mil. Surgeon, Wash., 1921, xlviii. 386-401.

Maxwell, J. P.

Obstetrics and gynecology in South China. — China M. J.. Shanghai.


1921. XXXV. 146-1. Intestinal parasiti XXXV, 377-382.


South Fliki


-China M. J., Shanghai. 1921,


Mei-er, a.

The contributions of psychiatry to the understanding of life problems. — In: A Psychiatric Milestone. Bloomingdale Hospital Centenary 1821-1921. N. Y,, 1921, 21-54. [Privately printed.] The integrative function of a hospital laboratory. Retrospect and prospect. — State Hasp. Q.. Utica, N. Y., 1921, vi, 445-451. I'sychopathology. By E. J. Kemp. St. Louis, 1920, C. V. Mosby Co., 762 p, 8°. (Book review.) — Arch, fiearol. d Psychiat., Chicago, 1921, v. 7.'<2-790.

Medizin und Kecht. Die Beziehungen der Medizin zum Recht, die Kausalitiit in Medizin und Recht und die Aufgaben des gerichtlichmediziuischen Unterrichtes, von Prof. H. Zangger, Art. Institut Orell Fiissli. (Book review.) — ./. .4. Inst. , . . Criviinol., Chicago, 1921-22, xii, 457-458.

Miller, M. K.

Splenic anemia of infancy, with report of a case occurring in an infant at six weeks of age. — Arch. Pcdiat., N, i',, 1921. xxxviii. 11 17, Polyneuritic syndrome in young children. — J. Indiana M. Ass., Kort Wayne, 1921, xiv, 144-145.

Miller, M. K., and Lyon, M. W., Jr.

Case of meningitis in an infant due to a thread-like diphtheroid organism. — Am. J. M. Sc, Phila,, 1921, clxii, 593-598.

Mills, R. G.

Abstract of .Tapanese medical literature, — China M. ./,. Shanghai,

1921. XXXV, 67-78; 171-180.

Pathological specimens (Correspondence). — China M. J., Shanghai,

1921, XXXV, 185-186,

A preliminary study of post-operative catheterization. — CAina M. J.,

Shanghai, 1921, xxxv, 217-241.

A study of post-operative catheterization in Korea. — China M. J.,

Shanghai, 1921. xxxv, 310-331.

Ecological studies in the Tang-Mai River basin. North Korea, — Tr.

Korea Branch Royal Asiatic Soo., 1921, xii, part 1.

MiNOT, G. R.

Two curable cases of anemia. — Med. Clin. X. Am., Phila., 1921, iv. 1733-1750.

MiNOT, G. R., and Smith, L. W.

The blood in tetrachlorethane poisoning 1921, xxviii, 687-702.

MooRE, J, E.

The genesis of neurosyphilis. — Arch. Dermatol, if Ki/ph., Chicago. 1921, iv, 55-61.

The cerebrospinal fluid in treated syphilis. — ./. Am. M. Ass., Chicago, 1921, Ixxvi. 769-773.

Moore, J. E., and Keidel, A.

Internal hydrocephalus in a syphilitic, probably due to intraspinal

treatment. — Am. J. M. Sc, Phila.. 1921, clxii, 209-215.

.Stomatitis and aplastic anemia due to neo-arsphenamin. — Arch.

Dermatol, d Syph., Chicago. 1921, iv, 169-176.

Dermatitis and allied reactions following the arsenical treatment of

syphilis, — Arch. Int. Med., Chicago, 1921, xxyii, 716-747.

Comparative results of colloidal mastic and colloidal gold tests. —

Arch. Xeiirul. ,1 I'siirhint., Chicago. 1921, vi, 163-172,

Studies in ;(s,\ iiiiiIorn;itic neurosyphilis. I. A tentative classification

of early asviiiptunuitic neurosyphilis. — .\rch. Neurol, d Psychiat.,

Chicago, 1921, vi, 286-291.

Studies in familial neurosyphilis. 1. Conjugal neurosyphilis. — J. Am.

M. Ass., Chicago, 1921, Ixxvii, 1-7.

Morris, R. S.

Some difficulties in the diagnosis of empyema complicating pneumonia.—,/. Am. M. Ass.. Chicago, 1921, Ixxvi. 1336-1337. A simple and inexpensive apparatus for diagnostic or therapeutic punctures,—^. Am. M. Ass., Chicago, 1921, Ixxvii, 286.

Mosenthal, H. O.

Diabetes mellitus. A system of diets. With list of diets in pad form 150 sheets in a pad). — N. Y., 1921, P. B. Hoeher. The clinicnl value of basal metabolism determinations in diseases of the thyroid glands. — .V. York M. J., [etc.], 1921, cxiv, 41-43.

Mosenthal. H. O., and Marks, H. E.

The clinical value of basal metabolism. 1921. iv. 1403.


-.\rch. Int. Med., Chicago,


-Med. Clin. X. Am., Phila..


Nixon, P. I.

Congenital hypertrophic pyloric stenosis in infants. — Med. Rec, N. Y., 1921. xcix, 433-435,

Acute dilatation of the stomach following g.ynecological operations. — Texas State J. M., Fort Worth, 1921, xvi, 4S1-483,

Norms, C. C.

Gynecological and obstetrical tuberculosis. — X. Y'.. J921. D. Apple

ton & Co. 348 p. S'.

Ovar.v containing endometrium, — Am. J. Obst. d Gynec, St. Louis,

1920-21, i. 831-834.

The treatment of gonorrhoea in the lower genito-urinary tract in

women. — Surg., Oyncc. d Obst.. Chicago. 1921, xxxiii. 308-311,


& Lond., 1921, D. Appleton & Am. M. Ass ,


Novak, E.

Menstruation and its disorders.—

Co., 357 p, S-,

Acute postoperative dilatation of the stomach. Chicago. 1921. Ixxvii. 81-88.

[The interpretation and clinical significance of uterine haemorrhage.] — .

Med. Weikbl.. Amst., 1921, xxvii. 563-573,

Nuttall. G. H, F., and Keilin. D.

On the nephrocytes of Pedicuhis hii 1921, xiii, 184192.


-Parasitology, Cambridge.


Opie, E. L., Rivers, T. M. [et a!.]

Epidemic respiratory diseases. The pneumon of the respiratory tract acc<inin.'inying inllu Louis, 1921, C. V. Mosby Co.. 402 p. 8\


February, 1922]


75


Oliver, J. R.

The results of the application of laboratory methods to the study

of delinquency. Read at Ann. Cong. Am. Prison Assoc, Columbus,

Ohio. — Proc. Am. Prison Assoc. 1921.

Emotional states and illegal acts in connection with schizophrenia. —

Proc. Am. Psychiat. Assoc, 1921.

The causes and cures of crime. Bv Thomas S. Mosby. St. Louis.

1913, C. V. Mosby Co., 354 p. 8°. — Ment. Uyg., N. Y., 1921, v, 873 Modern criminal procedure in Switzerland. By Prof. Hafter, of Zurich. [Transl. and comments.] — J. Am. Inst. . , . Crimmol., Chicago, 1921-22, xii.

P.^LMER, W. W.

•Metabolism. (Survey of literature from Sept. 1. 1920. to March 1, 1921.) — In: Nelson Loose-Leaf Med., Lond. & N. Y., 1921, iii, 36. Diabetes insifiidus. (Survey of literature from Sept. 1, 1920, to March 1. 1921.) — In: Nelson Loose-Leaf Med., Lond. & N. Y., 1921. iii, 54-55.

Palmer, W. W., Atchley, D. W., and Loeb, R. F.

Studies on the regulation of osmotic pressure. I. The effect of increasing concentrations of gelatin on the conductivity of sodium chloride solution. — J. Gen. Physiol., Bait., 1921, iii, 801-806.

P.-iLMER, W. W., and Jackson, H., Jh.

A modification of Folin's uric acid method. — Proc. Soc. Exper. Biol, d Med., N. Y., 1920-21, xviii. 126-127.

P.\LMER, W. W., and Ladd, W. S.

The carbohydrate fat ratio in relation to the production- of ketone bodies in diabetes mellitus. — Proc. Soc. Exper. Biol. <l Med., N. Y., 1920-21. xvili, 109-110.

Parfitt, C. D.

Summary of report. Board of Tuberculosis Consultants. Dept. Soldiers' Civil Re-Establishment. — Ottawa. Canada, 1921, 21 p. 8°. Experiences in classification of sanatoria. — Nat. Tiiherc. Ass., Tr., N. Y.. 1921. xvii.

Extensive rib resection in the treatment of pulmonary tuberculosis. Report of 4 cases. — Tr. Am. Climat. & Clin. Ass., Phila., 1921, xxxvii.

Park, E. A., McCollum, E. V., Simmonds, Nina, and Shipley, P. G. studies on experimental rickets. IV. Cod-liver oil as contrasted with butter fat in the protection against the effects of insufficient calcium in the diet. — Proc Soc. Exper. Biol. <£ Med., N. Y., 192()-21, xvili, 275-277.

Park, E. A., Shipley, P. G., McCollum, E. V., and Simmonds, Nina. Studies on experimental rickets. V. The production of rickets by means of a diet faulty in only two respects. — Proc. Soc Exper. Biol, li Med., N. Y., 1920-21, xviii, 277-280.

Park, E. A., McCollum, E. V., Simmonds, Nina, and Shipley, P. G. Studies on experimental rickets. VI. The effects on growing rats of diets deficient in calcium. — Am. J. Hyg., Bait.. 1921, i, 492-511.

P.\RK, E. A., Shipley, P. G., McCollum, E. V., and Simmonds, Nina. Studies on experimental rickets. VII. The relative effectiveness of cod-liver oil as contrasted with butter fat protecting the body against insufficient calcium in the presence of a normal phosphorus supply. — Am. J. Hyg., Bait., 1921, i, 512-525.

Paton, S.

Human behavior, in relation to the study of educational, social, and ethical problems. N. Y., 1921, C. Scribner's Sons. 465 p. 8".

Peabody, F. W.

Acute poliomyelitis. — In: Oxford Med., 1921, v, 107-123.

A report of the Harvard Infantile Paralysis Commission on the

diagnosis of acute cases in 1920 ; with special reference to the

incidence of cases without paralysis. — Boston M. if- S. J., 1921, clxxxv,

174-176.

The vital capacity of the lungs in heart disease. — Med. Clin. N. Am.,

Phila., 1921, iv, 1655-1671.

Peabody, F. W., and Sturgis, C. C.

Clinical studies of the respiration, vii. The effect of general weakness and fatigue on the vital capacity of the lungs. — Arch. Int. Med., Chicago. 1921, xxviii. 501-510.

Peabody, F. W., Stukgis, C. C, Tompkins, E. M., and We.^rn, J. T. Epinephrin hypersensitiveness and its relation to hyperthyroidism. — Am.. J. M. Sc, Phila., 1921, clxi, 508-517.

Peabce, Louise.

Studies on the treatment of human trypanosomiasis with tryparsamide (the sodium salt of N-phenylglycineamide-p-arsonic acid). — V. Exper. M., Bait.. 1921. xxxiv, 1-104. (Suppl. No. 1.)

Pearce, Louise, and Brown, W. H.

Latent infections with the demonstration of Spirochete pallida in lymphoid tissues of the rabbit. — Am. J. Syph., St. Louis, 1921, v. 1-8. Experimental production of clinical types of syphilis in the rabbit — • Arch. Dermatol, d Syph., Chicago. 1921. n. s., iii. 254-262. The defensive reactions of animals infected with Spirochaeta pallida — J. Am. M. Ass., Chicago, 1921, Ixxvii, 1619-1620.

Therapeutic action of N-phenylglycineamide-p-arsonic acid (tryparsamide) upon experimental infections of Trypanosoma rhodesiense — J. Exper. M., Bait.. 1921. xxxiii. 193-200.

Superinfection in experimental syphilis following the administration of subcurative doses of arsphenamine or neoarsphenamine.^-r/. Exper M., Bait. 1921, xxxiii, 553-567.

Experimental syphilis in the rabbit. VII. Affections of the eyes — J. Exper. Med., Bait.. 1921. xxxiv, 167-183.

Note on the preservation of the stock strains of Treponema pallidum and on the demonstration of infection in rabbits.— i/. Exper. Med Bait., 1921. xxxiv, 185-188.

The penetration of normal mucous membranes of the rabbit by Treponema pallidum. — Proc. Soc. Exper. Biol, d Med., N. Y., 1920-21 XTlii, 200-201.


Neoplasia in experimental syphilis. — Proc. Soc Exper. Biol. & Med .

N. Y., 1020-21, xviii, 201-202.

Superinoculation experiments with Treponema pallidum. — Proc. Soc.

Exper. Biol, d Med.. N. Y.. 1920-21, xviii. 255-257.

Multiple infections with Treponema pallidum in the rabbit. — Proc

Soc. Exper. Biol, d Med., N. Y., 1920-21, xviii, 258-261.

Pe.«ce, Louise, Brown, W. H., and Witherbee, W. D.

Experimental syphilis in the rabbit. VI. Affections of bone, cartilage, tendons, and synovial membranes. 1. Lesions of the skeletal system 2. Clinical aspects of syphilis of the skeletal system. Affections of the facial and cranial bones and the bones of the foreaim. 3. Syphilis of the posterior extremities with other affections of a miscellaneous type.— ^. Exper. M., Bait., 1921, xxxiii, 495-514 ; 515-524 ; 525-538.

Pearl, R.

The significance of biometry and vital statistics to the science of medicine. — In: Nelson Loose-Leaf Med., Lond. & N. Y., 1921, vii, 111 The biology of death : I. The problem. II. Conditions of cellular immortality. HI. The chances of death. IV. The cause of death V. The inheritance of the duration of life in man. VI. Experimental studies on the duration of life. VII. Natural death, public health, and the population problem. — Seient. Month., N. 1'., 1921, xii, 193-214 : 321-335 ; 443-456 : 489-516. xiii, 46-66 ; 144-164 ; 193-213. Influenza studies : II. Further data on the correlation of explosiveness of outbreak of the 1918 epidemic. III. On the correlation of destructiveness of the 1918 epidemic. IV. On the correlation between explosiveness and total destructiveness of the epidemic mortality. — Pub. Health Ren., Wash., 1921. xxxvi. 273-298.

A further note on war and population. — Science, N. Y. & Lancaster, 1921. liii, 120-121.

Variation in the rate of infant mortality in the United States birth rcRistration area. — Tr. Am. Child Hug. Ass., Bait, 1921, xi, 213-229. Biometric data on infant mortality in the United States birth registration area. 1915-1918. — Am. J. Hyg., Bait, 1921. i, 419-439. A biological classification of the causes of death. — Metron, Rovigo,

Pearl, IL., and Fairchild, T. E.

Studies on the physiology of reproduction in the domestic fowl. XIX. On the infiuence of free choice of food materials on winter egg production and body weight — Am. J. Hyg., Bait., 1921, i, 253-277.

Pe.^rl, R., and Kelly, F. C.

Forecasting the growth of nations. The future population of the world and its problems. — Harper's Mag., N. Y., 1921, cxlii, 704-713.

Pearl, R., and Schoppe, W. F.

Studies on the physiology of reproduction in the domestic fowl, xviii. Further observations on the anatomical basis of fecundity. — J. Exper. Zool, Phila.. 1921, xxxiv, 101-118.

Peters, L.

Report of a case of large meningocele producing dystocia, delivery by Porro operation. — .Im. J. Obst. d Oynec, St. Louis, 1921, ii, 636-639.

PiNCOFFS, M. C, and Boggs, T. R.

Diseases of the peritoneum. — In: Oxford Med., 1921, iii, 517-581.

VON PiRQUET, C.

Children's diet. (Correspondence.) — Lancet, Lond., 1921. i. 558. Der Begriff • Aequum." — Zt.'ichr. ). Kiuderh., Berl., 1921, xxx, 14-5 Plaggemeyer, H. W.

The medical aspect of prostatic surgery. — Grace Hoxp. Bull, Detroit 1921, V, 1-7.

Post, L. T.

A study of the etiology of periodic ophthalmia in horses. — Am J. Ophth., Chicago, 1921, s. 3, iv, 330-333.

Quantitative determination of cocain and atropin absorption by aqueous humor. — J. Am. M. Ass., Chicago, 1921, Ixxvii, 1323-1324.

Post, L. T., and Shahan, W. E.

Thermophore studies in glaucoma. — Am. J. Ophth., Chicago, 1921, s. 3. iv, 109-118.

Putnam, Mary.

A survey of the rural children of western Massachusetts — Com monwealth, Bost., 1921, vii. No. 6.

Putnam, T. J., and Wislocki, G. B.

Absorption from ventricles in experimentally produced internal hydrocephalus. — Am. J. Anat., Phila., 1921, xxix, 313.

QUINBY, W. C.

The transperitoneal approach to the kidney ; its indications and limitations. — J. Vrol, Bait., 1921, vi. 135-143.

The pathology of the renal pelvis in two cases showing hematuria of the so-called essential type. — Tr. Am. Urol. Ass., 1920, Bait 1921 xii, 234-241. *

Randall, A.

Congenital yalves of the posterior urethra. — Am. Surg.. Phila 1921

Ixxiii. 477-480.

Report of a vesical calculus weighing four pounds. — Tr. Am Vrol

Ass., 1920. Bait.. 1921, xii. 53-50.

Giant vesical calculus — J. Vrol., Bait, 1921, v. 119-125.

The varying types of prostatic obstruction. — J. Vrol., Bait., 1921,

Randall, A., and Moorhead, S. W.

Sterilizer for cystoscopes. — J. Urol., Bait., 1921, v, 265-266.

Randall, A., Small, J. C, and Belk, W. P.

Tropical inguinal granuloma in the Eastern United States. — J. Vrol.. Bait., 1921, V, 539-548.


76


[No. 3:3


Reichert, F. L.

On the fate of the primary lymph-sacs in the abdominal region of th-^ pig. and the devolpment of lymph-channels in the abdominal and pelvic regions. — Contrib. EmbriioL, Xo. 08, (Carnegie Inst. I. Wash., 191>1. xiii, 17-39.

Reid, M. R.

Drainage of the choledochostomy


ommon bile duct through the cystic duct : -.ijin. ^iirg., Phila., 1921, Ixxiii, 458-409.


histamine shock. — ./. Expcr. M., Bait.,


Reynolds, L., and McClure, C. W .

Observations on the behavior of the normal pyloric sphincter in man. — Am. J. Ilocntyenol.j Detroit, 1921. viii, 158-162.

Reynolds, L., McClure, C. W., and Wetmore, A. S.

New methods for estimating enzymatic activities of duodenal contents of normal man. — Arch. Int. Med., Chicago, 1921, xxvii, 706-715.

Rich, A. R.

Condition of the cipillaries 1921. .\x.xiii, 287-298.

Richards, Esther L.

Mental nursing. — Johns Hopkins y,urses' Alumnae Mag., Bait.,

.XX, 1211-125.

The role of situation in psychopathologieal conditions. — Ment.

X. v.. 1921, V, 449-467.

The elementary school and the individual child. — Ment. Hug.,

1921, V, 707-723.

RiGGS, T. F.

The Plnney pyloroplasty.


-Journid-F.ancet, Minneapolis, 1921, xli.


Am. M. Asx., Chicago,


Exper. M., Bait.,


Rivers, T. M.

(irowth of influenza bacilli without blood. 1921, Ixxvi, 1744-1745.

Rivers, T. M., and Kohn, L. A.

The biological and the serological reactions of influenza bacilli producing meningitis. — J. Erper. M., Bait,. 1921, xxxiv, 477-494.

Rivers, T. M., Opie, E. L. [el all

Epidemic respiratory diseases. The pneumonias and other infections of the respiratory tract accompanying influenza and measles. — St. Louis, 1921, C. V. Mosby Co., 402 p. 8°.

Robinson, G. C.

Special symptoms of chronic diseases of the heart. Valvular disease. — In: Xelson Loose-Leaf Med., Lond. & N. Y., 1921, iv, 359-381.

Robinson, G. C., and Herm.^nn, G. R.

Paroxysmal tachycardia of ventricular origin, and its relation to coronary' occlusion. — Heart, Lond., 1921-22. viii. 59-81.

Rosen, R.

The phvsiopathology of the verumontanum. — I'rol. iC- Cutan. Rev., St. I>ouis, 1921, XXV, 26-30.

Rots. P.. and L.wimore, Louise D.

ICt'liitiun of the portal hltnul to liver maintenance. A demonstration of li\t'r atro|ihv louditional on compensation. — Stud. liockefeUer Inst. M. Uencarch, X". Y.. 1921. xxxvi, 157-182.

Rous, P., and McMaster, P. D.

The concentrating activity of the gall-hladder. 1921. xxxiv, 47-73.

Physiological causes for the varied character of stasis bile. — ./. Exper.

.If.," Bait., 1921, xxxiv. 75-96.

Rous, P., Wilson, G. W., and Olfver, Jean.

Experiments on the production of specific antisera for infections of unknown cause. III. The effects of a serum precipitin on animals of the species furnishing the precipitinogen. — Stud. Rockefeller Inst. M. Research, N. Y., 1921, xxxvi, 99-111.

Rqwntree, L. G.

Diabetes insipidus. — In:- Oxford Med., 1921. iv. 179-193. The differenfi.il diaenosis of polyuria, with special reference to diabetes iii-iphiiis — Uirf. r(iM. N. Am.. Phila., 1921, v. 439-453. The rAle :i[p1 .1. \ ri.,|iment of drug therapy. — J. Am. M. Ass., Chicago, 1921, Ixxvii. liiiil 1065.

The spirit of iuvisiigation in medicine. — Science. X. Y. & I^ancaster. 1921, liv, 179 1S3.

Sampson, J. A.

Perforating hemorrhagic (chocolate) cysts of the ovary. Their importance and especially their relation to pelvic adenomas of endometrial type (' adenomyoma " of the uterus, rectovaginal septum, sigmoid, etc.) — .4rc/i. Surg., Chicago. 1921, iii, 245-323.

S.^NGER, B. J.

The glucose mobilization rate in hyperthyroidism.Biol. d Med., X. Y.. 1921, xviii, 117-120.

SCHLAEPFER, K. I'eber einen w Tiibing.. 1921. T'eher tildli. "


-Proc. Soc. ETper.


'iteren Fall von Gallensteinileus. — Bcitr. ;. kiln. Chir..

oxxi. 122-135.

N'ar-hhlntuntren nach Tracheotomie bei Larvnxdiphtherie. — Hcitr. - l.-liH chir liiliiii;.'.. 11121. cxxi. 212-227. l>ie inTr.ipl' iir:it.!i i;i il. \.' inni ihre Bedeutung bei operativen Eingriffen.- i:r<i,i,„. ,1 (),,,• „, (irth;p.. Berl., 1921, xiv. 797-905. I'eber die r.( li iii^Miiii^ d.r .Vuallistcl and der Hiimorrhoiden. (Beobachtunt;en :iiii si M nkSpital in London). — Med. Klin., Berl. & Wien. 1921. xvii. i_--7 Ii's'.i Reiseeindriicki' > in*1920.) Briete aii-^ 1 11, 281-285 ; 302-306.

Seem, R. B.

Relation of the dispensarv to the Hospitjil.1921, xvi, 78-79.


-Mod. Hasp., St. Louis,


Sellards, a. W.

Bacillary dysentery. In: Oxford Med.. 1921. iv, 767-782. Amebiasis. — In: O.xford med., 1921, v, 7U9-816.

Asiatic cholera. Survey of literature from Sept. 1, 1920, to March 1. 1921. — In: Xelson Loose-Leaf Med., Lond. & X. Y., 1921, ii, 104-105.

Sellards, A. W., and Bigelow, G. H.

Investigation of the virus of measles. — J. Med. Research, Bost.. 1921, xlli. 241-259.

Sexton, L. A.

A new in and out registering device for the attending staff. — Mod. Hasp., St. Ijouis, 1921, xvi, 552-553.

Selecting the personnel for the social service department, — Mod. Hasp., St. Louis, 1921. xvii, 43-44.

Sharps, W.

The pathology and treatment of chronic brain injuries, with and

without a fracture of the skull. — Am. J. Surg-, X. Y., 1921, xxxv,

147-152.

Observations regarding the diagnosis and treatment of brain tumors. —

Internat. Clin., Phila., 1921, 31. s., 11, 227-240.

Observations regarding the surgical treatment of selected cases of

purulent meningitis. — Med. Rec, X. ?., 1921. c, 709-713.

Medical impressions of South America. — Med. Rec., X. Y., 1921, c,

1002-1065.

Shaw, H. N.

A short account of the deaths occurring in the gynecologic service of the .Tohns Hopkins Hospital during the year 1919. — Arch. Surg.. Chicago, 1921, ii, 535-541.

Shipley, A. M.

A consideration of one hundred and ninety chest injuries. — Am. J. Surg., X. Y., 1921, xxxv, 221-223.

Shipley, P. G., McCollum, E. V., Simmonds, Nina, and P.^rk, E. A. Studies on experimental rickets. IV. Cod-liver oil as contrasted with butter fat in the protection against the effects of insufficient calcium in the_diet. — Proc. Soc. Exper. Biol. <£ Med., X. Y., 1920-21, xviii,

Shipley, P. G., Park E. A., McCollum, E. V., and Simmonds, Nina. Studies on experimental rickets. V. The production of rickets by means of a diet faulty in only two respects. — Proc. Soc. Exper. Biol, d Med., N. Y., 1920-21, xviii, 277-280.

Shipley, P. G., McCollum, E. V., Simmons, Nina, and Park, E. A. studies in experimental rickets. VI. The effects on growing rats of diets deficient in calcium. — Am. J. Hyg., Bait., 1921, i. 492-511.

Shipley, P. G., Park, E. A., McCollum, E. V., and Simmonds. Nina. .studies on experimental rickets. VII- The relative effectiveness of co<l liver oil as contrasted with butter fat protecting the body against ins\itBcient calcium in the presence of a normal phosphorus supply. — Am. J. Hyg., Bait., 1921, i, 512-525.

Shohl, a. T., and Mason, C. C.

Food at the front lines. — Mil. Surgeon, Wash., 1921. xlviii, 386-401.

SlEWERS, A. B.

Eye signs in intracranial tumors of the anterior fossa. With report of two cases. — Arch, yeurol. it- Psychiat., Chicago, 1921, vl, 424-428.

Simon, C. E.

Glardia enterica : a parasitic intestinal flagellate of man. — Am. J. Hyg., Bait., 1921. i, 440-491.

SissoN, W. R., and Denis, W.

Studies on the" inorganic constituents of milk.Chicago. 1921, xxi, 389-400.

Slack, H. R.

Diseases of the tonsils, pharynx and nasopharynx. (Survey of literature from Sept. 1. 1920, to March 1. 1921.) — In: Xelson Loose-Leaf Med., 1921, ii, 18-19.

Slemons, J. M.

The prospective mother. A handbook during pregnancy. 2d. ed. NewYork, 1921. D. Appleton & Co., 343 p. 12'.

Smith, W. H.

Address to the graduating class. Training School for Nurses. Peter Bent Brigham Hospital. — Boston M. d S. J., 1921, elxxxiv, 367-371, Adequate medical service for a communitv. Some factors of importance. — J. Am. M. Ass., Chicago, 1921. Ixxvi. 1053-1062.

Steiner, W. R.

Diseases of the muscles. — In: l).xford Med.. 1921. iv. ^53-366.

Stevens, R.

A procedure for the cure of prostatic abscess. — Tr. Am. t'rol. Ass.. 1920, Bait., 1921, xii, 19-24.

Stone, H, B.

The toxic agents developeil tion, and their action. — Su, 415-419.

Strong, R. P.

Tren<* fever. — In: Oxford Med., 1921. v, 423. 437.

Remarks made at the closing session of the Inter-Allied Sanitary Commission. Paris. — Boston M. d 8. J.. 1921. elxxxiv. 27-31. International public health problems. — Xorth Am. Rev., Concord N. II., 1921, ccxiii, 319-332.

Strong, R. P., and Councilman, W. T.

Plague-like infections in rodents. — Tr. Ass. Am. Physicians, 1921,


J. Dis. Child.,


i


February, 1932]


77


Strouse, S.

Dietetic therapeutics. — Med. Clin. X. Am., Phila., 1921, v, 229-244.

SwARTZ, E. O., and Davis, D. M.

Action of mercurochrome — 220 on tlie gonococcus. — J. Am. M. Ass.,

Ciiicago, 1921, l-xxvi, S44-840.

The action on the gonococcus of variotis drugs commonly used in the

prophylaxis and treatment of gonorrhea. — J. Urol., Bait., 1921, v,

235-253.

SwARTZ, E. O., Young, H. H., and White, E. G.

Further clinical studies on the use of mercurochrome — as a general germicide.—./. Urol., Bait.. 1921. v, 353-388.

Taylor, A. S.

The use of fine silii in surgery. — China J/. J.. Shanghai, 1921. xxxv, 4C7-472.

Thomas, H. M., Jr.

Recurrent type 1 pneumonia : serum treatment of t\yo attaclis one month apart. — .im. J. M. Sc, Phila.. 1921, clxi. 103-109.

TiBBETTS, M. H.

Some essential points in the treatment of infantile paralysis. — Minnesota Med., St. Paul, 1921. iy, 484-487.

TiSDALL, F. F., and Kramer, B.

A clinical method for the quantitative determination of potassium in small amounts of senim. — ./. liiol. Chem., Bait.. 1921. xlvi, 339-349. Simple method for the direct quantitative detennination of sodium in small amounts of serum. — ,/. Biol. Chcm., Bait.. 1921. xlvi, 467-473. A simple technique for the determination of calcium and magnesium in small amounts of serum. — /. Biol. Chem., Bait., 1921, xlvii, 475481.

Methods for the direct quantitative determination of sodium, potassium, calcium, and magnesium in urine and stools. — ./. Biol. Chem.. Bait., 1921, xlviii. 1-12.

The direct quantitative determination of sodium, potassium, calcium, and magnesium in small amounts of blood. — ./. Biol. Chem., Bait., 1921. xlviii, 223-232.

Tisdall, F. F., Kramer, B., and Howland, J.

Observations on infantile tetany. — Am. J. Dis. Child., Chicago, 1921,

xxii, 431-437.

The clinical significance of calcium concentration in the serum of

children and possible lurors in its determination. — Am. J. Dis. Child.,

Chicago, 1921, xxii, 5(')0-564.

The concentration of sodium and potassium as compared with that of

calcium and magnesium in the serum of patients with active Infantile

tetany. — Proc. Soc. Exper. Biol, d Med., N. T., 1921, xyiii, 252-253.

Trueblood, D. V.

Fracture of the carpal scaphoid.279.


-Northwest Med., Seattle, xx, 27


VanderHoop, D.

The significance of vertigo. — South. M. J., Birming., Ala., 1921, xiv,

182-186.

The electrocardiograph in diagnosis. — Tirginia M. Month., Richmond,

1921, xlviii, 212-214. Also: South. If. d 8., Chattanooga, 1921,

Ixxxiii, 428-430.

VoEGTLiN, C, Hooper, C. W., and Johnson, J. M.

Trinitrotoluene poisoning, its nature, diagnosis, and prevention. — J. Indust. Hi/g., Boston. 1921-22, ill. 239-254 ; 280-292.

VoEGTLiN, C, and Smith, H. W.

Quantitative studirs in rh.'motherapy. IV. The relative therapeutic value of arsiihinaniiiir .nid ni'o.-irsphen.-imine of different manuf.acture.— ./. PhurnuunI ,i ;/;„•)•. Thrrnp.. Bait.. 1920-21. xvi. 449-461. Quantitative stiidi'-s in chemotherapy. V. Intravenous versus intramuscular administration of arsphenamin ; curative power and minimum effective dose. — J. Pharmaeol. <C E.Tper, Therap., Bait., 1921, xvii, 357.

VoEGTLiN, C, Smith, M. I., and Johnson, J. M.

Therapeutic value of chaulmoogra oil and its derivatives in experimental tuberculosis. — J. Am. M. Ass., Chicago. 1921, Ixxvii, 10171020.

Walker, G.

Symphysiotomy as an aid to the removal of cancer of the prostate. A proposed new operation. — .Ijm. Surg., Phila.. 1921, Ixxiii, 609. The secretory pressure of the kidney as an index of pathologic conditions (Preliminary report). Ann. Surg., Phila., 1921, Ixxiii, 610612.

Apparatus to aid in differentiation between an obstruction in the urinary outlet and paralysis of the bladder. — J. Am. M. Ass., Chicago. 1921, Ixxvii, 2S6-2S7.

A proposed standard treatment of early syphilis. — South. M. J., Birmingham, Ala., 1921. xiv, 683-684.

W.\RFIELD, L. M.

Clinic of Louis M. Warfleld, M. D., Milwaukee Hospital. — Wisconsin

M. ./.. Milwaukee, 1920-21, xix. .541-545.

The use of stimulants in pneumonia. — Wisconsin il/. ./., Milwaukee,

1921-22, XX, 1-4.

Clinic of Dr. L. M. Warfleld at Milwaukee County Hospital. — TFis consin M. J., Milwaukee, 1921-22, xx, 242-244.

Waters, C. A., and Baetjer, F. H.

Injuries and diseases of the bones and joints : their differential diagnosis by means of the Roentgen rays. N. Y.. 1921, P. B. Hoeber. 349 p. 4".

Waters, C. A., and Young, H. H.

Pyelography. — Soc. Internat. d'Urologie, I'aris, Juillet, 5-7, 1921.


Watkins, S. S.

Indications for removal of the laucial tonsils. — Kentucky M. J., Louisvill... 19i'l, xix, 65-67.

Fift^-^tIlt jiii-ci- in the esophagus for three months, removal through moutli.- -/nH(»r;,-i/ M. J., Louisville, 1921, xix, 523-524. .Symiitonis, diasnosis and treatment of inflammatory lesions of the nasal accessory sinuses — from the viewpoint of the general physician and surgeon. — Kentucky M. J., Louisville. 1921. xix. 682-688. Primary scleroma of the larynx in a negro born in Maryland — Surg., Uynec. if Obst., Chicago, 1921, xxxiii, 47-52.

Watts, S. H.

Traumatic chylothorax.— .4hh. Snrg., Phila., 1921, Ixxiv, 691-696.

Webster, L. T.

Lymphosarcoma. Lymphatic leukaemia. Leucosarcoma. Hodgkin's disease. — Johns Hopkins Hasp. Rep., Bait., 1921, xx, 251-314,

Webster, L. T., and Lewis, W. H.

Migration of lymphocytes in plasma cultures of human lymph nodes. —

./. Exper. M., Bait., 1921. xxxiii, 261-269.

(Mant cells in cultures from human lymph nodes. — J. Exper. M., Bait.,

1921, xxxiii, 349-360.

\Vandcring cells, endothelial cells, and fibroblasts in cultures from

human lymph nodes. — J. Exper. M., Bait., 1921, xxxiv, 397-406.

Weed, L. H., and Hughson, W.

The skull as a closed box (Proceedings). — Anrit. Record. Phila., 1921, xxi, 88,

Welch, W. H.

Introduction [to American Jour, of Hygiene], 1921, i, pp, iii-iv.

The place of Dr. Meltzer in American medicine. — Proc. Soc. Exper.

Biol. <£- Med., N. Y., 1920-21, xviii, 37-42. Memorial Number for

S. J. Meltzer.

Wesson, M. B., and Young, H. H.

The anatomy and surgery of the 1921, iii, 1-37.

Wharton, L. R.


J. Hygiene, Bait.,


trigon. — Arch. Surg., Chicago,


V based on a series of seven hundred and sixteen liicago, 1921, ii, 246-314.

icrvix of the uterus of inflammatory origin, iloma. — Surg. Oyncc. d Obst., Chicago, 1921,


lal experimentation to nuinkind. — .int. J. Pub. Health,


diagnosis of heart failure. .\ hitherto unirregularity. — J. .im. M. .iss., Chicago, 1921,


xxxiii, 145-1. >:

Whipple, G. H. Value of anil N. Y^ 1921, xi, 105-107

Whipple, G. H., and Davis, N. C.

1. Liver regeneration following chloroform injury as influenced by the feeding of c^isein or gelatin. — Arch. Int. Med., Chicago, 1921, xxvii, 679-687.

Whipple, £i. H., and Delprat, G. D.

Studies of liver function. Benzoate administration and hippuric acid synthesis.— J^. Biol. Chem., Bait.. 1921-22, xlix, 229-240.

Whipple, G. H., and Robscheit, F. S.

Iron and arsenic as influencing blood regeneration ftdlowing simple anemia. — Arch. Int. Med., Chicago. 1921, xxvii, 591-603.

White, E. C., Young, H. H., and Swartz, E. O.

Further clinical studies on the use of mercurochrome — as a general germicide.—/. Urol.^ Bait.. 1921, v. 353-388.

Wiel, H. I.

Truer standards in the emphasized form of pulsi Ixxvii, 749-753.

Wight, O. B.

Practical medical publicit.v. — Proc. Alumni .Assoc. Med. School, Univ. Oregon, 1921, 9th Ann. Meeting.

Williams, J. W.

Report on graduate instruction in obstetrics and gynecology. — Am.

Med. Ass. Bull.. Chicago, 1921. xv, 52-56.

Report on undergraduate teaching of obstetrics and gynecology. — Am

Med. Ass. Bull.. Chicago. 1921, xv, 146-152.

The problem of effecting sterilization in association with various

obstetrical procedures. — .4 m. J. Ohst. d Gynec, St. Louis, 1921, i.

783-793.

Obstetrics and the general practitioner. — Pcnn. M. J., 1920-21, xxiv,

290-296.

Twenty-one years' experience with Caesarean section. — Med. Standard,

Chicago, 1921, xliv, 14-24.

Willis, H. S.

studies on tuberculous infection. VIII. Spontaneous pneumokoniosis

in the guinea-pig. (Introduction by A. K. Krause.) — Am. Rer. Tuberc, Bait.. 1921-22. v, 189-217.

WiSLOCKi, G, B., and Putnam, T. J.

Absorption from ventricles in experimentally produced internal hydrocephalus. — Am. J. Anat., Phila., 1921, xsix. 313.

Woods, A. C.

Immune reactions following iniuries to the uveal tract. — J. Am. M. Ass., Chicago, 1921, Ixxvii, 1317-1322.

Woollet, p. G.

Blood sugar tole /. Lab. S Clin M Coinpli<-:iti..r.K .if — ./. I.aii. ,1 I'Ini Syphilid


St.


192


ince in cancer and in hypertension. (Editorial.) — . St Louis. 1920-21. yi, 227-229.

li.' arsidirnamine treatment of syphilis. (Editorial.)

1/,, SI L.oiis. 1920-21. yi, 344-347.

ithesis. (Editorial.)— J'. Lab. cC Clin. M.,


169-471.


Superinfection in syphilis in its relation to subtreatment. — /. Lab. <£ CUn. M., St. Louis, 1920-21, vl, 717-719.


(Editorial.)


[No. 372


Wright, C. B.

Duodenal ulcer : medical treatment in ambulant cases. — JournalLancet, Minneapolis, Minn., 1921, xli, 396-397.

Yates, J. L.

.\a outline of twelve Tears' study of Hodgltin's disease and allied jilTcctions. — Wisconsin M. J., Milwaukee, 1920-21, six, 447-451.

Young, C. W., and Han, C. H.

AKjrIutination titer following repeated intravenous injections of tab vaccine. — China M. J., Shanghai, 1921, xxxv, 400-404.

Young, H. H.

Surgery of the prostate. — In: Surgery. Its Principles & Practice

(Keen). Phila. & Lond.. 1921, viii, 530-607.

The value of drugs in urology. — J. Am. M. Ass., Chicago, 1921, Ixvii,

1327-1332.

Success of the campaign for combating venereal disease In the

A. E. F. — Mil. Surgeon. Wash.. 1921, xlviii, 213-222.

Demonstration of a new combined cystoscopic and X-ray table. —

Tr. Am. Urol. Ass.. 1920, Bait., 1921, xii, 344-347. Also: J. Urol,

Bait., 1921, V, 391-404.

Young, H. H., and Cash, J. R.

A case of pseudohermaphrodismus masculinus. showing hypospadias, greatly enlarged utricle, abdominal testis and absence of seminal vesicles.^. Urol., Bait, 1921, v, 405-430.

Young, H. H., and Waters, C. A.

Pvelngraphy. — Rapport de la Soc. Inteniat. d'Vrologie, Paris. Juillet 5-7. 1921.

Young, H. H., and Wesson, M. B.

The nnatomv and surgery of the trigon. — .ireh. Surg., Chicago, 1921, iii, 1-37.

Young, H. H., White, E. C, and Swartz, E. O.

Further clinical studies on the use of mercurochrome — as a general germicide.— J. Urol., Bait, 1921, v, 353-3S8.

ZiMMERMANN, E. L.

A comparative study of syphilis in whites and in negroes. — Arch. Dermatol. <f Siiph.. Chicago, 1921, iv. 7o-S8.

S.vphilitic iritis, its racial incidence and its association with secondary svphilis and with neurosyphilis. — J. Am. M. Ass., Chicago, 1921, Ixxvi. 1818-1819.

RECEIVED TOO LATE FOR INSERTION IN ALPHABETICAL ORDER

Berry, J. M.. and Howard, W. P.

Reports from the Roentgen-ray laboratory of the Albany Hospital. — Albany M. Ann., 1921, xlii, 368-376.


Burrows, M. T.

Problems in cancer research.131-138.


Cancer Research, Bait., 1921, vi.


Dickson, E. C., and Burke, Georgina S.

Botulism. A method for determining the thermal death time of the spores of Bacillus botulinus. — Proc. Soc. Exper. Biol, d Med., N. Y., 1921-22, xi.v. 99-101.

DiEU.'HDE, F. R., and D.widson, E. C.

Terminal cardiac arrhythmias. Report of three eases. — .\rch. Int. Med., Chicago, 1921, xxviii. 603-677.

Halsted, W. S.

Ligations of the left subclavian arterv in its first portion. — Johns Hopkins Hasp. Rep., Bait.. 1921, xxi. 1-96.

Higgins. W. H.

Certain types of cerebral manifestations in cardio-renal diseases. — Yirginia M. Month., Richmond, 1920-21, xlvli, 598-601.

Levy, R. L.

Alterations in the cardiac mechanism after administration of quinidine to patients with auricular fibrillation. — Proc. Soc. Exper. Biol. £ Med., N. Y.. 1921-22, xix, 88-91.

Macht, D. I.

Isopropyl alcohol, a convenient lahoratorv anesthetic for cats. — Proc. Soc. Exper. Biol, cf- Med., N. Y.. 1921-22. xix. 85.

Pe.^RL, R.

The vitality of the peoples of America. — Am. J. Byg., Bait. 1921, 1, 592-674.

Parfitt, C. D.

The care and employment of the tuberculous ex-service man after discharge from the sanatorium. — Ottawa, 1921, F. A. Acland. 77 p. 8°.

Robinson, G. C.

Oreranizntion of the medical clinic of The Johns Hopkins Hospital. — Johns Hopkins Tfurses Alumnae Mag., Bait. 1921, xx, 226-227.

Schlaepfer. K.

Die Carrel-Dakin-Behandlung infizierter Wunden. — Miinchen. med. Wchnschr., 1921, Ixviil. 1490-1492.

Tisdall, F. F.

The etiology of rickets. — Oanad. M. Ass. J., Toronto, 1921, xi, 934-943.

Webster, L. T.

Experiments with B. enteritidls (murium) on normal and Immune mice. — Proc. Soc. Exper. Biol. & Med.j N. Y., 1921-22, xix, 71-72.


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Primary Carcinoma of the Liver. By M. C. Winteknitz, j\L D. 42 pages. Price 75 cents.

The Statistical Experience Data of The Johns Hopkins Hospital, Baltimore, Md., 1892-1911. By Frederick L. Hoffman, LL.D., P.S.S. 161 pages. Price, $2.00.

Venous Thrombosis During Myocardial Insufficiency. By Frank J. Sladen, M. D., and Milton C. Winternitz, M. D. Price, 75 cents.

The Origin and Development of the Lymphatic System. By Florence E. Sabin. 94 pages. Price, $2.00.

Leukaemia of the Fowl: Spontaneous and Experimental. By Harry C. Sohmeisser, M. D. Price, $2.00.


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The Operative Story of Goitre. The Author's Operation. William S. Halsted, M. D. Price, $3.50.


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Pathological Anatomy of Pneumonia Associated with Influenza. By William G. MacCallum. Price, $1.50. (This monograph will be on sale within a short time.)


CONTENTS

  • A Study of the Relation of the Adrenal Glands to Experimentally Produced Hypotension (Shock) ; with a Note on on the Protective Effect of Preliminary Anesthesia. (Illustrated.) By Ar.xold Rice Rich .......
  • A Clinical and Anatomical Study of Fifty-One Cases of Repeated Csesarean Section with Especial Reference to the Healinw of the Cicatri.s: and to the Occurrence of Rupture Through It. (Illustrated.) By Thomas O. G.\mble ......
  • Acute Lobar Pneumonia and Haematogenous Puerperal Infection. A Case Report. By R. A. JoHNSTO.v and H. .1. ilORGAX
  • Diplitheria Bacillus Carriers. Results of Re-E.vaminatiou of Apparently Negative Cultures. By B. C. Marshall and C. G. Guthrie
  • The Ilydrogen-ion Cuneentration of Tissue Growth in Vitro. (Illustrated.) By M. R. Li:wi.s and Lloyd D. Felto.n" ....
  • The Relation of H-ion Concentration to Specific Precipitation. By V. R. Masox
  • Notes on New Books


A STUDY OF THE RELATION OF THE ADRENAL GLANDS TO EXPERIMENTALLY PRODUCED HYPOTENSION (SHOCK)

WITH A NOTE ON THE PROTECTIVE EFFECT

OF PRELIMINARY ANESTHESIA

By Arnold Rice Eich {From the Deparlmerd oj Pathology, The Johns Hopkins Medical School)


In a study of the literature dealing with either surgical shock or the adrenal glands, one meets frequently the suggestion that shock may be the result of disordered function of the adrenals. This idea has been entertained especially as a corollary of the belief that, since epinephrin injected intravenously has such a remarkable effect upon the blood pressure, and removal of the glands brings about a condition of hypotension, the adrenals are therefore probably concerned in the maintenance of the blood pressure at the normal level. Since the condition of shock is characterized most strikingly by a marked fall in blood pressure, it has been suggested repeatedly that epinephrin exhaustion or adrenal fatigue may be causative factors.

Stewart and RogofE ' and others '• ' have shown that stimulation of the splanchnic nerves brings about an increased output of epinephrin from the adrenals. Hoskins and McClure * state that a similar effect can be produced by visceral exposure and operative trauma; Cannon and Hoskins' found that


sensory stimulation increases the output of epinephrin, and Elliott " showed that the glands can in this way be quite depleted of their epinephrin content. Thus, it might be supposed that excessive intestinal or peritoneal manipulation or any severe prolonged sensory stimulation accompanying trauma may exhaust the epinephrin content of the adrenals and so bring about the condition of low blood pressure characteristic of shock. Indeed, the experiments of Corbett ° and of Cannon and Hoskins " led them to make just this suggestion, although Cannon later ° stated his belief that during shock " the adrenal glands are, if anything, over-active rather than exhausted." Quite a number of workers have reported that the epinephrin content of the adrenals is diminished by prolonged anesthesia, and it has been suggested that surgical shock may be dependent in part upon this epinephrin depletion.'"

Numerous attempts have been made to determine whether the supply of epinephrin is actually exhausted during shock, and whether abnormal variations in the epinephrin output


so


[No. 373


from the adrenals can be related to the development of shock. The results of these investigations are somewhat conflicting. Bainbridge and Parkinson ' reported that they could find no epinephrin at all in the adrenals taken from fatal cases of postoperative shock, and this was confirmed by Elliott.' Short/ however, using a very delicate test, was unable to detect any reduction in the epinephrin content of adrenals taken from shock cases. Corbett' states that "the symptoms of shock fully develop only after the supply of epinephrin is greatly depleted," but he does not describe the experiments which led him to tliis conclusion. He does, however, report that animals which were subjected to prolonged sensory stimulation (sciatic) "very rapidly went into shock after a few minutes of peritoneal traiuna" when the intestines were subsequently exposed, and he explains this by assimiing that the preliminary sensory stimulation caused a reduction of the epinephrin supply. It is noteworthy in this connection that, in spite of numerous attempts, there is not a single convincing experiment on record in which true shock has been produced by the stimulation of sensory nerves. Sydenstricker, Delatour and Whipple '° state that in animals which had been brouglit into the condition of shock by the injection of contents from a closed duodenal loop, determination of the epinephrin content of the adrenals disclosed only one-fourth of the normal amount, or even less. Bedford " reported that during shock produced by intestinal manipulation the epinephrin content of blood taken from the adrenal vein is much higher than before the onset of shock, and that the concentration increases with the prolongation of the low blood pressure. Stewart and Eogoff," however, repeated Bedford's experiments with modifications in technique which they believe insure a greater accuracy in the estimation of the amount of epinephrin present in the blood, and they conclude that the output of epinephrin from the adrenals is the same during shock as it is under normal conditions.

Experiments such as these leave the question of the relation of the adrenals to shock in an unsatisfactory condition. In the first place, an altered epinephrin content of the adrenals or an abnormal output during shock might easily be a result rather than a cause of the condition. Thus Mann,"^ stating his belief that the adrenals enter as factors in the complex of shock, wrote that "it is quite difiicult to determine to what degree they participate as primary agents in producing the state or how much they are affected by the low blood pressure and the changes incident to the condition itself." It might be thought that the removal of both adrenals would throw immediate light upon the theory of adrenal exhaustion ; and indeed Crowe and Wislocki " have reported that following complete removal of both adrenals the animal gradually develops hypotension, rapid pulse, lowered body temperature, muscular weakness, apathy and dulled sensibility — all of which are characteristic of shock. On the other hand, it is claimed that adrenal extirpation does not really reproduce the condition of shock," and Vincent " has even stated that experiments carried out in his laboratory demonstrate that a fall of blood pressure is not a characteristic effect of complete adrenalectomy. Fui-thermore,


apart from the question of the effect of the mere removal of the adrenal glands, it is well known that there is a strong tendency at the present time to believe that the adrenals normally play no direct part in regulating vascular tone,"- "• " "• " or else that their activity is of value only in emergencies when an unusual strain is thrown upon the circulation. If the latter supposition be true, it is clear that exhaustion of the adrenals during the strain of an operation or severe trauma might deprive the animal of a protection of which the adrenalectomized animal, lying quietly in its corner, would have no need. Indeed, Abelous and Langlois" pointed out long ago that muscular exertion following adrenalectomy hastens the development of the characteristic sj-mptoms of adrenal deficiency. Thus it is evident that conclusions drawn from the effects of adrenalectomy alone cannot entirely satisfy the question of the relation of the adrenal glands to shock. The present experiments were undertaken in tlie belief that if normal animals, subjected to a standardized trauma, fall into shock in a reasonably definite period of time, then worthwhile information might be obtained from a comparison of the protocols of these normal controls with the time required for the production of shock in adrenalectomized animals, subjected to the same trauma. For if shock results from epinephrin exliaustion or adrenal fatigue, the condition would presmnably develop much more rapidly in the adrenalectomized animals than in the normal controls; if abnormal stimuli going to the glands cause an excessive output of some harmful material which produces shock, removal of the glands would remove the possibility of an outpouring of any noxious secretion and the adrenalectomized animals would be very resistant to the trauma; and finally, if the development of shock is independent of the activity of the glands, the adrenalectomized animals should react as normal animals to the same trauma. The fact that bits of accessory adrenal and chromaffin tissue are present in animals has not been overlooked, but it was believed that the removal of both adrenal glands (the great bulk of such tissue) would produce a deficiency sufficient to be detected by this method of attack if adrenal function actually plays an important role in the production of shock.

EXPEEIMEN'TS

Certainly one of the most confusing things that one meets throughout the literature concerned with shock is the failure of many workers to standardize their experiments. Often, in a series of experiments where such precautions would be of value, no attempt is made to treat each animal.in as nearly the same manner as possible either as regards the degree of trauma inflicted or (what is exceedingly important) the amount of anesthetic administered during the experiments. But especially is there a lack of a criterion as to what shall be regarded as shock. Thus, while most clinical and experimental observers incorporate a marked fall of blood pressure into their definition of shock, there are some whose experiments indicate that they do not regard such a fall of blood pressure as a necessary part of tlie picture. Wiggers " considers a fall of blood pressure a characteristic of "shock" produced by intestinal exposure.


March, 1922]


81


but he does not consider it an essential part of the "central nervous system shock " which he produced by sensory stimulation." This " central nervous system shock " is merely a condition of apathy and dulled sensibility and can occur vi^ith no important blood pressure fall. Doubtless some of the confusing experiments in the literature have arisen from a failure to recognize that this condition of apathy is not true sliock. Mann " some years ago called attention to the need of a criterion for the condition of an experimental animal wliicli should be regarded as shock, and his requirements followed admirably the signs which make up the clinical condition designated as shock.

In the present experiments, an animal was considered to be in shock when the blood pressure, having fallen to 60 mm. of mercury or below, sliowed no tendency to recovery and was accompanied by a permanent dulling of the sensibility, so that the ether could be discontinued ^nthout discomfort to the animal during operative procedures performed after a lapse of time (half an hour) sufficient to allow the anesthetic effect of the ether to wear off. Strong sensory stimulation applied to such an animal might cause struggling, but the animal would at once sink back into its quiet, apathetic state as soon as the stimulation Was discontinued, although the abdominal contents were exposed and the neck opened for tracheotomy and cannulation of the carotid artery. A low blood pressure was required in these experiments because clinical shock without hypotension, although spoken of by a few writers,^ is certainly an anomalous condition except in cases following head-wounds, and it is questionable whether such conditions are fundamentally the same as ordinary shock with hypotension. The level of 60 millimeters of mercury was arbitrarily chosen as a standard after a study of the average unrecoverable fall of blood pressure usually accompanied by a permanent dulling of the sensibility. The pulse of the animal in shock was usually rapid and feeble after the condition had persisted for some time, but frequently a marked slowing of the pulse was a prominent feature of the early stages, and periods of bradycardia have been observed during deep shock. The character of the respirations was very variable in the different experiments. The superficial reflexes remained active throughout, and the body temperature always fell several degrees centigrade more than did that of normal animals merely kept under ether anesthesia for the same period of time.

In all of the experiments described below apparently healthy, full grown cats were used and a careful attempt was made to subject each animal to conditions as nearly identical as possible as regards the trauma inflicted to produce shock and the amount of anesthetic used in each experiment. It must be stated here that during the progress of this work a number of animals were encountered which exhibited a peculiar sensitiveness to ether, so that the most careful administration of the anesthetic did not prevent them from behaving in a most anomalous way. The respirations would cease, and the blood pressure would fall abruptly shortly after anesthetization, and although artificial respiration would soon restore them, such lapses would occur repeatedly, and shock would appear much


sooner than in normal animals. At autopsy no reason for tlie peculiar reaction was ever discovered. The number of such animals was relatively small, and it is felt Justifiable to exclude them entirely from consideration, since they were clearly hypersensitive to the effect of ether alone.

Reaction of Normal Animals to Intestinal Maniijulation. — The first series of experiments was carried out to determine whether normal animals would exhibit any constancy in tlie time required for the development of shock if they were subjected to a uniform trauma. Each animal was anestlietized with ether and tracheotomized. The tracheal cannula was connected through a reservoir witli a tube through wliich ether vapor could be blowai from an ether bottle by means of a foot bellows. As soon as the carotid artery could be cannulated and connected with a mercury manometer, a blood pressure tracing was made and the rectal temperature was recorded. Then the abdomen was opened at once along the midline, and the small intestines were lifted out of the abdominal cavity and spread upon gauze pads, care being taken to prevent torsion of (lie mesentery from interfering with the circulation. Every five minutes a blood pressure tracing was made, tlie temperature was recorded, the intestines were pinched firmly between the thumb and forefinger all the way from the duodenum to the caecum once, and the animal received the amount of ether vapor which was forced into the reservoir by a single pump of the bellows. In the intervals between these procedures the animal was left to lie quietly. Usually a single pump of ether vapor sufficed to keep the animals anesthetized, and a second dose between the five-minute periods was administered only when necessary. The anesthesia was never deepened enough to abolish tlie corneal reflex. A careful control of the anesthetic is of great importance in such experiments, as may be seen from a consideration of Fig. 1. In this experiment a relatively large amount of ether was administered, with the result that the blood pressure fell to the shock level within a short while, and would have remained there, simulating the condition of shock, had not the ether been discontinued. Fifteen minutes after stopping the anesthetic the blood pressure began to rise and 10 minutes later had risen 40 millimeters more and the animal liad regained full consciousness and sensibility. This effect of ether was repeated several times in the same animal, and demonstrated the necessity, in every experiment, of observing the animal for at least half an hour following the discontinuance of the anesthetic when shock is believed to have developed, in order to be certain that the low blood pressure and dulled sensibility are not ether-effects.

By following carefully the method outlined above, eacli animal received as nearly as possible the same degree of trauma and the same amount of anesthetic.

Inspection of the protocols and blood pressure tracings of this series of experiments shows that normal animals react to these conditions in a strikingly constant way. The blood pressure falls abruptly during the first five minutes after exposing the intestines and then, either continuously or after a temporary rise, it sinks gradually, with some irregularity, to the shock level, and the characteristic signs of shock make




CaT ;Z3


flHer stopped.


3^s ^'^35



Fig. 1. — Careless anesthesia resulting in condition resembling J


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I I I I I I I I I I I I I I H I I I U I I ! J-IJ-LLIU-UJXI-U-I JJ-UJ U I I I I I I I I H 111 li-U


Fig. 2.— Blood pressure tracing of normal animal durmg production of shock by mtestm^n

pressure falls, a not mlrequeff <





Ether Stopped


J-A=>»l 6-^- >37o


h recoverj' when ether is discontinued ; not true shock.



Shock present 1^ hours after exposing intestines. Note slowing of pulse as blood c 1 the early stages of shock.


84


|Xo. 37:5


their appearance. The time required for the development of complete shock in these 10 animals was remarkably constant, varying between the extremes of an hour and a half and two hours, the average time being an hour and 50 minutes. Fig. 2 represents a typical experiment from this group.

ReacHon of Animals Immediately Following Adremlectomy. — Having determined the time required to bring normal animals into shock, w-e next carried out a series of experiments to study the reaction of adrenalectomized animals subjected to the same conditions. Eacli animal was anesthetized, tracheotomized and a blood pressure tracing taken Just a.s in the first series. Then both adrenals were carefully removed extraperitoneally, and the lumbar incisions were closed with sutures. This procedure required 30 or 40 minutes and was performed with no more ha?morrhage than would stain part of a small piece of gauze. Kymographic tracings were taken every five minutes for about 20 minutes following removal of the glands in order to observe the immediate effect of the operation. During tliis time the blood pressure invariably either remained at tlie' original level or, if anything, occasionally rose a few millimeters above it. The intestines were then exposed and manipulated exactly as in the normal controls, the metliod of anesthesia being the same throughout.

The reaction of these animals was most interesting. Kymographic tracings taken every five minutes, as before, showed tliat there occurred the usual slight fall of blood pressure immediately following the opening of the abdomen. This fall was promptly recovered from in several experiments ; but in every experiment, during observations continued as long as two and a half and three hours following the exposure of the intestines, the blood pressure exhibited no further fall than the negligible depression which prolonged light anesthesia alone produces ; and cessation of the anesthetic at the end of this time was invariably followed by prompt and complete recovery, although these animals were subjected to exactly the same conditions as the normal controls which were in deep sliock, with tlie blood pressure at 60 millimeters or below, after only an hour and 50 minutes of intestinal manipulation. Fig. 3 is typical of this series. It will be seen that the blood pressure at the end of two hours and a half of intestinal manipulation stands at 110 millimeters, and has fallen from the original level no more than the blood pressure of normal animals kept quietly under ether anesthesia for the same length of time. The pulse is of splendid quality ; and when the anesthetic was discontinued the animal promptly recovered full consciousness and sensibility. Not one of these 10 animals went into shock. Autopsy showed in every case complete removal of both adrenals.

This series of experiments seemed to favor strongly the idea that shock might be the result of some abnormal secretion of tlie adrenals, since there appeared no tendency for the condition to develop in adrenalectomized animals subjected to the same conditions which regularly produced it in the normal animal. There were, however, aside from the lack of adrenals, two points in which the adrenalectomized animals differed from tlie control animals at the beginning of the intestinal ex


posure — they had undergone an operation, and they had l)e('ii kept under anesthesia for about an hour during the operation and the subsequent blood pressure observations made before the abdomen was opened. The fact that they liad been subjected to an operation before beginning the shock trauma would be expected to increase their tendency to shock, if anything, and so too would a preliminary hour's anesthesia, A third series of experiments was carried out, however, to study the reaction of animals to intestinal manipulation following a preliminary hour's anesthesia alone.

Reaction After a Preliminary Hour's Anesthesia. — After tracheotomy and measurement of the blood pressure, the animals of this series were kept quietly under light anesthesia for an hour, kymographic tracings being taken every five minutes. Then the intestines were exposed and handled just a-s before. These animals reacted precisely as those of the adrenalectomized series ; they showed no tendency to fall into shock during three hours of intestinal manipulation. Fig. 4 is a tracing from a typical experiment. The blood pressure shows no more tendency to fall after intestinal exposure than if the animal were merely kept anesthetized for tlie same length of time. Two hours and a half after the abdomen was opened the systolic pressure was 120 millimeters, the pulse was good, and when the anesthetic was discontinued, the animal regained full consciousness and sensibility and had to be killed. It will be noted that, in this particular experiment, besides the usual manipulation of the intestines every five minutes, considerable further trauma was inflicted without effect. The kidneys were roughly handled, several inches of intestine were resected at intervals, and the parietal and visceral peritoneum was stretched severely, with only a slight temporary eifect upon the blood pressure.

In order to be certain that the resistance of these animals to shock was brought about by the preliminary hour's anesthesia rather than by some unrecognized technical divergence from the method used in the normal controls, two control experiments were now repeated, the intestines being exposed and liandled immediately following the first blood pressure reading, instead of after an hour's anesthesia. These animals beliaved exactly as the original normal controls. They were in deep shock in an hour and 55 minutes and two hours, respectively.

The 12 experiments with preliminary anesthesia were clearcut, and demonstrated that an animal which has been kept anesthetized with ether for an hour immediately before opening the abdomen becomes, in some way, much, more resistant to the shock-producing effects of intestinal manipulation than are animals in which the intestines are exposed more promptly after anesthetization. No explanation of the mechanism of this protective ether-effect can be offered here other than the suggestion that during the early stages of anesthesia the circulation appears to be in a rather unstable condition, and tlie added strain of visceral trauma at tliis period can, perhaps, bring about circulatory failure more easily than in an animal which has had time for its circulation to become accommodated to the state of anesthesia, and more stabilized. It is note


Maech, 1022]


85


worthy, however, that although an hour's light anesthesia before opening the abdomen had this protective effect upon the circulation, in other experiments it was clear that, once the blood pressure had begun to fall toward the shock level after intestinal manipulation had been begun, ether exerted a marked depressant action upon the circulation and distinctly favored the development of shock.

It is evident that no conclusions regarding the relation of the adrenals to shock could be drawn from the experiments in which the glands were removed immediately before exposing the intestines. The complicating factor of the protective ether-effect precluded that. It was, therefore, decided to remove the adrenals, to allow the animal to recover completely from the anesthetic, then to reanesthetize it afresh and proceed at once with the intestinal trauma.

Reaction Seven to Seventeen Hours after Adrenalectomy. — In tliis series of experiments both adrenals were removed extraperitoneally through lumbar incisions under strict aseptic precautions. Tlie animals were then allowed to recover from the anesthetic and were left in their cages for periods ranging between 7 and 17 hours. None of the animals displayed any sign of adrenal insuiEciency on inspection. They appeared active and not asthenic. Each animal was then anesthetized, tracheotomized, a blood pressure tracing taken and the intestines at once exposed and handled as usual. It was found at the outset that these adrenalectomized animals had a very low blood pressure, the first readings averaging 82 millimeters. The pulse was more rapid than normal. All of the animals of this series fell into deep shock within 30 or 35 minutes after exposure of the intestines, the blood pressure falling rapidly to 20 millimeters in most cases. At autopsy the operative sites were found in splendid condition, and dissection of the splanchnic nerves showed injury only to the small branches which necessarily must be severed in removing the adrenals.

Fig. 5 is a tracing from a typical experiment in this series.

Although these adrenalectomized animals went into shock in one-third the time required to bring a normal animal into shock, it was evident from the low blood pressure and rapid pulse that the circulation was already affected by the removal of the adrenals before intestinal manipulation was begun. It was not possible, therefore, to draw conclusions from this series concerning tlie relation of the adrenals to shock, and it was realized that experiments would have to be carried out upon adrenalectomized animals which had been allowed to recover from the anesthetic after adrenalectomy, in order to eliminate the protective ether-effect, but which had not been left long enough for circulatory signs of adrenal deficiency to appear before the animals were exposed to the shock-trauma.

Reaction One Hour After Adrenalectomy. — Since the blood pressure does not begin to decline until several hours after adrenalectomy, a series of experiments was carried out in which, foUo^nng removal of both adrenals under aseptic precautions, each animal was allowed to recover fully from the anesthetic imtil it was able to walk about and to react nonually to stimuli. In this way the protective effect of the anesthesia administered during tlie operation was avoided, since it had been determined


in two experiments that if an animal were reanesthetized immediately after recovery from an hour's anesthesia, it retained none of the protective effect of the ether, but reacted to intestinal manipulation exactly as a normal animal. Complete recovery from the anesthetic administered during adrenalectomy required usually about 30 minutes; then the animal was reanesthetized at once, tracheotomized, and a blood pressure tracing made. The blood pressure was normal in every ease, averaging 145 millimeters. The intestines were then exposed at once and handled as usual. These adrenalectomized animals reacted exactly as normal animals, requiring an average of an hour and 45 minutes to fall into shock. Fig. 6 is a typical tracing from this group.

This series of experiments indicates clearly that shock develops in the absence of the adrenal glands precisely and characteristically as it does in normal animals. Disordered function of the adrenals cannot be invoked as the cause of shock produced by peritoneal trauma. If the adrenals were causative factors of any importance, there would have appeared some difference between the reaction of the adrenalectomized animals and that of the normal controls. It might be objected that perhaps some of the secretion of the adrenals remains active in the body for a while after the glands are removed, so that adrenalectomy does not create an immediate deficiency of the secretion. It must be clear, however, that the same objection would be more applicable to any theory postulating disordered adrenal function as a cause of shock. The importance of the accessory chromaffin tissue is recognized fully, but since the adrenalectomized animals enter the experiment with complete absence of the adrenal glands, surely if epinephrin exhaustion or adrenal fatigue were causative factors in shock, these animals should fall into shock distinctly quicker than normal animals in which a longer time would be necessaiy to exhaust the greater epineplirin supply or bring the adrenal glands as well as the accessory tissue into a condition of dysfunction.

Condition of the Heart and Medullary Centers in Shock. — During the progress of these experiments advantage was taken of the opportunities to study the condition of various body functions during shock. Most observers have found that the heart functions normally during shock. Erlanger and his coworkers ^ acquired the impression that the heart in shock has not the normal reserve power and this might be expected, since Markwald and Starling have shown that weakening of the cardiac contraction accompanies low blood pressure.^^ Boise " attributed the circulatory failure in shock to impairment of cardiac function. In the present experiments the heart during shock has invariably worked splendidly both iinder the strain of large doses of adrenalin, and when normal saline was run rapidly into the jugular vein. In every experiment in whicli an animal was allowed to die in shock, respiratory rather than cardiac failure was the immediate cause of death.

It is almost universally stated that the pulse is rapid during shock. Wiggers, however, mentions slowing of the heart during the late stages of the condition ° and Dupuytren °° wrote from clinical observations " Le pouls est d'une lenteur et d'une


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Fig. 4— Intestinal manipulation after an hour's preliminary anesthesia. Besides the usual manipulation ever}' 5 minutes, sev(

^ • Condition oi animal^"





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Condition of animal normal after 2^- hours of intestinal trauma. No shock.


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leritoneum and mesentery was made at A'; kidneys roughly handled at }'; resection of several inches of intestine at Z and Z' . l\ hours. No shock.


[Xo. 373


mollesse telles que la pliis legere pression le sufflamine," and that, if recovery takes place, " le pouls est plus fort et moins rare." In a number of the present experiments periods of pronounced slowing of the pulse were observed during deep shock. This slowing of the heart-beat is a result of the action of the cardio-inhibitory center. It has been held that inhibition or paralysis of this center is a characteristic and perhaps a cause of shock; on the other hand, the experiments of numerous workers indicate that the cardio-inhibitory center "functions normally during shock. Mann reported that stimulation of the central end of one cut vagus caused reflex cardiac inhibition " even in the most extreme degrees of shock." " Jackson and Ewing wrote that they were able to obtain the reflex only while the blood pressure remained above 60 millimeters of mercury.^ I have never failed to obtain slowing of the heart-beat during shock by this means, and the reflex has been elicited repeatedly when the blood pressure was as low as 20 millimeters (Fig. 7). In these experiments a further proof that the heart, in shock, is under control of the cardioinhibitory center was obtained by observing the efEeet of cutting the vagi. If the vagi are sectioned during one of the periods of bradycardia which occur occasionally when an animal is in deep shock, a most striking acceleration of the pulse always results immediately, as can be seen in Fig. 8, where the heart-rate increased from 80 to '200 per minute. Section of the vagi when the pulse is rapid during shock will always increase the heart-rate unless it is already too rapid to be influenced by removal of vagus inhibition; and in the cases in which Vagus section could not increase the rate, stimulation of the central end of one cut vagus has already produced slowing of the heart immediately before section of the remaining vagus, showing that the cardio-inhibitory center was capable of responding to stimuli during shock. In three experiments both vagi were sectioned before exposing the intestines; these animals went into shock no more rapidly than the normal controls, and their blood pressure curves were characteristic of the normal shock curves. It is clear that failure of the cardioinhibitory center is not a causative factor in shock.

The question of the condition of the vasomotor center has been the subject of many investigations. On the basis of their well-known experiments, Crile," Lockhart-Mummery " and others have maintained vigorously that surgical shock represents an exhaustion of the vasomotor center ; others '"• "• • '^ have produced splendid evidence that it is not exliausted. In the present experiment, a pressor response could always be elicited by stimulation of sensory nerves during shock. In most of the experiments the rise of blood pressure was less prompt and less marked than before the onset of shock, although in some cases the rise was quite as marked. Fig. 9 shows a definite pressor response obtained by sciatic stimulation during deep shock, when the blood pressure was only 22 millimeters. I can entirely agree with those who hold that exhaustion of the vasomotor center is not a primary factor in shock.

Janeway and Ewing" have stated that in shock produced by intestinal manipulation " there is absolute paralysis of every


tissue of the intestines, of the muscles, of the intestinal walls and of the arterioles," and they stress the importance of a local peripheral paralysis of the splanchnics as a factor in shock. I have never observed such a complete paralysis of the intestines during shock. Abortive peristaltic movements occur throughout the duration of shock, and not infrequently persist for a short while after death. Janeway and Ewing wrote that in some of their experiments the intestinal manipulation was quite violent, causing rhexis of the peritoneum. It is possible that excessive trauma might produce such a paralysis, but surely it cannot be regarded as a characteristic part of shock.

The Hypotension Resulting from Adrenalectomy. — It may not be entirely out of place here to describe several observations upon the group of adrenalectomized animals which may have some bearing upon the function of the adrenal glands.

In the first place, although we have the clinical evidence of Addison's disease and certain experimental evidence besides, indicating some relation of the adrenal glands to the maintenance of normal blood prressure, Vincent " reports that continous blood pressure tracings, taken up to the moment of death, after complete removal of the adrenals from the circulation, show no greater faU. in blood pressure than would have occurred in a normal animal subjected to ether anesthesia alone for the same period of time, and he concludes that " these experiments appear to show conclusively that the secretion of adrenalin into the circulation is not to be regarded as a factor in the maintenance of the normal blood pressure." The present experiments do not at all agree with such an observation. Howell " and others have pointed out that adrenalectomy has no effect upon the blood pressure for several hours, and I have observed this fact repeatedly. But the blood pressure of an adrenalectomized cat begins to fall gradually about four hours after the glands have been removed, and within the first 12 hours after adrenalectomy the blood pressure in the unanesthetized animal invariably falls to a very low level and continues to decline progressively until death. In these experiments it was demonstrated that the marked hypotension, observed in every case as early as seven hours after adrenalectomy, is a characteristic result of adrenal deficiency and not merely a condition of lowered tone in an animal several hours after an operation. For in a number of experiments both adrenals were exposed aseptically and handled even longer than woiild have been necessary to remove them. They were then left in their normal positions and the incisions closed. Seven to twelve hours later these animals were reanesthetized and the blood pressure in every case was normal and exposure of their intestines produced shock in the length of time characteristic of normal animals. In another series, under aseptic precautions, one adrenal was removed and the other exposed and handled, the operation taking as long as if both had been removed and the operative trauma being made purposely more severe. When these animals were reanesthetized after a lapse of from 7 to 15 hours, tlieir blood pressure was found to be normal and they reacted to intestinal manipulation exactly as normal controls. Autopsy showed complete removal of one


March, 1932]


89


adrenal in every case. Thus, the trauma of the operation alone is not responsible for the low blood pressure which developed in every completely adrenalectomized animal ; nor has removal of one adrenal any effect upon the blood pressure during the period of these experiments ; but when both glands are removed the blood pressure invariably falls strikingly. The splanchnic nerves were dissected at autopsy in every case in order to make certain that injury was confined only to the little branches which must be cut in order to remove the adrenals. I cannot agree with those who have claimed that the low blood pressure developing after adrenalectomy is merely tlie hypotension to be observed in any moribund animal, nor does the contention of Hoskius and McClure/' " that the hypotension is a result of asthenia, seem reasonable; for these adrenalectomized animals were by no means in a moribund condition, nor was there evident asthenia seven hours after removal of the glands. One of these adrenalectomized animals, a rather wild male, escaped from its cage just before it was anesthetized for the blood pressure reading and ran swiftly about the room, evading capture. It was caught, anesthetized, and the systolic pressure found to be 75. It seems more reasonable to believe that the hypotension leads finally to asthenia because of the impairment of the circulation.

The fact that hypotension is a characteristic effect of adrenalectomy and that it appears to be the result neither of the trauma of the operation nor of the asthenia, supports the belief that the adrenals are, after all, directly concerned in some way in the maintenance of normal blood pressure. This idea is strengthened by observations upon the powers of resistance of the circulation of adrenalectomized animals to strains (such as prolonged anesthesia and trauma) imposed upon it. These experiments are as yet incomplete, but they strongly suggest that the circulation of an adrenalectomized animal, after the development of hypotension but before the appearance of asthenia, is distinctly more unstable than tliat of a non-adrenalectomized animal with the same degree of hypotension.

Discussion

It may possibly appear inconsistent that any observations supporting the belief of the activity of the adrenals in maintaining normal blood pressure are presented here together with evidence that these glands are not concerned in the circulatory failure characteristic of shock; but certainly, even though the adrenals be concerned with blood pressure regulation, they are not by any means the only factor involved. The demonstration of the lack of any causal relation between the adrenals and the acute hypotension of shock merely points more distinctly to a disturbance of some other part of the intricate circulatory mechanism.

At the present time, the idea that the adrenals may be concerned with the maintenance of normal blood pressure is widely repudiated, and the work of Cannon, Gley, Stewart and Rogoff, Hoskins and McClure, and Vincent, in this connection is familiar enough to everyone who has given any attention to the function of these glands. The objection has been repeatedly brought forth that if adrenalin were necessary for the


maintenance of normal blood pressure, removal of the glands should result in an immediate fall of pressure; but no fall occurs during several hours after adrenalectomy."- '"• "• ^' Much has been made, also, of the fact that adrenalin can be detected in normal blood in minute amounts only; so minute, indeed, that numerous writers do not hesitate to declare that it is physiologically useless in affecting vasomotor tone, especially since the intravenous injection of small amounts of adrenalin can produce a depressor instead of a pressor effect." ^- *• ™ Perhaps these, and certain other similar obsen^ations which cannot be discussed here, deserve more critical consideration and study before they can be accepted as reasons for believing that adrenalin plays no part in the maintenance of normal arterial pressure, or that " I'adrenaline ne doit plus etre consideree comme un produit de secretion vraie." "

In regard to the objection that the blood pressure does not fall immediately after adrenalectomy (and some have actually been content with blood pressure observations continued less than two minutes after compression of the adrenal veins)" it does not seem permissible to ignore completely the fact that there is a definite supply of physiologically active epinephrin left in the accessory chromaffin tissue after removal of the adrenal glands," ^ and tliat this supply may be sufficient to sustain the blood pressure for several hours, but not indefinitely. It is true that Stewart and Rogoff have considered this point to the extent of writing : " No account is here taken of the possibility that the sporadic chromaffin tissue may discharge a certain amount of epinephrin, since, although it has been shown to contain that substance, nothing is known as to its liberation " ; ^ however, the fact of our ignorance as to the mode of liberation of epinephrin from this accessory chromaffin tissue is not a satisfactory reason for taking no account of it. The well known protective effect of minute residual fragment* of parathyroid," thyroid and adrenal " tissue left, either intentionally or by accident, in extirpation experiments demonstrates strikingly enough the role that a relatively insignificant amount of such tissue can play. It is curious, in any event, that those who feel that the adrenals have nothing directly to do with vascular tone, because hypotension does not occur the instant tlie adrenal veins are clamped, nevertheless appear to accept the belief that the glands are concerned in some way with the maintenance of muscular tone, although asthenia develops, after adrenalectomy, distinctly later than hypotension. It seems to the writer that the important fact may be, not that the blood pressure fails to fall immediately after removal of tlie glands, but rather that a primary effect of adrenalectomy is hypotension.

Concerning the minute amount of epinephrin that can be detected in the blood, it is difficult to be sure that it has no physiological significance in relation to normal blood pressure simply because the amount collected at any given moment is too small to affect the contractions of a strip of smooth muscle in vitro, or to exert a pressor effect upon the blood pressure when introduced into the circulation of an animal. Unquestionably, such procedures appear to be surprisingly delicate quantitative tests for the presence of epinephrin within wide


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Fig 5— Intestinal manipulation 11 hours after complete adrenalectomy. April 2. 11-50 p.

about, apparently normal. Intestmes exposed IO.00.


both Deep


idrenals removed; April 3, 10.40 a. ra.li! shock 30 minutes later.




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Fig. 6.-Intestinal manipulation 1 hour after adrenalectomy. June 23, 3.30 p. m„ operation for removal of both adrenals begun; o


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Fio. 7.— Slowing of the heart produced (iuring deep shock by stimulating central end of cut left vagus (B.P. 25).


Fio. S. — Shows activity of cardio-inhibitory center producing spontaneous bradycardia during deep shock. Heart rate increases from 80 to 200 per minute on section of vagi.



Fig. 9. — Pressor response elicited by stimulation of .sciatic nerve during deep shock (B.P. 22).


^*^ Sto


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Complete recovery from anesthetic at 4.40 p. m. Intestines exposed at 5.05 p. m. Shock 1 hour 35 minutes later. Compare with Fig. 2.


93


[No. 373


limits; however, they have limitations, a point which Stewart and liogoft' did not ignore when they concluded from certain experiments that the " liberation of epinephrin from the adrenals is not indispensable for life or health, nnless indeed tlie necessary quantity is, even in the adrenal vein blood, below the limits of detection by the methods used." "^ The fact that the quantity of epinephrin in the blood, at any given time, is minute or even indetectable by present methods ( Stewart and Rogoff, however, do detect it unfailingly in normal adrenal vein blood) does not entirely satisfy the question of the relation of this substance to normal blood pressure. In the first place, the methods of collecting and assaying the blood samples and the conditions under which the blood is obtained are apparently so subject to error that almost each worker has devoted some space in the literature to a more or less caustic criticism of the other's technique : and in the second place, it is highly probable that there may be a difference between the physiological potentialities of minute amounts of a substance secreted continually, and the effects which can be demonstrated when the amount that is withdrawn from the circulation at any one cross-section of time is used in an acute experiment. Otherwise, we would have to conclude from a study of small samples of normal arterial blood that the secretion of the thyroid gland has no effect upon body oxidations, and that of the para-thyroids no relation to calcium metabolism.

The most elaborate and important recent work on the adrenals *• '^^ "• "' has been concentrated upon the question of the existence of a physiological adrenalinaemia, and has been concerned not at all with the fact that the most striking primary effect of the lack of the adrenal secretion is hypotension. Unquestionably, the painstaking and ingenious experiments of these workers hold a very great interest. Because of the ease with which epinephrin can be detected, the study of the epinephrin content of the blood promised much, and it was most important that it be carried out. However, it is perhaps permissible to feel that this study, even in the hands of such able investigators, has nst led to conclusions as concordant or decisive as one might have hoped for. It must be remembered that there still exists an active controversy between these workers over the amount of epinephrin normally present in the blood, and also over the more important question of whether the epinephrin content of the blood is increased during the various reflex pressor reactions which have been assumed to be conditioned or influenced by an outpouring of this secretion. The only point of agreement, in fact, seems to be the opinion that the amouat of epinephrin circulating normally is not great enough to exert an influence upon normal blood pressure. It is quite possible that epinephrin has nothing to do with the maintenance of normal pressure, as these observers insist; it may, indeed, be that the lack of the unknown cortical secretion is responsible in some way for the hypotension that follows adrenalectomy. Nevertheless, the observations leading to a denial of any relation of epinephrin to normal arterial pressure have certainly not yet settled this important question, which must remain incompletely answered until we are aV)le to understand why the blood pressure falls


so characteristically when the adrenal glands are removed from the body.

Summary

I. Adrenalectomized animals, subjected to uniform intestinal manipulation before the blood pressure has begun to decline as a result of adrenalectomy, fall into shock exactly as do normal controls — the time required for the production of shock and the character of the blood pressure curves being the same in both series. It is therefore concluded that disordered adrenal function is not a factor in the production of shock.

II. Hypotension invariably results from removal of the adrenal glands, and with the development of hypotension the circulation of adrenalectomized animals appears to become more unstable than that of normal animals even before the appearance of asthenia. The blood pressure begins to fall several hours after adrenalectomy and becomes progressively lower until death. The fall in blood pressure is shown to be independent of the operative trauma and begins before asthenia has appeared. This is offered in support of the idea that the adrenals are concerned in the maintenance of the blood pressure at the normal level, and certain objections to this belief are briefly discussed.

III. Animals that are kept lightly anesthetized with ether, for an hour immediately before the abdomen is opened, become very resistant to the shock-producing effect of intestinal manipulation. Even when subjected to severe peritoneal trauma for a period of three hours, the blood pressure shows practically no tendency to fall and sensibility is retained. In contrast, if identical intestinal manipulation is begun more promptly after anesthetization, the blood pressure invariably begins to decline progressively within an hour, has fallen to 60 mm. o: below an hour and a half to two hours after opening the abdomen and the animal is in complete shock. An hour's ether anesthesia preliminary to opening the abdomen has proved to be a striking protective against shock, under the conditions of these experiments. If an animal is kept anesthetized for an hour, permitted to recover from the anesthetic, and at once reanesthetized and intestinal manipulation begun, the protective effect of the hour's anesthesia will have dis


IV. Ether has a distinct tendency to hasten the onset of shock once the blood pressure has begun to decline after the abdomen is opened.

V. Cardiac failure is not a factor in the production of shock.

VI. Failure of tlie vasomotor center is not a primary factor in shock.

VII. The cardio-inhibitory center is shown not only to respond to stimuli but also to function independently during deep shock. Its failure cannot be regarded as a cause of the condition.

BIBLIOGRAPHY

1. Stewart and Rogoff: Jour. Pharm. and Exp. Thera., 1916, VIII, 205.

2. Elliott: Jour. Physiol., 1912, XLIV, 374.

3. Annep: Jour. Physiol., 1912, XLV, 307.


Maech, 1922]


93:


4. Hoskins and McClure : Arch. Int. Med., 1912, X, 343.

5. Cannon and Hoskins: Am. Jour. Physiol., 1911, XXIX, 274.

6. Cannon: Natl. Health Ins., Med. Research Com., Special Rep., Series No. 25, p. 109.

7. Bainbridge and Parkinson : Lancet, 1907, 1296. S. Short: (Quoted by Cannon)."

9. Corbett: Jour. Am. Med. Assoc, 1915, 380.

10. Sydenstricker, Delatour and Whipple: Jour. Exp. Med., 1914, XIX, 536.

11. Bedford: Am. Jour. Physiol., 1917, XLIII, 235.

12. Mann: Jour. Am. Med. Assoc, 1917, 371.

13. Crowe and Wislocki: Beitr. zur klin. Chirurgic, 1914, XCV, 8.

14. Vincent: Endocrinology, 1917, I, 140.

15. Hoskins and McClure: Am. Jour. Physiol., 1912, XXXI, 59.

16. Abelous and Langlois: Arch, de Physiol, norm, et path., 1892, IV, 269.

17. Janeway and Ewing: Ann. Surg., 1914, LIX, 158. IS. Wiggers: Am. Jour. Physiol., 1918, XLVI, 314.

19. Gley : " Quatre legons sur les secretions internes," Paris, 1921, 48.

20. Wiggers: Jour. Am. Med. Assoc, Feb. 23, 1918.

21. Mann: Johns Hopkins Hospital Bulletin, July, 1914.

22. Keith: Natl. Health Ins., Med. Res. Com., Special Rep., Series No. 26, p. 36.


23. Boise: Am. Jour. Obs., 1917, LV, 1.

24. Crile: Boston Med. and Surg. Jour., 1903, CXLVIII, 247.

25. Lockhart-Mummery : Brit. Med. Jour., 1910, II, 759.

26. Seelig and Joseph : Proc Soc Exp. Biol, and Med., 1914, XII, 49.

27. Seelig and Lyon: Jour. Am. Med. Assoc, Jan. 2, 1909.

28. Porter: Am. Jour. Physiol., 1907, XX, 399.

29. Erlanger, Gesell, Ga.sser and Elliott: Jour. Am. Med. Assoc,. 1917, 2089.

30. Dupuytren: "LeQons orales de chnique chirurgicale," Paris,, 1832, Vol. II, 493.

31. Jackson and Ewing: Am. Jour. Physiol., 1914, XXXIII, 31.

32. Markwald and Starling: Jour. Physiol., 1913, XLVII, 275.

33. Hoskins and McClure: Am. Jour. Physiol., 1912, XXX, 192.

34. Hornowski: Biochem. Centralbl., 1909-10, IX, 572.

35. Cannon: Am. Jour. Physiol., 1919, 428.

36. Gley: "Quatre legons sur les secretions internes," Paris, 1921, p. 63.

37. Gley and Quinquaud: Compt. rend. Soc biol., 1919, 1175.

38. Bazzett: Jour. Physiol., 1920, LIII, 333.

39. Stewart and Rogoff : Jour. Pharm. and Exp. Ther., 1917, X, 1.

40. Fulk and McLeod: Am. Jour. Physiol., 1916, XL, 21.

41. MacCallum: Jour. Exp. Med., 1909, XI, 118.

42. Stewart and Rogoff: Am. Jour. Physiol., 1919, XLVIII, 22.


A CLINICAL AND ANATOMICAL STUDY OF FIFTY-ONE CASES OF

REPEATED CESAREAN SECTION WITH ESPECIAL REFERENCE

TO THE HEALING OF THE CICATRIX AND TO THE

OCCURRENCE OF RUPTURE THROUGH IT

By Thomas 0. Gamble (From the Obstetrical Department oj The Johns Hopkins Hospital and U nivcrsity)


In 1917 Dr. J. Wliitridge Williams published in the Bulletin OP The Johns Hopkins Hospital the results of a histological study of 50 uteri removed at Caesarean section. Included among them were 10 uteri which had been incised at a previous similar operation. They were only briefly described, as Dr. Williams stated that "the details concerning this series will be published in full later." Since that time 11 additional specimens have been added to our collection, and it is my purpose to report the results of a careful clinical and anatomical study of this material.

It is but a natural sequence that, while investigating the histories of the women from whom the uteri were removed, we should likewise consider the closely allied cases in which a conservative Ccesarean section was repeated at a subsequent pregnancy, or delivery was effected through the natural passages. It is only by a careful consideration of snch cases that we may be enabled to draw definite conclusions concerning the truth or fallacy of the oft-quoted dictum — " Once a Cesarean, always a CEesarean."

Our discussion is based tipon the study of 63 pregnancies occurring in 51 women who had previously been subjected to Cassarean section. Fifty-five of these pregnancies followed a single Caesarean section, while in eight there were two opera


tions. The manner in wliieh the pregnancies were terminated' was as follows :

A second or third Cesarean section was done in 45 cases.

Vaginal delivery occurred in 17 cases.

Rupture of the old Caesarean scar occurred in one case.

That the woman who has once been subjected to a Cssarean section, and who again becomes pregnant, presents an interesting and at the same time a somewhat puzzling problem has been generally recognized and frequently commented upon. Prior to 1876 the mortality following Cesarean section was appalling, from 50 to 60 per cent of the women dying from infection or hEemorrhage, but at the present day elective Caesarean section, performed under proper surroundings by men trained to recognize its indications, as well as its contraindications, should be attended by an immediate mortality of not more than one per cent. From an obstetrical point of view we cannot, with fairness, rest content with the immediate results, but must also consider what may happen in the subsequent pregnancies of such women. Eongy has estimated that approximately three per cent of all Caesarean section scars eventually rupture, and that at least one-half of such accidents terminate fatally. ISTotwithstanding this very definite menace, and completely ignoring the frequent warnings, which have been sounded by many vrriters in recent years, surgeons, gynecolo


94


[Xo. 373


gists, and obstetricians throughout the country are performing the operation with increasing frequency.

Proof that the uterine scar constitutes a locus minoris resistentuE in a cei-tain proportion of cases may be obtained by reviewing the hterature on rupture of the Cffisarean scar in subsequent pregnancies. Approximately 103 such cases have been reported and summarized by numerous authors. Thus, Wyss, in 1912, analyzed 4,3 cases, while four years later Bell, Schroeder, and Findley collected 79, 63, and 63 cases, respectively. To the 63 cases reported by the latter, Spalding, in 1917, added 12 others, which he had collected from the literature. Furthermore, Losee reported nine instances of partial or complete rupture in 1918, and since then Davis, DeCourcey, Howson, Freund. Novak, Baisch and Holland have reported additional cases. In addition to tlie occurrence of actual rupture, a number of cases have been reported in which the uterine scar was so attenuated that rupture would probably have occurred had the uterine distention not been relieved by operation, not to speak of those in which the rupture or extreme thinning of the scar was not recognized, or, if recognized, was not reported.

Naturally, the questions arise : Wliat is the causative factor in the production of the thin, imperfect scar, and what are the conditions predisposing to its rupture ? Numerous explanations and theories have been offered which may be roughly classified under the following headings :

1. Infection.

2. Improper suture technique.

3. Unsuitable suture material.

4. Location of the uterine incision.

5. Involvement of the placental site at operation.

6. Implantation of the placenta over the scar, with inversion of foetal elements.

7. Miscellaneous.

We shall consider each group in some detail.

1. Infection'

The important role played by infection in the production of an imperfect scar is so generally recognized that a case of rupture or extreme thinning is seldom reported without emphasis being laid upon the presence or absence of fever during tlie preceding puerperium. Losee asserts that " when infection takes place and the cut surfaces are infiltrated with leukocytes and serum, associated with more or less necrosis, then only that muscle tissue remains which has not become necrotic." The extreme necrosis which may take place in an infected wound is well illustrated by Fig. 1. Findley states that failure to secure perfect healing is partly due to septic infection of the wound, and calls attention to the possible existence of a latent gonorrhoeal infection, which " may defeat tlie most painstaking efforts to secure perfect wound healing." The character of the puerperium was noted in 66 of the 97 eases of rupture studied by Holland, who found that 51 were febrile and 13 afebrile. In commenting upon these figures, he asserts that "infection of the uterine wound may occur without rise of temperature, or with only a very slight one ; for example, few


will deny that the presence of extensive adhesions to the scar at a subsequent operation is evidence of sepsis duiing the healing process."

In our opinion the latter part of Holland's statement does not necessarily hold true, and it is permissible to suppose that adhesions may form in the absence of infection, whenever there is a rough, raw surface, such as occurs when the uterine wound has been improperly sutured. For example, in the first case reported in our series the wound had been closed with interrupted silkworm-gut sutures and the stiff knots had been left upon the surface. The puerperiimi was normal and the scar well-nigh perfect, but nevertheless was covered by dense adhesions. Consequently, if, as is likely, such adhesions resulted from irritation by the stiff knots, it behooves all operators to secure accurate coaptation between the cut edges and to do as little damage as possible to the covering serosa. It must be admitted that imperfect scars may result when the temperature has not been elevated, and in such cases some causative agent other than infection must be sought, wliile, on the other hand, a scar sufSeiently strong to withstand the strain of labor may result even thougli the record of an elevated temperature indicates that infection had occurred. Here it must be assumed that the infectious process did not involve the uterine incision, or, if it did, that no extensive necrosis resulted.

In the 183 Cesarean sections, which were done in the service up to the end of December, 1920, there was only one instance of actual rupture of the scar and in this patient the temperature had been elevated for 10 consecutive days following the primary operation. In 15 cases in which the uterus was removed later and studied, and in which we had definite information as to the character of the preceding puerperia, seven were found to be febrile and eight afebrile. In the first group, no trace of the sear could be found in six specimens, while it was only shghtly thinned in one. In the second group, the sear could not be located in three instances, while the healing was good in three, fair in one, and poor in one case. Jloreover, in three cases the uterus was removed at the third section, so that in such specimens we can only speculate as to which operation had given rise to the scar in question. Of the 13 patients in whom delivery was effected through the natural passages following Csesarean section, six presented a preceding febrile puerperium.

Such figures, while suggestive, only serve to show how difficult it is to predict the strength or weakness of the Cfesarean scar in the individual case. However, it would seem permissible to infer that the uterine scar will be weak rather than strong if frank infection has occurred in the preceding puerperium, and that such patients should not be subjected to the strain of a prolonged labor, particularly if the original section has been performed on account of pelvic dystocia. On the other hand, when it has been done for some temporary indication, or if there is only moderate pelvic contraction with a small child, even though fever has complicated the former puerperium, we believe that tlie patient may be allowed to go into subsequent labor, provided that she be kept under careful observation. This view is at variance with that recently expressed by Newell : " It [repeated Csesarean section] should, however.


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95


be considered obligatory in patients who give a history of a febrile convalescence, since this points to the probability of uterine infection, and it is in these patients that the rupture of tlie scar in subsequent pregnancies and labors is most to be feared."

3. SuTUEE Technique

HuiTied closure of the uterine incision without due care in accurately approximating the cut surfaces, the placing of the sutures too widely apart, and the inclusion of the decidua in the line of sutures may all be factors in producing an imperfect scar. In this clinic the incision is closed with two layers of catgut, the first consisting of deep buried interrupted sutures placed at intervals of about 1 cm., while a superficial running suture brings together the serosal edges. If, after the first layer of sutures has been placed, there is still considerable gaping of the niuscularis, an additional continuous buried suture is used. The decidua should be avoided, since any bits of it inverted into the wound may proliferate and develop into areas of unusual friability. Figs. 3 and 3 illustrate the technique.

Fischer, in 1912, directed attention to the danger of suturing the uterus before it has firmly contracted and retracted, and consequently Green, in 1916, advised that the operation should not be undertaken until labor had been in progress for several hours. " It stands to reason," he says, " that the scar will be thicker and stronger if the closing sutures are applied to a uterine wall thickened by several hours of contractions, than when placed in the thin, comparatively flabby wall of a uterus incised before labor has begun." Apparently the rationale of this theory rests upon a mechanical basis; for, if the sutures be laid and tightly tied while the musculature is lax, it is conceivable that when the uterine wall increases in thickness as the result of subsequent contraction, the sutures will either become untied, or else they will tear through the thickened tissue. In the latter event a gap may be left on the inner surface of the uterus, which is invaded by endometrium. Unfortunately, however, it is not always possible to wait for the occurrence of firm contraction, as brisk hsemorrhage may necessitate immediate suturing of the wound. Figs. 4 and 5 illustrate the assumed sequence of events.

Analysis of the operative notes concerning the first operation in our patients, from whom the uterus was removed at a subsequent section and its scar studied microscopically, shows that the cliaracter of the uterine contraction was recorded in 11 instances. In eight, firm contraction had occurred before the sutures were placed, and in five of these the scar was normal or could not be found, while in the three others definite thinning was evident. In one of the latter the convalescence was complicated by an outspoken infection, so that it is difficult to determine whether the infection or the slow muscular contraction had played the more important part. On the other hand, in the three instances of poor contraction, there was no trace of the scar in two, while it was very thin in the third case. Moreover, in the one instance in which the old scar ruptured in a subsequent pregnancy, it had been necessary to suture the uterus while its musculature was lax.


It must be admitted that the figures available are too few to permit final conclusions, but they clearly indicate that suture of the uterus after firm contraction has occurred does not necessarily insure an ideal scar.

3. Suture Mateeial

The fact that a wide variety of suture material has been used in closing the uterine incision is sufficient proof that none is entirely satisfactory from every point of view. Plain, formic, and chromicized catgut, silkwonn-gut, kangaroo tendon, ordinary black or white silk, metallic wire, etc., have been employed at one time or another. Plain catgut has generally been discarded on account of its rapid absorbability, although Carstens advocates it for that very reason.

In our hands chromic catgut has given relatively satisfactory results. Holland, on the contrary, believes that it is also absorbed more rapidly than is desirable, particularly in the presence of infection. Upon the analysis of a large number of cases, he found that subsequent rupture occurred two and a half times more frequently after the use of catgut than of silk. Consequently, he is of the opinion that non-absorbable sutures are preferable, and holds that silkworm-gut fulfills the requirements most satisfactorily. Plain silk is a notorious harborer of bacteria, as is admirably illustrated by Case X below. Prusmann objects to non-absorbable sutures on the ground that small channels may be formed about them, and as these may be invaded by bacteria or even by endometrium, necrosis or weakening of the scar may result. Eckstein, in 1904, indicated the advantages of metallic sutures, and suggested the use of thin flat bands of lead, wliich, in addition to being non-absorbable, would serve as supports for the scar in future pregnancies.

In our series of operations silkworm-gut was used only in a single instance, and the resulting scar was ideal. Wlien the uterus was removed a year later, the sutures were still in situ (Fig. 6), but, as has been mentioned previously, dense adhesions had developed along the entire length of the scar, probably as the result of the knots irritating the surrounding tissues. It would seem, therefore, that when this material is used the sutures should be buried, and the superficial muscle layer and peritoneum brought together with catgut over it.

Obviously, the choice of suture material is at present a matter of personal preference, and no definite statement can be made as to which possesses the greatest merit. To my mind, the technique used in laying the sutures is of greater importance than the material of which they are composed.

4. Situation" of the Incision At a Caesarean section the uterus may be incised in any one of the following four locations; (1) in the midline of the anterior wall; (3) in the posterior wall; (3) in the fundus, either transversely or longitudinally; and (4) in the lower uterine segment, likewise either longitudinally or transversely. In the last group may be included those cases of vaginal hysterotomy, in which the lower uterine segment becomes involved in the incision. "Without doubt, in the great majority


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[No. 373


of cases the median anterior wall is the site of election, although in recent years there has been a growing tendency to advocate incision of the lower segment. The uterus is opened through the posterior wall only in the rare instances in which its body has undergone extreme forward displacement.

Varying results have been obtained with the several modes of incision and a voluminous literature has accumulated upon the subject. In 1897, Fritsch described his transverse fundal incision, which has been discarded after a number of years of experimentation. Eckstein, in 1904, reported the first case •of rupture of a fundal scar in a subsequent pregnancy, and following it mmierous similar reports have appeared in the literature. SchefEzek called attention to the fact that in order to secure perfect healing of any wound, its edges should be immobilized, and admitted the impossibility of keeping the actively contracting and relaxing uterus entirely at rest. Furthermore, he pointed out that the fundal incision approached the ideal even less than one through the median wall or the lower uterine segment. In proof of this he reported the findings at autopsy upon a woman who had died from a toxEemia several days after a Caesarean section, which showed that the sutures in the fundal wound had become loosened or entirely untied, presumably as the result of active uterine contractions. By way of contrast, he also reported his observations upon 10 women who had been subjected to extraperitoneal Caesarean section. In five of them repeated section was necessary ; while of the other five, one was delivered spontaneously, and in four labor was induced in the latter part of pregnancy. In each instance the uterine scar remained intact, although in one the pains continued for 68 hours before delivery was effected. Consequently, he concluded that the scar is much more resistent after extraperitoneal section than after either the fundal or median incision. Spalding holds that " the extraperitoneal Caesarean section carries with it a better prognosis than the classical abdominal operation." Furthermore, Holland concluded that transverse fundal scars are especially liable to rupture, and Offerman, in 1916, collected 21 such accidents from the literature. In addition to this unusual liability to rupture, he feels that adhesions are especially prone to occur, and that in those cases in which infection of the uterine wound occurs, drainage will be into the abdominal cavity rather than through the abdominal wound, thereby materially decreasing the chances for recovery.

Fischer has described in detail two uteri which had been subjected to previous fundal incisions. In one a complete rupture of the scar occurred in a subsequent pregnancy, while in the other it was so thin that rupture would have undoubtedly occurred had labor not been terminated by operation. From this experience he is convinced that the fundal incision should not be used. Findley has gone even further, and says that " the transverse fundal, extraperitoneal, and cervical incisions have not lessened the liability of rupture in subsequent labors, but on the other hand have probably increased the hazard."

Since our experience with the extraperitoneal and cervical incisions has been limited to four cases, we do not feel quali


fied to make definite statements concerning their comparative merits. In none of our cases did rupture occur, although in one instance the woman subsequently went tlirough a moderately severe spontaneous labor. Moreover, in two of the uteri which were removed at a subsequent section, examination revealed no trace of the old scar. Kohrbach claims that the scar following the cervical operation practically insures against danger of rupture in future pregnancies, and it is undoubtedly true that only a few instances of this accident have been reported. It must, however, be admitted that this is too favorable a verdict, as Wolff, Franz, Freund and Labhardt have each described cases of rupture. Baisch, in 1920, reviewed the results following 170 Caesarean sections in the Stuttgart clinic, and attributed the good results to the emploj-ment ot the transperitoneal cervical operation. Twenty-four of hu patients had repeated sections, and in three rupture of the uterus occurred. In the first case it was vmquestionably through the old cervical scar; while in the other two he attempts to prove that it was not. In the second case, although the rupture involved the scar, he argues that it had originated in the body of the uterus, as at the first operation the incision had torn upward during extraction of the child so that only a part of it could be covered with the bladder. In the third case the patient had a bicomate uterus in which three pregnancies had occurred. The first was terminated by a transverse fundal incision, the second by a cervical incision, while the third terminated by rupture. On opening the abdominal cavity at that time he found that the cicatrix of the first operation had yielded, while the one in the lower segment was intact. He considers this an excellent demonstration of the superiority of the cen'ical operation. We, however, cannot agree with his contention; for, even accepting his argument concerning the second case, which appears questionable, he admits one cervical rupture in 24 repeated sections, which is somewhat higher than the 3 per cent incidence following the usual incision.

Before concluding the consideration of the location of the uterine incision, I shall refer to two cases of repeated Caesarean section wliich possess points of unusual interest, one reported by Planchu, the other by Harrar. In the former, the three linear scars were plainly visible in the anterior median line at the time the fourth consecutive section was performed. They were equidistant and approximately at the same level and were included in a broad band of thinning — 2.5 cm. wide, where the muscle had been replaced in large part by fibrous tissue. An explanation of the mode of production of such a condition is afllorded by Harrar's case. Here the uterus had ruptured after a third Caesarean section, and upon examining the specimen he found that the line of rupture did not involve an old incision but had occurred between two of them. As the distance between the scars was one centimeter, he inferred that a danger zone had been created at the previous operations by cutting the tropliic nerves and tliereby interfering with the blood supply in that neighborhood. To obviate such a danger he urges that in a repeated Cajsarean section the inci


March, 1922]


siou should be made some- distance away from the old scar, or else that the latter should be completely resected.

5. Incision into the Placental Site Writers upon the subject have advanced two reasons for assuming when the uterine incision involves the placental site that the resulting scar will, in some cases, be thinner than it would have been had the incision been elsewhere. First, as the thickness of the uterine wall at the placental site is several millimeters less than elsewhere, it is argued that the scar will be thinned in like proportion. Second, the foetal elements may interfere in the healing process. The latter view is held, in a general way, by Spalding, Fischer, and others. However, they, and practically all observers, believe that this is only an accessory factor of no great importance.

For the purpose of determining what relation, if any, incision through the placental site has upon the healing of the uterine wound, I have divided our cases into two groups. The first includes those in which the location of the placenta was noted at the first operation and in which the uterus was removed at a subsequent section and studied histologically; while the second includes patients who were delivered by the natural passages following a preceding section in our clinic. The former group includes 17 specimens, and the latter six cases. In the first group the incision involved the placental site in seven instances, and of these, two uteri showed no trace of the scar, in two there was very slight thinning, while in the remaining three the thinning was quite marked. In the 10 instances in which the placental site was not involved, no trace of the scar was visible in three, it was satisfactory in six, and was poor in one uterus. In the second group the placental site had been incised in two patients, and not in the other four. It may be added that in the single instance of rupture occurring in our series and reported below, the operative note concerning the first section failed to mention the location of the placenta.

From these figures the hasty observer might be led to the conclusion that a definite relation exists between imperfect healing of the scar and the involvement of the placental site at operation. Our observations do not prove it. and in addition it should be remembered that such few figures as are available do not justify binding conclusions. Consequently, we can only say that our findings merely point to the necessity of closer ol)servation of the influence of this factor in the future.

6. Implantation of the Placenta over the Old Scar Eckstein, in reporting his case of ruptured Cfesarean scar, laid particular emphasis upon the insertion of the placenta over the old scar as a probable cause of the rupture. He assumed that the foetal elements invaded and weakened the scar, much in the same way as occurs in tubal pregnancy. Spalding states that "while it is plausible that the placental elements might lead to poor healing of the wound in cases where the incision is into the placental site, there is hardly


sufficient evidence available to uphold the idea that the syncytium will attack sound scar tissue any more than it will attack sound uterine tissue." Consequently, he believes that the placenta, when inserted over the scar, may even act as a splint and serve to support the weakened uterine wall. At the same time he calls attention to the possibility that the formation of small retroplacental ha?matomata may eventually cause the rupture of the scar as they grow larger. In 1906, Couvelaire reported a series of nine cases of ruptured Csesarean scar and stated that in five of the six instances in which the placental attachment had been noted, implantation had occurred over the old scar. He therefore concluded that the insertion of the placenta over the scar cannot be considered the sole cause of rupture, but that when it is already thin its invasion by foetal elements must lead to further thinning and accentuation of its friability.

Our observations concerning the bearing of this factor indicate that it is not important. Strange to say, in each of the three uteri which had been removed after two previous sections the placenta at the third operation was implanted over the old scar. In the first case, both scars were seen and were quite thin; in the second, only one scar was visible and it, likewise, was considerably thinned ; while in the third, neither scar was visible. In 17 uteri which had been removed at the second operation, the placenta was implanted over the scar in three. In two of these the scar of the previous section could not be found, while it was well healed in the third uterus. Furthermore, in the one case of rupture the placenta was inserted over the old scar, but in none of these cases was there any evidence that the scar had been invaded by foetal elements.

7. Miscellaneous

(a) Excessive Distention of the Uterus. — In the cases of rupture reported by Woyer, Couvelaire, and Scheffzek, the presence of hydramnios or multiple pregnancy had subjected the uterus to abnormal distention, which was assumed by these authors to have been a predisposing factor. On the other hand, Gilles, in 1916, reported the successful termination of a twin pregnancy without rupture of the uterus, in spite of two previous Cajsarean sections. The combined weight of the two children was 4950 grams.

(b) Forcible Intrauterine Maniiiulations. — Version and extraction, the use of hydrostatic bags, uterine tampons, etc., have been mentioned as possible causes of rupture, although it is only in cases of marked thinning of the scar that such factors are of any importance.

(c) " Water-Wedge Theory." — Spalding, in attempting to explain the rupture of the scar in a certain percentage of cases, advanced the theory that a small water-wedge formed by the amniotic sac may serve to dilate the thinned-out scar in exactly the same way that the bag of waters brings about dilatation of the cervix in normal labor.

Before passing on to the consideration of the anatomy of the Caesarean scar, we shall refer to several investigations concerning its resistance, and then say a few words concerning the incidence of adhesions and their mode of production. In 1910,


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Mason and Williams studied the resistance of the Cassarean scar experimentally. They subjected a number of pregnant animals to Caesarean section and some time later removed the uterus for investigation. They attached weights to a strip of muscle containing the scar and increased them until rupture occurred, when it was found that it took place through the sound muscle and not through the scar itself. Their experiments gave such uniform results that they drew the conclusion " that a firmly united scar is even stronger than the intact uterine muscle and should easily be able to withstand any strain which the latter is capable of bearing." Moreover, the case reported by Harrar demonstrated that the normal muscle tissue may jaeld before the cicatrix, although the myometrium in that instance was regarded as having become " devitalized." As anatomical study of the well healed scar shows no reason why it should rupture before the surrounding muscle, it is probable that the statement of Mason and Williams holds true for a " firmly vmited scar." In the thin, poorly healed wound, on the other handj there can be little doubt that the rupture occurs directly through the scar and not through the adjacent tissue.

Adhesioxs

That adliesions are quite common following Caesarean section is clearly demonstrated by the following observations made in our clinic at repeated operations.



TABLE I






Atlhe


sions



Dense Filmy


Moticr- [ No ate 1 note



17 7 6 1


1 4


After two previous sections


(I



TABLE II






I'lierperium



Febrile


Afebrile


Dense adhesions


S

1







Xo note


1


Table II illustrates the fact that while adliesions are more commonly formed after a febrile puerperium, yet the absence of fever does not necessarily mean that adhesions have not formed.

It is difficult to determine just how much harm such adhesions do. Doubtless, they may give rise to considerable abdominal discomfort, which may occasionally be so great as to necessitate a second operation; and Humpstone reports an instance in which it was necessary to remove the uterus


before the symptoms disappeared. In other patients the adhesions may be so dense that the subsequent operation is performed under tremendous difficulties, and in rare instances regrettable accidents, such as injury to the gut or bladder, can be directly traced to their presence. For example, in Case XIX described below, after the adhesions had been freed, the anterior surface of the uterus presented such an extensive raw bleeding area that hysterectomy was necessary.

Can the formation of adhesions be prevented? Davis has suggested that the hability toward their development may be lessened by resorting to the high abdominal incision. This, however, is a mere supposition, and inasmuch as the advantages of the lower incision are so great, we do not feel that it should be discarded in favor of the higher one. Consequently, it seems that the only way at present by which we can lessen the incidence of adhesions in the absence of infection is by securing as perfect coaptation between the cut surfaces as possible, thereby leaving a minimal area which can excite mechanical irritation.

Anatomy of the Cesarean Section Scab For convenience in description Csesarean scars may be divided into two main groups: first, those which have ruptured or were on the point of rupturing when the abdomen was opened; second, those in wliich satisfactory union has persisted.

As Losee and also Couvelaire have pointed out, the first step in the mechanical healing of the uterine scar is the deposition of fibrin between the edges of the wound, wliich acts as a framework for the ingrowing tissue which will eventually determine the character of the cicatrix. In spite of the constant contraction and relaxation of the uterus, if infection is absent, and the sutures have been so laid that the edges of the incision remain approximated, firm union will almost invariably result. On the other hand, if there is necrosis due to infection, or if the sutures tear through, leaving a gaping wound, there is an ingrowth of endometrium, which may involve almost the entire thickness of the wound. Such a scar, when seen in a subsequent pregnancy, is made up almost entirely of decidua and peritoneum, and its gross appearance in the unopened distended iiterus is characteristic. Instead of the vertical shallow depression seen in the more perfectly healed scars, it is represented by a glistening translucent band of tissue which bulges outward beyond the general surface; while after the uterus has been emptied, hardened, and cut in ci'oss-section, the scar is marked by very deep depressions on both the outer and inner surfaces of the uterus. On microscopical examination it will be found that it is composed almost entirely of thickened and highly vascularized serosa overlying the decidua. There is an increase in the fibrous tissue and a definite decrease in the elastic fibres, while occasionally a few muscle fibres may be present.

Between a scar of this nature and one wliich has healed perfectly all gradations may be observed in so far as gross appearance and thickness are concerned. Nor is the microscopical picture markedly different in the thin scnr as con


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trasted with the well healed one, except when there has been no muscular union. Figs. 7, 8 and 10 depict the characteristic appearance of well healed scars, while Fig. 9 (A) shows a scar which is considerably thinner than the other three. In all there is a slight depression both upon the inner and outer surfaces of the uterus, while the cicatrix itself appears as a white Une joining the two depressions. Unless such scars are examined under the microscope, one is apt to infer that they are made up of fibrous tissue alone. Such, however, is not the case, and in all four specimens muscle fibres can be seen running directly across the old line of incision, with no break in their continuity and with only slight distortion of their course.

Jolly and others have reported instances of partial rupture of the old Cesarean scar. Upon studying such specimens in detail, they found that one portion of the scar was composed solely of decidua and peritoneum, while the rest was made up of normal muscle. Similar variations in thickness throughout the length of the same scar are well illustrated by Figs. 15 and 16, which are photographs from the two sides of a block one centimeter thick, and show that within so short a distance the thickness of the scar may be doubled.

Briefly then, Csesarean section scars vary considerably in thickness, depending upon the accuracy with which the margins of the wound have been brought into apposition. Under ideal conditions the muscle unites perfectly, and its fibres cross the site of the incision as if it had never been made. Wlien, however, there has been no muscle union, the scar is made up solely of decidua and peritoneum. Most scars are marked by funnel-shaped depressions on the outer and inner surfaces of tlie uterus. The muscle bundles are not much distorted, the formation of fibrous tissue is much less than would be anticipated, and when it is increased, the elastic fibres are correspondingly diminished in number. (See Fig. 7.)

Fig. 7, which represents a low power microscopic picture, gives an idea of the characteristic conditions obtaining in a wfeU healed wound, while the photographs accompanying some of the case reports clearly depict some of the gross variations which may be encountered.

In the case reports which immediately follow, we shall describe the gross appearance and microscopic structure of the Cesarean scar as it appeared in 21 uteri which were removed at a second or third section, and which will serve to amplify and reinforce what has already been said.

Case Eeports and Description of Specimens' In the following cases the uterus was either removed at operation or secured at autopsy. A brief clinical synopsis precedes the more detailed description of the specimen.

Case I.— House No. 1274, 24 years, black. Generally contracted rhachitic pelvis, D. C. 10.5 cm.

Three vaginal deliveries. Fourth labor ended by Cassarean section after a second stage of three hours. Child dead — 41S0 gm. Incision through the placental site; closure with interrupted silkworm-gut sutures. Puei-perium normal except for a rise of temperature to 100.4° on the second day. Fifth labor ended by Csesarean section followed by supravaginal amputation of the uterus. Uneventful recovery.


Omental adhesions, placenta on posterior wall. No note as to the condition of the cicatrix before the incision of the uterus.

Description of Specimen. — After hardening, the uterus measures 14x12x10 cm. On the midline of its anterior surface is a mass of adhesions, evidently over the scar of the previous operation. On removing them, a linear depression 8 cm. long becomes visible, with the silkworm-gut sutures still in place. (Fig. 6.) On cross-section through the uterus the scar is represented by depressions on its inner and outer surfaces, which are joined by an irregular whitish line (Fig. 8). The uterine wall is 11 mm. thick at the site of the scar, as compared with 17 mm. adjacent to it, a ratio of 1 to 1.54. On microscopical examination no trace of the scar is seen except for the funnel-shaped depressions and a slight irregularity in the arrangement of the muscle fibers. The external depression is filled with highly vascularized connective tissue, while the inner one is lined with decidua containing many glands. In the center of the scar is a small island of characteristic decidual tissue. The Weigert stain shows a decrease in the elastic tissue at the site of the scar.

Case II. — House No. 7145, 27 years, black. Generally contracted rhachitic pelvis, D. C. 8.75 cm.

First labor, spontaneous, premature; second, pubiotomy. The third and fourth pregnancies were ended by Csesarean section with silk sutures. Ideal recovery after both operations except for a mastitis in the fourth puerperium. The placenta was on the posterior wall in the first, and on the anterior wall in the second section. The fifth pregnancy ended in a 3 months' abortion, and the sixth was terminated by a Porro section. Puerperium normal except for a mastitis. There were dense adhesions between the uterus and anterior abdominal wall, but no traces of the cicatrices of the previous Caesarean sections were visible. The placenta lay anteriorly.

Description oj Specimen. — After hardening, the uterus measures 10 X 11 X 8 cm. Numerous dense adhesions are present over its anterior surface. On either side of the present incision is a vertical depression apparently corresponding to the scars of the previous operations. On cross-section, the scars are clearly visible and are represented by the typical funnel-shaped depressions on the outer and inner walls (Fig. 9). The scar on the left is 6 mm. thick, while the adjacent uterine wall is 23 mm., a ratio of 1 to 4. On the right side the scar is 11 mm. thick, a ratio of 1 to 2. On microscopical examination, except for the thinning of the uterine wall, there is little to indicate the location of the former incisions. There is no scar tissue and the muscle fibers run directly across from side to side. The inner depressions are lined with decidua, while the outer ones are filled with thickened scar-like peritoneum. The elastic fibers are decreased. It is impossible to say which scar belongs to the first and which to the second operation. One is almost twice as thick as the other, and the only factor noted at either operation that might have influenced the healing was the poor contraction of the uterus at the first section.

Case III. — House No. 6540, 22 years, black. Generally contracted rhachitic pelvis, D. C. 9.5 cm.

The first pregnancy ended by a conservative Caesarean section. The placenta lay posteriorly, the uterus contracted firmly, and the incision was closed with catgut. The puerperium was febrile for five days. The second labor was spontaneous premature at seven months. The third pregnancy was terminated by a Porro section, the operation being performed 19 hours after the onset of labor. There were Several dense adhesions between the uterus and the anterior abdominal wall; the placenta was inserted posteriorly; the puerperium was normal. The scar of the previous section was not noted at the time of the operation.

Description oj Specimen. — After hardening, the uterus measures 17x12x9 cm. The posterior surface is free except for a few velamentous adhesions. On its anterior surface the scar of the previous operation is indicated by a deep depression, from the lower angle of which a broad adhesion extends down to the free peritoneal margin. On cross-section (Fig. 10), the scar is represented by funnel-shaped


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depressions on both the outer and inner surfaces of the uterus. Joining them is a white puckered hne resembling scar tissue. The uterine wall measures 28 mm. in thickness, while the scar is 22 mm. thick, a ratio of 1 to 1.36. On microscopical examination the only trace of scar consists in a slight irregularity in the arrangement of the muscle fibers and some increase in the fibrous tissue. The elastic tissue is decreased.

Case IV.— House No. 6S47, 24 years, black. Generally contracted rhachitic pelvic, D. C. 9.25 cm.

First pregnancy ended by a conservative Cffisarean section. The placental site was incised, the uterus contracted firmly, and closure was with catgut. Puerperium uncomplicated.

Second pregnancy ended by a Porro section. Two broad adhesions attached the anterior surface of the uterus to the abdominal wall. The placenta lay posteriorly. No sign of the old uterine scar. Puerperium normal.

Description oj Specimen. — After hardening, the uterus measures 19 X 13 X 10 cm. Two broad bands of adhesions on the anterior surface. Careful examination, grossly and microscopically, shows no trace of the old scar. '^

Case V. — House No. 6939, 18 years, black. Generally contracted rhachitic pelvis, D. C. 1025 cm.

First pregnancy • ended by a conservative Csesarean section. Placental site not incised; closure with catgut. The character of the uterine contraction not recorded. The puerperium was complicated by wound infection, the temperature being elevated for 15 days. The second pregnane}' was terminated by a Porro Csesarean section. In attempts to extract the child through too small an incision, the uterus was torn down to the bladder and into the right broad ligament, necessitating its removal. The placenta was inserted posteriorly. The puerperium was normal except for a simple mastitis.

Description oj Specimen. — After hardening, the uterus measures 15 X 12 X 10 cm. The surfaces are free from adhesions and there is no trace of the old scar.

Case VI. — House No. 6076, 23 years, white. Generally contracted rhachitic pelvis, D. C. 10 cm.

The history of the first two labors is meagre. Both were instrumental and both children were stillborn. The third labor was terminated by a conservative Csesarean section. The placental site was not involved, the uterine contraction was poor and closure was with catgut. Puerperium febrile for six days. Fourth labor ended by a Porro section. There were a few filmy adhesions on the anterior surface of the uterus. The placenta lay posteriorly. Puerperium normal.

Description of Specimen. — After hardening, the uterus measures

14 X 14 X 8 cm. A few thin adhesions cover its anterior surface, in the midline of which is a smooth shallow depression indicating the site of the previous operation. On cross-section, except for this depression and a similar one on the inner surface, there is no trace of the scar. The uterine wall measures 26 mm. in thickness, the scar 23 mm., a ratio of 1 to 1.13. Microscopically there is no sign of scar tissue, and the muscle bundles show complete regeneration.

Case VII. — House No. 8087, 29 years, black. Generally contracted rhachitic pelvis, D. C. 8.5 cm.

The first and second labors were ended by a conservative Cesarean section and a destructive operation, respectively, both operations being performed elsewhere. The third pregnancy was terminated by a Porro section. Several loops of gut were densely adherent to the anterior abdominal wall and anterior surface of the uterus. The placenta was inserted posteriorly. Puerperium normal.

Description of Specirnen.—Aiter hardening, the uterus measures

15 X 16x7 cm. On the anterior surface is a depressed raw area, representing that portion which was covered by adhesions, the latter overlying the scar of the previous operation. On cross-section, the scar is represented by shallow depressions on the outer and inner surfaces of


the uterus. The thickness of the scar is 19 mm., that of the adjacent wall, 25 mm., a ratio of 1 to 1.3. Microscopically, the inner depression is seen to be filled with decidua, while the outer is fined with vascular serosa. The muscle fibers run directly across the line of the former incision without break in continuity. There is no increase in fibrous tissue.

Case VIII. — House No. 8137, 31 years, black. Generally contracted rhachitic pelvis, D. C. 9.5 cm.

The first pregnancy was terminated by a conservative Csesarean section. The placental site was incised, the uterus contracted firmly; closure with catgut. Puerperium normal. Second labor ended by a destructive operation elsewhere. Third labor ended by a Porro section. There were a few thin omental adhesions on the anterior surface of the uterus, but no sign of the previous scar. Placenta inserted posteriorly. Puerperium normal.

Description of Specimen. — After hardening, the uterus measures 16 x 14 X 6.5 cm. Upon the anterior wall, half a centimeter lateral from the present incision is a slight vertical depression, 2 mm. deep, and 4 cm. in length. On cross-section, the usual funnel-shaped depressions are seen on the outer and inner surfaces. The scar measures 18 mm. in thickness, the adjacent uterine wall, 22 mm., a ratio of 1 to 12. Microscopically, no trace of the scar can be found except for the depressions just mentioned. There is no increase in fibrous tissue.

Case IX. — House No. 8332, 21 years, black. Generally contracted rhachitic pelvis, D. C. 9.5 cm.

The fii-st pregnane}' was terminated by a conservative Csesarean section. The site of the placental attachment was not recorded; uterine contraction poor; closure with catgut. Puerperium normal. A conservative Csesarean section ended the second pregnancy. There were numerous filmy adhesions near the fundus, while lower down was a single broad adhesion running to the old abdominal scar. The placental site was incised, the uterus contracted firmly, and closure was with catgut. The scar of the previous operation was not visible. The puerperium was complicated by infection. The abdominal wound broke down on the sixth day, and a sinus developed which extended well down into the uterus. A pelvic abscess was opened and drained through the vagina on the seventeenth day. The third pregnancy was terminated by a Porro section. Dense adhesions covered the entire anterior siirface of the uterus. The placenta lay anteriorly. Puerperium normal.

Description of Specimen. — After hardening, the uterus measures 15 X 13 X 6 cm. The present incision lies to the left of the midline. The entire anterior surface presents a ragged appearance due to adhesions. In the midline the scar of one of the previous operations is marked by a slight depression. On cross-section there is a similar, though much deeper, depression on the inner surface, but there is no trace of a second scar. The scar is 20 mm. in thickness; of this, 4 mm. is made up of fibrous peritoneal tissue, 4 mm. of muscle, and 12 mm. of a triangular wedge of decidua. The adjacent uterine wall measures 22 mm. On microscopical examination it is seen that, although there has been some muscle reunion, the individual bundles are greatly distorted, with a marked increase in the fibrous tissue in its locality. Presumably this scar is the result of the second operation.

Case X. — House No. 8679, 24 years, black. Generally contracted rhachitic pelvis, D. C. 1025 cm.

The first pregnancy was terminated by a conservative Cssarean section elsewhere. Three years later the patient was admitted to the surgical service of this hospital with the following historv-: Fifteen months after the Csesarean section the abdominal wound began to drain, the discharge consisting mainly of blood, but occasionally containing pus. A sinus tract, which extended down into the uterine cavity, was dissected out, and a number of hea\'>- black silk sutures were removed from the uterine wall. Recovery was uneventful. Sixteen months later the second pregnancy was terminated by a Porro section. A few thin omental adhesions were attached to the anterior


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surface of the uterus. Placenta posterior. Puerperium normal except for a simple mastitis. ,

Description of Specimen. — After hardening, the uterus measures 13 X 10 X 7 em. To the left of the present incision is a slightly depressed area 2 mm. wide, and 7 cm. long, which apparently represents the old scar. On cross-section, however, there is no depression on the inner surface of the uterus, and microscopically no trace of the scar can be found.

Case XI. — House No. SS26, 24 years, black. Generally contracted rhachitic pelvis, D. C. 9.75 cm.

The first labor ended spontaneously after 30 hours, the child being stillborn. The second pregnancy was terminated by a conservative Caesarean section. The placental site was incised, the character of the uterine contraction was not recorded; closure with catgut. Temperature elevated to 101.4° on the second day. The third pregnancy was also ended by a conservative Caesarean section. Several loops of small intestine were adherent to the abdominal scar and one of them was accidentally opened. It was repaired at once with silk. The omentum was adherent to the fundus. The placental site was incised, the character of the uterine contraction was not recorded; closure with catgut. Puerperium normal. The fourth labor was terminated by a third Caesarean section and sterilization effected by resection of the tubes. Again an adherent loop of gut was opened, and was repaired with silk. A few omental adhesions covered the fundus. The placental site was incised, the uterus contracted well; closure with catgut.

On the third day of the puerperium the abdominal wound broke down, exposing the anterior surface of the uterus. This was covered with small glistening blebs filled with gas. A drain was inserted and the wound closed with silkworm-gut. Cultures of the wound showed B. aerogenes capsulatus, B. coli, Staphylococcus aureus, and streptococci. The patient died 12 hours later and autopsy showed a generalized peritonitis (gas bacillus) ; haemorrhage from the uterine incision; diphtheritic colitis; epithelial necrosis of the kidneys.

Description oj Specimen. — After hardening, the uterus measures 17x12x6 cm. On the anterior surface is a depressed area, more or less covered by fine adhesions, corresponding to the partially broken down wound of the recent operation. Several of the catgut sutures have become untied, and for a distance of 2 cm. at the upper angle of the incision there has been complete separation of its edges. On cross-section (Fig. 11), it is seen that the cut surfaces are covered by necrotic tissue and at no place is there any sign of firm union. The scars of the previous operations are not visible. Microscopical examination shows that the decidua has been replaced, in large part, by fibrin, leucocytes, and necrotic tissue. The musculature presents a pecuhar areolated appearance due to the formation of gas pockets. In many places the individual muscle cells are undergoing degeneration. There is no evidence of beginning muscular union.

Case XII. — House No. 9115, 19 years, black. Pelvis normal.

The first pregnacy was terminated elsewhere by a Cxsarean section, the indication being " convulsions." The patient was admitted to this hospital in the eighth month of her second pregnancy. Blood pressure 180; the urine contained 3 gm. of albumin per hter. Labor was induced by means of a bougie. After pains had lasted for 12 hours, the patient had her first convulsion, followed in a few minutes by a second. As the cervix was still undilated and the child dead, it was decided to remove the uterus unopened. The omentum was adherent to the anterior surface of the uterus. Puerperium febrile.

Description oj Specimen. — After hardening, the uterus measures 22x16x13 cm. On the anterior surface are a few thin adhesions. No trace of the old scar. On crosa-section the placenta is found to lie anteriorly. There is no sign of the usual depressions which so frequently represent the hne of former incision. Microscopical examination shows no increase in fibrous tissue. The Weigert stain shows the presence of large numbers of streptococci in the decidua, thus


demonstrating the wisdom of removing the uterus, rather than being content with a conservative operation.

Case XIIL— House No. 9323, 33 years, black. Generally contracted rhachitic pelvis, D. C. 9 cm.

The first pregnancy ended in a spontaneous abortion at the third month, the second in a conser\'ative Caesarean section. The placenta lay posteriorly; character of the uterine contractions not recorded; closure with catgut. The puerperium was febrile for the first three days. The third pregnancy was terminated by a Poito section, the vulva being so completely covered by a foul, sloughing mass of condylomata that a conservative operation would have almost certainl}' been attended by infection. There were no adhesions, and the placenta lay beneath the old scar. Puerperium normal.

Description oj Specimen. — After hardening, the uterus measures 15x11x6 cm. Extending from the fundus downward for a distance of 10 cm., and with its lower half slightly to the right of the present incision, is a vertical depression, representing the scar of the previous operation. It averages 1.5 cm. in width, and its deepest portion lies 0.5 cm. beneath the general surface (Fig. 11). It presents a number of transverse depressions, w-hich are apparently due to the individual sutures. On cross-section (Fig. 12) the scar is marked by typical depressions on both surfaces, whose tips are 16 mm. apart, as compared with 25 mm. in the adjacent uterine wall, a ratio of 1 to 1.59. On microscopical examination there is considerable distortion of the individual muscle bundles, although they definitely run from side to side without break. There is no increase in the fibrous tissue.

Case XIV. — House No. 9539, 24 years, black. Generally contracted rhachitic pelvic, D. C. 8 cm.

First pregnancy terminated by conservative Caesarean section elsewhere. She was admitted in her second pregnancy, after having been in labor for 30 hours with numerous vaginal examinations. A Porro section was done with difficulty on account of dense adhesions between the lower angle of the abdominal scar and the anterior surface of the uterus. The scar of the previous Caesarean was not visible before the uterus was opened. The placenta lay posteriorly. Puerperium febrile.

Description oj Specimen. — After hardening, the uterus measures 15 X 12 X 8 cm. Externally the scar is represented by a very slight depression along the upper third of the anterior wall. On cross-section (Fig. 13) the uterine cavity presents a triangular appearance, the base being formed by the posterior wall and the apex by the fimnel-shaped depression on the inner surface of the anterior wall. Upon microscopical examination no trace of scar tissue can be found and the muscle fibers at the site of the scar are only very slightly disarranged.

Case XV. — House No. 9530, 31 years, white. Simple flat pelvis, D. C. 10.75 cm.

In the first pregnancy the child was delivered by pubiotomy after a second stage of 14 hours, the D. C. at that time measuring 9.75 cm. The second pregnancy was terminated by a conservative Caesarean section with an uneventful recovery. Incision into the placental site ; character of uterine contraction not recorded; closure with catgut. The third pregnancy was ended by supravaginal amputation of the uterus. There were several large adhesions extending from the old abdominal scar to the anterior surface of the uterus. Placenta posterior; puerperium normal.

Description oj Specimen. — After hardening, the uterus measures 17 X 13 X 7 cm. There was no trace of the old Caesarean scar either on gross or on microscopical examination.

Case XVI.— House No. 10243, 21 years, black. Flat rhachitic pelvis, D. C. 10 cm.

First pregnancy terminated by a conservative Caesarean section. The placenta lay posteriorly ; character of the uterine contractions not recorded; closure with catgut. Puerperium febrile for two days. In her second pregnancy the patient was admitted to the hospital after having been in labor 15 hours with numerous vaginal examinations. Caesarean section with supravaginal amputation of the uterus, followed


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by a febrile puerperium for five days. The placenta was posterior. There was a single broad adhesion attaching the omentum to the anterior surface of the uterus.

Description oj Specimen. — After hardening, the uterus measures 14x11x7 cm. In its contracted condition a number of transverse markings are visible on the anterior surface, apparently corresponding with the sutures of the former operation. On cross-section there is no trace of the scar, except immediately adjoining the uterine cavity where there is an infolding, 7 cm. in length, perhaps indicating that union had been faulty in that location. Microscopical examination shows no trace of scar tissue.

Case XVIL— House No. 10792, 28 years, black. Generally contracted rhachitic pelvis, D. C. 9 cm.

The first two pregnancies were terminated by Cesarean sections, the first being performed elsewhere. At the second the utei-us was found loosely adherent to the anterior abdominal wall. The placenta was inserted on the anterior wall; the uterus contracted slowly, and closure was with catgut. Puerperium febrile for seven days. The third pregnancy was ended by a Porro section. Broad adhesions extended from the abdominal wall to the anterior surface of the uterus. Placenta posterior; puei-perium normal.

Description oj Specimen. — After hardening, the uterus measures 16x11x5.5 cm. The entire anterior surface presents a raw surface which was covered by adhesions at the time of operation. A single scar is visible as a shght linear depression not more than 1 mm. in depth on the external surface, while on the inner surface there is a deeper depression, 7 cm. in length (Fig. 14). Cross-sections made at various levels reveal considerable variation in the thickness of the scar (Figs. 15 and 16). On microscopical examination there is a sHght increase in the fibrous tissue, but the musculature has regenerated completely and there is no distortion of the individual fibers.

Case XVIIL— House No. lOSOS, 25 years, black. Generally contracted rhachitic pelvis, D. C. 10.5 cm.

First pregnancy ended by an extraperitoneal Csesarean section, after 24 hours of labor. Incision closed with catgut. There was a wound infection and the puerperium was febrile for six days. Second pregnancy terminated by a conservative Caesarean section. Placenta posterior; uterine contraction fair; closure with catgut. The puerperium was febrile for seven days. The third pregnancy was ended by a Porro section. There were dense adhesions between the abdominal wall and uterus and also about the site of the extraperitoneal section. The placenta lay posteriorly; puerperium normal.

Description oj Specimen. — After hardening, the uterus measures 15x12x9 cm. To the left of the present incision is the scar of the second operation. It is marked by a shallow depression on the outer surface and a much deeper one on the inner surface. The scar measures 25 mm. as compared with a thickness of 35 mm. in the adjacent uterine wall, a ratio of 1 to 1.4. On microscopical examination there is complete regeneration of the musculature at the site of the scar, but there is also a considerable increase in the fibrous tissue. There is no trace of the scar of the extraperitoneal operation.

Case XIX. — House No. 10758, 26 years, black. Generally contracted rhachitic pelvis, D. C. 10 cm.

First pregnancy terminated by a consei'V'ative Csesarean section elsewhere. The patient was in bed with a discharging wound for two months following it. At the second section done at the onset of labor. the lower two-thirds of the uterus was broadly adherent to the old abdominal scar, and after the adhesions had been released such an extensive raw bleeding area was left that it was thought best to remove the uterus. The placenta was inserted on the posterior wall; puerperium normal.

Description oj Specimen. — After hardening, the uterus measures 15 X 12 X 6 cm. On the anterior surface is the ragged area referred to above. On cross-section, the scar of the previous operation is visible as an irregular whitish line joining funnel-shaped depressions on the outer and inner surfaces (Fig. 17). On microscopical examination


there is little to mark the hne of any former incision, the muscle fibers running from side to side without break in continuity and with only very slight increase in the fibrous tissue.

Case XX. — House No. 11047, 19 years, black. Generally contracted rhachitic pelvis, D. C. 8.75 cm.

The first pregnancy was ended by a conservative Caesarean section at the onset of labor. The placental site was incised, the uterus contracted firmly, and closure was with catgut. Puerperium normal except for a rise of temperature to 101° on the second day. In second pregnancy the patient was admitted 36 hours after the onset of labor with intrapartum infection. A Porro section was performed with eventual recovery after a stormy convalescence. Dense adhesions bound the anterior surface of the uterus to the abdominal wall. Placenta posterior.

Description oj Specimen. — After hardening, the uterus measures 15x12x8.5 cm. In the midline is a slight longitudinal depression which corresponds with the scar of the previous operation. Crosssections show almost perfect healing. The thickness of the scar is 21 mm., that of the adjacent uterine wall 29 mm., a ratio of 1 to 1.4. Microscopical examination shows normal muscle nmning directly across the line of former incision, with no increase in fibrous tissue.

Ruptured Cesarean Scar

The foUovdng is tlie clinical historj' and the description of the amputated uterus from the single instance in our series in which the scar of a previous Csesarean section ruptured. Figs. 18 and 19 show the anterior surface and cross-section of the uterus.

Case XXI. — Hou.se No. 7570, 24 years, black. Generally contracted rhachitic pelvis, D. C. 9.5 cm.

The first pregnancy was terminated by a prolonged spontaneous premature labor. The second was ended by a Csesarean section, seven hours after the onset of labor. The uterus retracted poorly, and the incision was closed with two layers of chromic catgut. The puerperium was febrile for 10 days.

The third pregnancy progressed normally to the seventh month. On November 29, 1915, the patient complained of much abdommal pain, and was seen in her home by the out-patient service. As she was not in labor, and as the result of the examination was unsatisfactory, she was instructed to report to the dispensary on the following afternoon. This she did, walking a distance of six or eight blocks each way. She returned the next day with slight vaginal bleeding and was admitted to the hospital. On examination the cervix admitted one finger easily, while just above the internal os is a firm, rounded tumor apparently projected from the posterior wall of the uterus into its cavity, so that a tentative diagnosis of myoma was made. The abdomen was shghtly distended but not sensitive, and it was impossible to map out the foetus or to hear the foetal heart. Temperature normal, pulse 100.

Under the supposition that we had to deal with a myoma, which would interfere with the birth of the dead child, laparotomy was decided upon. When the abdomen was opened with a median incision, a small amount of bloody fluid escaped and the dead fcetus surrounded by the placenta and membranes lay free in the abdotninal cavity. The uterus was tightly contracted, ft'ith a large, jagged, irregular, blood-stained opening occupying its anterior wall. There was no bleeding, and what had appeared to be a myoma on vaginal examination was found to be the firmly contracted posterior wall of the utenis. The organ was then removed by supravaginal amputation, and the patient made an ideal recovery.

The interesting points in connection with the case are :

(a) the impossibility of determining when the rupture occurred ;

(b) the trifling clinical signs connected with it;

(c) the difficulty of diagnosis;

(d) the absence of serious haemorrhage, and

(e) the total absence of shock both before and after the operation.


Maecii, 192-3]


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Description of Specimen. — The uterus, after hardening, measures 10xSx6 cm. On its anterior surface, just to the left of the midhne, is a jagged, irregular blood-stained opening 6 cm. in length. There are practically no adhesions. Upon cross-section, it is seen that the muscle had failed to unite after the first section, as is shown by the fact that the edges of the opening are smooth and show no sign of recent tear. Evidently the scar had been composed only of decidua and peritoneum, which had yielded when the uterine distention had become pronounced. Microscopical examination bears out this supposition, as the decidua is found to cover the entire inner surface of the rupture, and to extend up to the peritoneum, which is quite thick and scar-like. Nowhere is there any suggestion that muscle union had occurred. The placenta had been implanted anteriorly over the scar.

Unfortunately, in a small number of our repeated Csesarean sections, which were followed by removal of the uterus, the specimen was lost or mislabeled. Likewise, in a number of instances the patient was sterilized at the subsequent section by resection of the Fallopian tubes, and consequently anatomical studies of the scar could not be made. The main


interest in such cases lies in the fact that they serve to demonstrate that the uterine scar was able to withstand the strain incident to a second or third pregnancy. Tables III and IV have been so arranged as to give the salient points in each case Avithout making a detailed report necessary.

Delivery by the Natural Passages, Following CESAREAN Section In 1904, Von Leuwen was able to find in the literature 32 instances in which a previous Caesarean section had been followed by deLivei7 through the natural passages, and since then Brodhead, J. T. WilHams, Willson, Harrar, Davis, Breitstein, Mason, Himipstone and others have reported similar cases. Study of their material shows that pelvic dystocia had only occasionally afforded the indication for the original operation, but that it had generally been undertaken for some such temporary complication as eclampsia or placenta praevia. Naturally, this is what one would expect, for had the pelvic contrac


TABLE III

Outline of the Clinical History of Patients Whose Uterus Was Lost, or of Patients Sterilized at Second

Section by Eesection of the Fallopian Tubes


Case


First operation


Second operation



number


Indication


Placental site


Closure


Uterine contraction


Puerperium


Placental site


Adhesions


Puerperium


Remarks


1530 1548


Pelvic di'stocia.


Posterior. Anterior. Posterior.


Deep silk superficial catgut.

Deep silk superficial catgut.

Catgut.

Catgut.


Not recorded. Not recorded. Firm.

Not recorded.


Febrile.


Not recorded.

Anterior.

-interior. Posterior.


Moderate. Not recorded. Dense.


Febrile.


Uterus removed but lost. No record of scar. Excessive bleeding. Patient died on table.

Death on Sth day, infection. Uterus secured

at autopsy but losi. First operation in Pittsburgh. No admissions

after 2d section. No subsequent admissions. No note of scar.


1611


Febrile. Afebrile, Febrile.


6787


Anterior.


Febrile.


8243



Not recorded.



Not recorded.


Afebrile.



Moderate.


Afebrile.


Tubal sterilization. Old scar not seen.


8303


"


Anterior.



Firm.




Dense.



Tubal sterilization. No mention of old scar.


85.37



Anterior.


1 Not recorded.



Anterior.


Not recorded.



Tubal sterilization. No mention of old scar.


8666


"


Posterior.


I Poor.



Posterior


Filmy.



Tubal sterilization. Old scar not seen.


916D



"



Febrile.



Film.v.

Not recorded.

Filmy


Febrile.


First operation extraperitoneal. No subsequent admissions.

First operation done elsewhere. Tubal sterilization. Old scar-depressed line 5 cm. long.

No subsequent admissions. Old scar not



9647


Posterior.


Catgut. Not recorded.


Afebrile.





1

Catgut. Poor.




Dense. Filmy.


Afebrile. Afebrile.


First operation in Washington, D. C. No

subsequent admissions. Old scar not seen.

No subsequent admissions. Old scar not


10212


Posterior.


Febrile.


1()?79 10631


"


Anterior. Posterior.


Good. Poor.


Febrile. Afebrile.


Anterior.


Dense. Dense.


Febrile. Afebrile.


No subsequent admissions. Old sear visible

as thin depressed line. No subsequent admissions. Old scar not


TABLE IV

Outline of Two Cases in Which Two Cesarean Sections Preceded the One in Which Tubal Sterilization

Was Effected


1


First operation


Second operation


Third operation


q O


Indication


Placental site


Closure


Uterine contraction


Puerperium


Placental site


Uterine contraction


Closure


Adhesions


Puerperium


Placental .^^hesions


[ Remarks Puerperium



Pelvic dystocia.

Pelvic dystocia.






Anterior. Posterior.


Firm.

Not recorded.


Catgut. Catgut.


Dense.

Filmy.


Afebrile. Febrile.


P t 1 ^-1


Afebrile. {First operation else where-extraperitoneal.

Tubal sterilization. Febrile. Tubal sterilization. One

of old scars visible as

depressed line.


9254


Anterior.


Catgut.


Not re- Febrile, corded.;


Anterior.


Dense.


104


[Xo. 373


tion been sufficiently pronounced to demand a CEesarean section at the first labor, those subsequent to it would not be likely to terminate spontaneously. Occasionally, however, even in such cases, the patient may not realize the gravity of her condition, and may fail to summon medical aid until labor has been in progress for a number of hours, when examination may show that the head has become sufficiently molded to permit its descent into tlie pelvis, and the labor may terminate spontaneously, or at most by a simple forceps delivery. Doubtless had most of these women been seen before the onset of labor, they would have been subjected to a repeated section, for in the presence of a definite degree of disproportion we should not allow a vigorous test of labor, although, as has already been pointed out, if the disproportion is slight, we may allow labor to progress under careful observation.

The points of interest in our 13 cases of delivery by the natural passages following a Csesarean section are outlined in Table V. It will be noted that in 11 there was a varying degree of pelvic contraction, although in three it had not been sufficiently pronounced to furnish the indication for the previous


section. Moreover, in Cases 5959, 6927, and 8572 the child was so premature that the question of pelvic dystocia did not enter into consideration at the subsequent labor. They are of interest, however, from the fact that active labor pains persisted for 18, 15, 7.75 hours, respectively, and thus adduced additional proof of the ability of the well healed scar to withstand the strain of a moderately severe labor.

As 48 of our patients were subjected to one or more repeated Csesarean sections, the 13 cases just mentioned, to which may be added four other deliveries elsewhere, indicate that 25 per cent of the women who have had a previous section possess a uterine scar sufficiently strong to withstand the strain of delivery through the natural passages. Naturally, this does not represent the entire truth, as it must be supposed that many of the uteri which were incised at the second or third section would have proven equally as strong had the disproportion not been so marked as to contraindicate any test of labor.

The gross mortality attending tlie repeated sections was 5.9 per cent. The death of one patient from haemorrhage was due to tlie inexperience of" the operator ; but the other two


TABLE V

Outline of the Clinical History of 13 Cases Delivered Through the Natural Passages Following a Previous

C.issAREAN Section


Name

Number

Color


Obstetrical history

previous

to operation


Operation


Placental Closure of


Place I«<'--»™iattachSent ^'^r."' Pu"P"ium


Labor


1899. Podalic ver- 1901.

sion. Johns

Hopkins. 1908. Abortion-4, 1909.

months. j (.'leveland.

1911.

Johns Hopkins.


1913,

Johns Hopkins.

Four labors. AUI '913. spont. at term. Chicago.

1914.

Johns Hopkins.

Two abortions. 1912. Spontaneous. One Johns tubal abortion. ; Hopkins.

1913.

, Philadel1 phia. 1909. Spont. Still- 1915.

born. Johns

'1910. Spont. Seven Hopkins.

mos. Stillborn. 1911. Spont. Term.

d. 6 weeks. 1914. Spont. Seven mos. Stillborn.

1917.

Washington.

1916.

Johns Hopkins.

1917.

Church Home, Baltimore. 1917. Spontaneous 1919. at term. Difficult .Tohns labor. Child died Hopkir from intracraniali hcemorrhage. '


Eclampsia rigid cervix.


Eclampsia rigid cervix.

Eclampsia,


2 layers.

1, Deep s

2. Chr. c


Posterior 2 lavers chr. «all. c. g.


Posterior 2 layers chr.


Febrile. Febrile.


,,G. C. funnel. 'D. C. 11 cms.

T. T. 8 cms.

Simple flat.

D. 0. 101 cms.


G. C. funnel. D. C. ll-i cms. T. I. 7} cms. G C. typical. ^. 11 cms.


7i hrs, 6Jhrs. 18 hrs.


Podalic ver- 3335 gm.

sion. 2dstage B. p. 10 cm.

2] hrs. 1

Low forceps. ;3149 gm.

2d stage 1 hr. JB. p. 9J cm.

Spontaneous. ,1400 gm. Premature. jB, p. 73 cm.


1 Manual dil. of 2080 gm. cervix from B. p. 8 cm. 5 cm. Mid j forceps. Spontaneous. 12780 gm.

B. p. 83 cm.


Spontaneous. 1980 gm.

Frank breech. B. p. — ?

Premature. Mid forceps. 2620 gm.

2d stage l-J ,B. p. Sj cm.

hrs.


Low forceps. S230 gm.

2d stage 11 |B. p. SJ cm.

hrs. Induced- 12450 gm.

Bougie. B. p. Si cm.

Convulsions.

Spontaneous. Spontaneous. 2750 gm.


In 1905 delivered in another hospital by forceps.


Uterus removed after Cesarean in 1914. Scar— Excellent. Wound healed per' primam. Fever six days.

Wound healed per


Wound prtmaii days.


Extra peritoneal Cesarean. In 1916 spont. at 7 months. Private doctor. Fever


Patient said she had

'* milk-leg." In bed

man.v weeks. Wound healed per

primam. Fever for

eight days. In 1919 spontaneous at


Wound healed per primam. Fever for nine days.


THE JOHNS HOPKINS HOSPITAL BULLETIN, MARCH, 1922



Fig. 1. — Cioss-SLctiuu of utoriis removed at autopsy from patient dying from peritonitis (gas bacillus) on third day after operation. Case XI. (A) Site of recent incision. XVi.



Fig. 6. — Scar from Case I, showing original silkworm gut sutures in situ at second section. X 1


\


Figs. 2 and 3. — Illustrating the two layers of sutures employed in closing the uterine incision. (A) Buried layer. (B) Superficial layer.



Figs. 4 and 5.— Illustrating a possible cause of imperfect scars. In 4 the sutures have been tightly tied in a relaxed musculature. In 5 contraction has taken place with resulting indentations on both uterine surfaces.


Fig. 7, — Drawing of cross-section of a comparatively well healed Csesarean scar. It demonstrates. the typical depressions on both surfaces of the uterus and the regenerated muscle fibres, with almost no fibrous tissue. X 6.


THE JOHNS HOPKINS HOSPITAL BULLETIN. MARCH. 1922


PLATE XIII


,.J^Pr^


r

K


^aJlsi^"i^


Fig. 8. — Cross-section of uterus from Case I, X yi- (A' recent incision. (B) Dense adhesions covering the old scar (C).



Fig. 9. — Croiss-section of uterus from Case II. (A) Old scar. (B) Recent incision. (C) Old scar. X Vi



Fig. 11. — Uterus from Case XIII, showing (A) old scar with transverse indentations, and (6) recent incision. X Vi


Fic. 12. — Cross-si ition of utiM\is irom Case XIII. (A) Old scar. X -A



FiG. 10. — Cross-section of uterus from C;i.s(' III. (A) Recent incision. (B) Old scar. X :'.;•


Fig. 13. — Cro.<.-i-section of uterus from Case XR (A) Old scar. X yi


THE JOHNS HOPKINS HOSPITAL BULLETIN. MARCH, 1922


PLATE XIV


A



Fig. 14.— Inner surface of uterus from Case X\I1. (A) nuJ (li) upper ;md luwer angles of old. scar. X Vs





Fig. 17. — Cross-section oi uterus nom Case XIX. (A) Recent incision. (B) Old scar. X -A


Fig. 15.


V ^~j-) i\r


Figs. 15 and 16.— Opposite sides of block 1 cm. in thickness from uterus in Case XVII. Shows variation in diameter of scar at various levels. X 1


Fig. is. — Uterus from Case XXI, showing the rupture of old Caesarean scar. X 1


Fig. 19. — Cross-section of uterus from Case XXI. Note the smooth edges of the muscle at the site' of rupture. X Vs'


March, 1923]


105


fatalities were directly attributable to the repeated abdominal operation. Our 48 •n-omen went through 63 pregnancies, 13 of which ended by vaginal delivery under our supervision, and 4 elsewhere, with only one instance of rupture of the uterine scar- — an incidence of 1 to 63 or 1.5 per cent. It might be argued that this unusually low incidence is due to the fact that We are accustomed to sterilize our patients at the third operation, instead of doing five or six repeated Caesarean sections, as is the practice of some obstetricians. Doubtless this may play some part in reducing the incidence of ruptured scars, as there can be no question that the greater the number of Caesarean sections done upon one woman, the greater will be the number of imperfect scars. Consequently, we feel that, after a patient has been subjected to the risk of three major operations, she should be relieved of the added danger of a ruptured scar, which must inevitably become increased after each subsequent operation.

COXCLUSIOXS

1. The weak Caesarean scar may be due to a single factor or to a combination of factors, the most important of which is infection.

2. An afebrile puerperium does not give an absolute assurance of perfect wound healing.

3. The perfection of technique in suturing the uterine incision will undoubtedly lessen the incidence of weak scars.

4. Chromic catgut, in our hands, has proved to be a satisfactory suture material.

5. The uterine wound should not be closed, if possible, until firm contraction of the musculature has occurred.

6. As a rule foetal elements do not invade the uterine scar.

7. Adhesions follo'wing Caesarean section are common. They are not necessarily the result of coexisting infection, and may give rise to serious complications at subsequent operations.

8. The dictmn " once a Cfesarean, always a Caesarean " cannot be accepted without considerable reservation.

9. A patient who has once been subjected to a Cffisarean section should enter the hospital several weeks prior to the expected date of confinement, so that she may have the benefit of immediate operation should rupture occur.

REFERENCES

Baisch : Die Gefahren der Kaiserschnittsnarbe und ihre Verhiitung. Monatsschnft f. Geb. u. Gyn., 1920, LIII, 57-70.

Bell, J. X.: Rupture of the Uterus in Caesareanized Women, With a Review of the Literature on This Subject to Date. Amer. Jour. Obst., 1916, LXXR', 950-954.

Breitstein, L. 1.: Rupture of the Uterus Following Caesarean Section. Jour. Amer. Med. Assn., 1914, LXIl, 6S9-691.

Brodhead, G. L.: Normal Labor Following Caesarean Section. Amer. Jour. Obst., 1917, LXXV, 702-703.

Carstens. J. H.: Abstract of Discussion — " Caesarean Section Scars " —Spalding. Jour. Amer. Med. Assn., 1917, LXIX, 1854.

Couvelaire, A.: Rupture de la cicatrice d'une ancienne operation cesarienne. Sur\'enue a la fin d'une grossesse compliquee d'hydramnios. Ann. de gynec. et d'obst., Paris, 1906, 2. s., 148-164.

Idem: Considerations sur la technique de I'operation cesarienne conser\'atrice. Ann. de gj-nec. et d'obst., Paris, 2. s., 557-586.


Davis, A. B. : A Study of Repeated Caesarean Section. Cleveland Med. Jour., 1914, XIII, 51-94.

Idem: Two Cases of Rupture of the Uterus Following Csesarean Section with Living Children. Amer. Jour. Obst., 1918, LXXVII, 136-145.

DeCourcey, J. L.: Spontaneous Rupture of the Caesarean Scar. Jour. Amer. Med. Assn., 1018, LXX, 840.

Eckstein, E.: Die erste Spontanruptur des graviden Uterus im Bereiche der alten Kaiserschnittsnarbe nach querem Fundalschnitte nach Fritsch. Zntrlbl. f. Gyniik., 1904, LXIV, 1302-1309.

Findley: Rupture of the Scar of a Previous Caesarean Section. Amer. Jour. Obst., 1916, LXXW, 411-432.

Fischer: Ueber die Ruptur des graviden Uterus in einer alten Kaiserschnittsnarbe. Ztschr. f. Geb. u. Gyn., 1912, LXX, 838-857.

Franz: Ueber Kaiserschnitt. Ztschr. f. Geb. u. Gyn., 1915, LXXVII, 215-223.

Freund, H. : Spontane Uterusruptur in einer cervicalen Kaiserschnittsnarbe. Zntrlbl. f. Gyniik., XLIII, 73-75.

Gilles, R.: Troisieme cesarienne conser\-atrice chez la meme femme. Ann. de gynec. et d'obst., Paris, 1916, 2. s., 42-43.

Green, C. M.: Caesarean Section; A Consideration of Indications, Technique, and Time of Operating. Boston Med. and Surg. Jour.,

1915, CLXXIV, 41-50.

Harrar : A Study of the Integrity of the L^terine Scar After Caesarean Section. Amer. Jour. Obst„ 1912, LXV, SOS-820.

Holland, E.: Rupture of Caesarean Section Scar in Subsequent Pregnancy or Labor. Lancet, 1920. CXCIX. 591-598.

Howson, C. R.: Report of a Case of Rupture of Caesarean Scar During Pregnancy. Jour. Amer. Med. Assn., 1918, LXXI. 728-729.

Humpstone. C. P. : The End Results of Caesarean Section. Amer. Jour. Obst., 1917, LXXV, 372-378.

J0II3', R.: Uterusruptur in der alten Kaiserschnittsnarbe. Arch. f. Gyn., 1912. XCVII. 229-236.

Labhardt, A.: Ueber Uterusruptur in Narben von friiheren Geburten. Ztschr. f. Geb. u. Gjm., 1904, LIII, 478-488.

von Leuwen: Openation cesarienne repctee. .-Vnn. de g\Tiec. et d'obst.. Paris, 1904, 2. s.. 576-580.

Losee. J. P.: The Caesarean Scar, an .Anatomical Study. Bulletin Lying-In Hosp., New York. 1918. II, 228-240.

Mason. E. R.: Vaginal Deliverv After Caes.arean Section. Boston Med. and Surg. Jour.. 1917. CLXXVI, 127-133.

Mason. E. R., and Williams. J. T.: The Strength of the Uterine Scar After Caesarean Section. \r\ Experimental and Clinical Study. Boston Med. and Surg. Jour.. 1910. CLXII. 66-72.

Newell. F. S.: Caesarean Section. G>Tiecological and Obstetrical Monographs. New York. 1921.

Novak, E.: Rupture of the Utenis Through the Caesarean Section Scar. Jour. Amer. Med. Assn., 1918, LXXI. 105-106.

Offcrmann, J. J.; Heilung und Spatfolgen der Narbe beim queren Fundalschnitt beim Kaiserschnitt nach Fritsch. Monatsschr. f. Geb. u. Gyn.. 1916. XLW, 173-186.

Planchu : Operation cesarienne pratiquee pour la quatrieme fois chez la meme femme. Ann. de gynec. et d'obst., Paris, 1916, 2. s., 45-46.

Prusmann, F.: Die Spontanruptur in der alten Kaiserschnittsnarbe. Ztschr. f. Geb. u. Gyn.. 1905, LV. 415-425.

Rohrbach, W.: Nachuntersuchungsresultate nach extra- imd transperitonealem Kaiserschnitt. Ztschr. f. Geb. u. Gyn., 1914, LXXV, 530-547.

Rongy. \. J.: Rupture of the Caesarean Scar. Amer. Jour. Obst.,

1916. LXXn^ 954-961.

Scheffzek : Die Uterusnarbe des korporealen und cervikalen Kaiserschnitts und ihre Chancen bei spiiteren Schwangei"schaften und Geburten. Ztschr. f. Geb. u. Gyn.. 1910, LXVII. 752-772.

Schroeder, E.: LTaer Rupturen der Kaiserschnittsnarbe bei nachfolgenden Schwangerschaften. Monatsschrift f. Geb. u. Gyn., 1916, XLTV. 191-231.


106


[No. 373


Spalding, A. B. : CiEsarean Section Scars. A Histological Study of Four Specimens. Jour. Amer. Med. Assn., 1917, LXIX, 1847-1853.

Williams, J. T.: Delivery by the Natural Passages Following Cesarean Section, With a Report of Two Cases. Amer. Jour. Obst., 1916, LXXIII, 425-428.

Idem.: Delivery by the Natural Passages Following Csesarean Section. Amer. Jour. Obst., 1919, LXXX, 435-441.

Wilhams, J. Whitridgc : A Histological Study of 50 uteri Removed at Cesarean Section. Bull. Johns Hopkins Hospital, 1917, XXVHI, 335-343.


Willson, P. ; Report of Four Cases Dehvered by Vagina Following a Previous Csesarean Section. Amer. Jour. Obst., 1916, LXXIV', 701703.

Wolff: Ztschr. f. Geb. u. Gyn, 1914, LXXIV, 740-746.

Woj'er, G.: Ein Fall von Spontanruptur des Schwangeren Uterus in der alten Kaiserschnittsnarbe. Monat.schrift f. Geb. u. Gyn., 1897, VI, 192-200.

Wyss, A.: Beitriige zur Uterusruptur nach Kaiserschnitt. Beitrage f. Geb. u. Gyn., 1912, 337-368.


ACUTE LOBAR PNEUMONIA AND HiEMATOGENOUS PUERPERAL INFECTION

A CASE REPORT

By R. A. Johnston and H. J. Morgan {Frovr the Department of Obstetrics and the Biological Division of the Medical Clinic of The Johns Hopkins Hospital and University)


The demonstration of a ftsed type pneumococcus as the causative agent of a puerperal infection developing during the course of acute lobar pneumonia is unusual, and makes the following case report one of considerable interest both to the obstetrician and internist.

Summary of Case.— Thirty-five year old primipara; difficult labor with prolonged second stage; chloroform anceslhesia; low forceps delivery; immediate development of signs of acute lobar pneumonia; pneumococcus (type 1) septiccemia; pneumococcus (type 1) endometritis; anti-pneumococcus (type 1) serum therapy followed by rapid disappearance of organisms from the blood stream; prolonged course suggesting "delayed resolution" ; recovery from endometritis; serum sickness; suggestive evidence of empyema; terminal hemolytic streptococcus septicoem-ia ; no autopsy.

Case Report. — A mulatto housewife, 35 years old, was admitted to the Obstetrical Service February 27, 1921. The family history was negative. The general health of the patient had always been poor. When a child she had had measles, mumps, chicken-pox and possibly pneumonia. At the age of 13 she had recovered after two months from an illness characterized by severe diarrhoea with tenesmus, blood and mucus. She denied having had diphtheria, scarlet fever, rheumatic fever, malaria or typhoid fever. She had suffered from frequent colds and occasional attacks of epistaxis. " Sore throat " had been a frequent winter complaint, and had been usually associated with enlarged tender cer\'ical glands. Six months before admission to the hospital she had suffered for a short time from pollakiuria and dysuria, which had disappeared promptly under medical treatment. Two months before admission to the hospital she had had bilateral suppurative inguinal adenitis, which had been cured by incision and drainage.

Menstruation had begun at the age of 13 ; it had been regular and of three or four days' duration. The flow had been normal in amount and accompanied by a moderate amount of pain. She had been married in April, 1920, and the last menstrual period had occurred in May, 1920.

Present Illness. — The patient was first seen by one of us in the prenatal clinic on January 11, 1921. At that time she was eight months pregnant with the child lying in the right-occipito-transverse position. The examination of the heart and lungs was negative. The sj'stolic blood pressure was 110; diastolic 70. The incisions in the inguinal regions were healing satisfactorily. The urine was normal and the blood Wassermann reaction was negative. The pelvic measurements were normal. At subsequent visits to the prenatal clinic, on January 26 and February 8, her condition was regarded as satisfactory.


She was admitted to the obstetrical ward in the first stage of labor at 9 p. m. February 27, after having walked several city blocks in a drenching rain. As the temperature was normal and examination of the heart and lungs was negative, she was regarded as being in good condition. Labor progressed satisfactorily until 3 a. m., February 28, when the second stage began. The head soon reached the outlet in the right occipito-posterior position, but as it made very little advance during the next three hours, operative delivery was decided upon. Under chloroform anaesthesia a deeply asphyxiated child was delivered at 620 a. m. after a difficult low forceps (Scanzoni) operation — the total duration of labor being 29 hours and 40 minutes. The child was revived by sensory stimulation, and weighed 3380 gm. After the repair of a vaginal laceration, the placenta was expressed from the vagina. Approximately 300 c. c. of blood were lost. The routine microscopic examination of the placenta revealed no abnormalities and a subsequent study of sections stained for bacteria was negative.

The patient left the delivery room in excellent condition. Two hours after delivery, the temperature was 99.4° F., but from then on it rose steadily until it reached 104° F. at 8 p. m. At that time the patient complained of slight pain in the right side of her chest. No chill had occurred, and there was no cough nor sputum, nor were the respirations accelerated. On examination of the lungs a soft pleural friction rub was heard over the right lower lobe, without other changes. Abdominal examination was negative. During the night the temperature fell to 102.5° F., but rose to 104.2° F. the following morning. At that time (March 1) the patient was quite toxic. The respiratory rate was 36 per minute but there was no cough. The breath sounds were slightly suppressed over the right lower lobe and an occasional fine rale could be heard. The abdomen was quite distended and sensitive, but there was no muscle spasm or evidence of fluid. A blood culture was made and 24 hours later was reported positive for the pneumococcus (type 1).

On March 2, the general condition was essentially unchanged. The temperature remained elevated and the patient was quite toxic. Frank signs of consolidation of the lower lobe of the right lung were present. Lochia for intrauterine culture were obtained (by Little's tube), and consisted of a few cubic centimeters of dark serosanguineous fluid. On microscopic examination numerous diplococci were seen, which by culture and by serological examination proved to be pneumococci (type 1). A urine culture, made on March 1, was sterile. The patient was transferred to the medical ser\'ice March 2, when examination revealed a fairly well nourished tired looking mulatto woman with high fever, tachj-cardia, moderate cyanosis, and rapid, shallow respiration. There was no cough or sputum. The pulmonary signs were those of consolidation of the right lower lobe. There was


Maech, 1922]


107


marked distention of the abdomen with considerable tenderness over its lower third, but no muscle spasm or evidence of fluid. Rectal examination revealed a large firm tender uterus. Blood pressure; systolic 125, diastoHc 65.

Blood Ex.\mi>;atiox

R. B. C. count 3,393,000

Hemoglobin (Sahh) 67%

W. B. C. count 6,250

Differential W. B. C. count (300 cells)

Pm. N 91.4%

Pm. B 00 %

Pm. E 0.77o

S. M 3.0%

L. M. and T 2.6%

Myelocytes 2.0%

Myeloblasts 0.3%

With the exception of a trace of albumin and a few white blood cells the urine examinations were negative. The Wassermann reaction (blood) was negative.

Immediately following admission to the ward, anti-pneumococcus (type 1) serum treatment was instituted, as follows: March 2, 1921, 200 c. c. antipneumococcus type 1 serum intravenously. March 3, 1921, 200 c. c. antipneumococcus type 1 serum intravenously. March 5, 1921, 100 c. c. antipneumococcus type 1 serum intravenously. March 7, 1921, 100 c. c. antipneumococcus type 1 serum intravenously. March 8, 1921, 100 c. c. antipneumococcus type 1 serum intravenously. A quantitative blood culture was made (blood-agar plate method) before the first serum treatment, and showed 173 colonies per c. c. of blood, whereas a culture made eight hours later and prior to the second treatment contained but three colonies per c. c. Specimens of blood were likewise taken before the third (March 3, 1921) and subsequent treatments, but were all sterile:

There was no systemic reaction to the intravenous administration of the serum, and the usual chill, leukopoenia and the subsequent abrupt leukocytosis did not appear. However, there was a gradual rise in the number of leukocytes and two days after the first injection they reached 18,000 per c. mm.

With the termination of the septicaemia the patient's general condition improved greatly. The toxsemia became much less evident, and the abdominal distention less distressing. However, the temperature remained elevated and the physical signs in the lungs were essentially unchanged. -There was still a moderate degree of pelvic tenderness on abdominal and rectal examination, as well as a fairly profuse, greenish, muco-purulent vaginal discharge. On March 6 (three days after the disappearance of the septicjemia) , lochia were obtained for a second intrauterine culture. Smears showed a moderate number of leukocj-tes and lancet-shaped diplococci, and pneumococci (type 1) were again demonstrated in pure culture.

For two weeks the patient's condition remained practically unchanged, with the exception of a moderate degree of serum sickness, which first manifested itself on March 11. The temperature remained elevated (102.5° F.-104° F.), and the pulse varied between 110-130. The leukocyte count was between 18,000 and 35,000 per c.mm. There was very slight cyanosis and only a moderate degree of abdominal distention. The tenderness over the lower abdomen became much less marked and the \'aginal discharge diminished in amount. On March 14 a third intrauterine culture was made and proved to be sterile. It was the opinion of those attending the patient that the prolonged course of the pneumonia was a manifestation of " delayed resolution," and that the uterine complication no longer formed a part of the picture.

On March 16, examination of the lungs showed signs suggestive of encapsulated pleural fluid high in the right axillary region; while the X-ray showed " remains of consolidation at the right base with suggestion of encapsulated fluid in the right axilla." The same


equivocal physical and radiographic findings were present the following day. On that day, the 17th, the patient complained bitterly of headache. She was very weak and slept most of the time. Ophthalmoscopic examination showed slight oedema and hyperaemia of the discs, but no other changes. Kernig's sign and neck rigidity were absent, and the reflexes were normal. Nevertheless, a lumbar puncture was performed. Clear cerebrospinal fluid under normal pressure was obtained, and contained ten cells per c.mm. The colloidal gold curve, Wassermann reaction and tests for globuhn were negative. The patient's general condition rapidly became worse. The temperature remained high, the rapid pulse became weak and thready, and drenching sweats occurred. Cyanosis became marked and delirium developed. A pelvic examination revealed no abnormalities, and exploratory thoracentesis performed at several different sites in the right axilla and back was negative. A blood culture taken on the evening of this day (March 17) revealed the presence of an overwhelming streptococcus septicaemia — approximatelj^ 7600 colonies per c. c. of blood. The patient gradually sank into coma. The pulse and respirations became irregular, and she died on the evening of March 18, the nineteenth day after delivery. Unfortunately permission for an autopsy could not be obtained.

Discussion

The case presents several interesting clinical aspects. The sequence of events seems relatively clear. A primipara, 35 years old, with a past history of numerous infections, and on the whole of rather poor health, entered the hospital for delivery. At the onset of labor she was esposed for a short time to inclement weather. Labor was completed by forceps delivery under chloroform anaesthesia. Immediately following the birth of the child an acute lobar pneumonia developed. As evidence of the lowered resistance of the patient to infection at this time, one might cite the absence of chill and letikocytosis at its onset, and particularly the existence of the septicemia with large numbers of bacteria in the blood stream (173 colonies per cubic centimeter). Intrauterine culture demonstrated the presence of the same organisms in the postpartum uterus. That this finding was not accidental, or due entirely to the normal oozing of blood infected with pneumococci, is clear; for a greenish mucopurulent uterine discharge developed, which contained numerous white blood cells and organisms. Furthermore, this condition persisted for several days after the disappearance of the bactersemia, and was accompanied by clinical evidence of uterine infection. Accordingly, it seems clear that we were dealing with a metastatic pneumococcus (type 1) puerperal endometritis, wliich developed in a woman suffering from acute lobar pneumonia.

When one considers the anatomical and physiological conditions present in the fresh post-partiun uterus, one is struck by the fact that it presents a locus minoris resistentiae, as Bondy has aptly pointed out, and offers an ideal site for the growth of bacteria seeded into it by a heavily infected blood stream. In our case the puerperal infection obviously did not spread, but remained localized to the interior of the uterus, and had entirely disappeared clinically and baeteriologically by the 14th day. The disappearance of the type 1 pneumococcus bactersemia following administration of the specific anti-pneumococcus type 1 serum in this case conforms in every respect to the observations of AveiT, Chickering, Cole and Do


108


[No. 373


chez, and others. That the serum had a beneficial effect upon the local intrauterine infection seems probable ; for the process, although caused by a virulent organism and occurring in a patient with obviously lowered resistance, was in itself of rather benign type and of short duration. We feel therefore, that the use of the serum gave the patient a much better opportunity to overcome the infection, as was evidenced by the disappearance of the baeterfemia, the markedly improved general condition, the leukocytosis, the failure of the pneumonic process to spread to other lobes of tlie lung, as well as by the disappearance of the endometritis.

In concluding the discussion of the case from a clinical point of view, we feel that we may speculate with a fair degree of certainty concerning the terminal process. Although there was suggestive evidence, both from physical findings and radiographic examinations, that an empyema was developing and the prolonged course of the disease confirmed this possibility — repeated exploratory punctures of the pleural cavity failed to substantiate such a surmise. The normal cerebrospinal fluid almost conclusively rules out meningitis as a complication, and there was no evidence of the existence of pericarditis or endocarditis. The pelvic examination made the day before death was normal and the lochia negative. In the absence of a post-mortem examination, we accordingly have to fall back upon the assumption that we were dealing with a patient whose powers of resistance were such that she could not, in the usual length of time, overcome the local puhnonary infection, and as a result of the general debilitation resulting from it became a fit subject for a terminal generalized haemolytic streptococcus infection, the respiratory tract sei-ving as the portal of entrance. Moreover, it seems likely that the streptococcus pulmonary infection engrafted itself upon the slowly resolving pneumococcus pneumonia, causing an exacerbation of the pulmonary condition and resulted in an overwhehning generalized infection. MacCallum (4) who noted the occurrence of hsemolj^tic streptococci in three cases of lobar pneumonia studied in army camps, in which pneumocoeci had been isolated from the blood during the life or from the lungs at autopsy, has suggested the possibility that the latter organisms may have acted as the predisposing agents to infection with the streptococcus.

Pneumococcus Pueepeeal Infection The infrequency with wliich the pneumococcus is encountered as the etiological factor in puerperal infection no doubt explains, to a great extent, the interest with which instances of this condition have been regarded. Bondy, in 1912, published an excellent article upon the subject, which included a report of his own eases and a review of the literature. It is necessary to recall, however, that until comparatively recently the differentiation of the pneumococcus from certain strains of non-hfemolytic streptococci was fraught with difficulty, and that even to-day occasional strains are met with in which the differentiation is not sharply defined. It is, therefore, suggested that statistics compiled before the more recent advances


in bacteriological diagnosis were made should be accepted with some reservation, particularly as concerns the reported instances of " primary infection " — or infection by pneumocoeci gaining access to the uterus by way of the vagina.

It is manifest that two routes are open for the introduction of organisms into the uterus: (1) haematogenous (metastatic or secondary), (2) vaginal (primary). We shall consider each in some detail.

Hwrnatogenous Pneumococcus Puerperal Infection. — Aufrecht, in 1SS4, was the first to produce an experimental pneumococcus endometritis by the introduction of living organisms into the blood-stream of rabbits shortly after labor. At autopsy a localized uterine pneumococcus infection was found with but insignificant changes elsewhere. Thus he proved experimentally that the puerperal uterus in that animal is a Jocus niinoris resistentite, and that the pneumococcus is capable of producing pathological lesions in it. Orthmann (referred to by Bondy) confirmed this work. Weichselbaum, in 1888, reported the first instances of hfematogenous pneumococcus infection of the puerperal uterus in women. Both occurred in patients suffering from acute pulmonary infections (exudative pleurisy and pneumonia). At autopsy the pneumococcus was found in the endometrium, as well as in the primary focus in the respiratory system. Bondy refers to the instance reported by Bumm in 1899, in which a pneumococcus peritonitis was associated with pneumonia and empyema. At autopsy pneumocoeci were demonstrated in the uterine blood vessels and secretions. Burchardt, in 1901, reported an instance of premature labor during the course of acute lobar pneumonia, in which pneumocoeci were found in the placental site and lymphatics of the litems. Furthermore, Foa and Bordoni-Uffreduzzi cite two instances of abortion in pneumonic women in which pneumocoeci were found in the uterine veins. Stanoskiadis, in 1913, reported 12 cases of lobar pneumonia in which pneumocoeci were found in the vaginal secretions : He considered that they represented instances of hsematogenous^ infection which was favored by the changes incident to abortion or delivery. Haller, Oertel and others have likewise discussed the subject.

In the light of the above reports, as well as of our own case, is seems profitable to recall that in 728 cases of acute lobar pneumonia reported by Cole, in which blood cultures were made, 27.8 per cent were positive for the pneumococcus. In other words, he demonstrated that a pneumococcus bacterEemia is probable in approximately every fourth case of pregnancy which is complicated by lobar pneumonia. Under these conditions the possibility of a pneumococcus infection of the uterus at the time of, or shortly after, its evacuation does not seem remote, and should be borne in mind by obstetrician and internist alike.

Vaginal (Primar;/) Pneumococcus Puerperal Infection. — Although our case obviously does not fall into this group, a brief survey of the subject may prove of interest. As has been intimated, our knowledge of the pneumococcus has been considerably extended during recent years, and the organism can now be more easily and accurately differentiated from other cocci. Allowances must, therefore, be made in interpret


March, 1932]


109


ing the results of investigators who worked prior to the acquisition of this knowledge.

It would seem that the pneumococcus is very rarely an inhabitant of the normal vagina. Bondy, in 1913, failed to find it in a search of the vaginal flora of 30 non-pregnant women, but found it once in the same number of normal pregnant women, as well as once in the lochia from 30 postpartum patients. Fabret and Bourret called attention to this work in 1913, and regarded it as affording conclusive evidence that the pneumococcus is not an inhabitant of the normal vagina. On the other hand, Schottmiiller in 1910 found pneumococci in the lochia of three per cent of his cases of septic abortion ; while Foulerton and Bonney in their examination of 54 puerperal women found pneumococci in four instances.

That the pneumococcus may occasionally be present in the vagina of the pregnant and puerperal women cannot be doubted, and in the light of the cases collected by Bondy and by Fabret and Bourett it seems certain that it may, under certain conditions, find its way into the postpartum uterus and there give rise to puerperal infection. The reader is referred to articles by the last named authors for an extended discussion of this question.

Treatment Beside the general supportive measures usually employed in puerperal infections, Moore has suggested that uterine irrigations with ethylhydrocuprein (optochin), 1 : 10,000, may possibly be used to advantage, on account of the powerful antipneumococcus properties of the drug. If the infection is due to a type 1 organism, intravenous serum therapy may be indicated. In our case the uterine infection was of moderate degree so that local treatment was not attempted, but all of our efforts were directed to the relief of the pulmonary condition. However, we feel that the serum treatment was perhaps incidentally responsible for the mildness of the uterine infection and its early disappearance.

Summary We have reported a case of acute lobar pneumonia (pneumococcus type 1) with septicaemia as a complication of the puerperiimi, and have shown that a hsematogenous (pneumococcus, type 1 ) endometritis developed and disappeared under, observation. Death occurred on the 19th day from generalized hsemolytic streptococcus infection.


A brief review of the literature upon pneumococcus puerperal infections is given, and instances of both secondary (hematogenous) and primary (vaginal route) types are cited. It is suggested that the possibility of a pneumococcus puerperal infection should be borne in mind when dealing with patients in whom abortion or term delivery has occurred during the course of acute lobar pneumonia.

BIBLIOGRAPHY

Aufrecht : Die crupose Pneumonia. Nothnagel, Specielle Pathologie und Therapie, Wien, 1899, XIV, Bd. I, 37.

Avery, Chickering, Cole, and Dochez: Acute Lobar Pneumonia. Prevention and serum treatment. Monogr. Rockefeller Inst. Med. Research, No. 7, 1917.

Bondy: Die Bedeutung der Pneumokokken fiir die puerperale Infektion. Ztschr. f. Geburtsh. u. Gyniik., 1912, LXXII, 631.

Burchardt: I. Die endogene Puerperal-infektion. II. PuerperalInfektion mit Pneumococcus. Frankel, Beitr. z. Geburtsh. u. Gynaek., 1901, V, 327.

Cole : Acute Lobar Pneumonia. Kelson's System of Medicine, 1920, I, 248.

Fabret and Bourret : Infection puerperale a pneumoeoque, etc. Bull. Soc. d'obst. et de gynec. de Paris, 1913, XVI, 474.

Foa und Bordoni-Uffreduzzi : Uber die Aetiologie der Meningitis cerebro-spinalis epidemica. Ztschr. f. Hyg. u. Infectionskrankh., IV, 67.

Foulerton and Bonney: An Investigation into the Causation of Puerperal Infection. J. Obst. & Gynaec. Brit. Emp., Lond., 1905, VII, 121.

Haller: Tiber Pneumonie in der Schwangerschaft. Ludwigsburg, 1911, Ungeheuer & Ulmer, 31 p., 8°.

Little: A simple method of obtaining uterine lochia for bacteriological examination. Johns Hopkins Hosp. Bulletin, 1904, XV, 250.

MacCallum : The Pathology of the pneumonia in the United States Army Camps during the winter of 1917-18. Monogr. Rockefeller Inst. Med. Research, No. 10, 1919.

Moore : The action of Ethylhydrocuprein (optochin) on type strains of pneumococci in vitro and vivo and on some other micro-organisms in vitro. J. Exper. Med., 1915, XXII, 269.

Moore: A furthei- study of the bactericidal action of ethyldydrocuprein on pneumococci. J. Exper. Med., N. Y., 1915, XXII, 551.

Oertel: Hiimatogene Puerperal-sepsis. Wiirzburg, 1913, F. Standenraua.

Schottmiiller: Zur Pathogenese des septischen Abortes. Munchen. med. Wchnschr., 1910, LVII, 1817.

Stanoskiadis : Uber die Veriinderungen des Uterus bei akutert Infektionskrankheiten. Monatschr. f. Geburtsh. u. Gynaek., 1903, XVII, 1 and 187.

Weichselbaum : Beitriige zur Aetiologie und path. Anatomie der Endocarditis. Beitr. z. path. Anat. u. allg. Path., 1899, IV, 125.


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110


rjS'o. 373


DIPHTHERIA BACILLUS CARRIERS

RESULTS OF RE-EXAMINATION OF APPARENTLY NEGATIVE CULTURES

Bj' B. C. MAESHAiL and C. G. Gi'thkie (From the Division of Clinical Pathology of the Medical Clinic, The Johns Hopkins University and Hospital)


As ordinarily practiced, the examination of throat cultures for the presence of diphtheria bacilli is made after incubation of the tubes for from 13 to 24 hours, usually over night. Not infrequently a positive result may be obtained much sooner than this and in special cases examinations are made at intervals beginning at six hours, but it is quite generally the custom to discard as negative all cultures in which diphtheria bacilli have not been found after 24 hours in the incubator. This custom has probably arisen from several causes, as for instance :

1. The desire to secure positive information as early as possible.

2. The belief that the growth of the diphtheria bacillus on a suitable medium is relatively rapid during the first 24 hours, but that after that time it is apt to be overgrown by the other organisms encountered in cultures from the throat.

3. The belief that the morphology of the diphtheria bacillus is characteristic only during the earlier period of its growth on even the best of media; that after 24 hours its staining properties are lost to a greater or less degree and involution forms may appear, thereby rendering its recognition difficult or impossible in mixed cultures from the throat.

4. The belief that if diphtheria bacilli have not been found in the examinations of a throat culture during the first 24 hours they will not appear subsequently in that culture and therefore that search made at a later time would merely entail useless routine work.

We have no criticism to offer concerning examination of throat cultures " early and often," but we believe that the practice of regarding as negative all cultures which have not shovm diphtheria bacilli up to 24 hours may yield results which are misleading, particularly in the case of bacillus carriers.

In the course of some experimental work with the so-called " elective medium " of Drigalski and Bierast.* we found that a number of the tubes, considered negative at 24 hours, were positive when re-examined a day later. It naturally occurred to us that the apparent delay in development of the diphtheria organisms might be due to the inhibitory effect of the bile in the special type of medium employed, but it soon became evident that a similar retardation might occur with other sorts of media as well. After this point was established we made it a rule to examine all cultures both at 24 and at 48 hours.


v. Drigalski and Bierast: Nachweis der Diphtheriebazillen und seine praktische Bedeutung. Deutsche med. Wchnschr., 1913, XXXIX, 1237. This medium consists essentially of LoefBer's blood serum to which a certain amount of bile has been added prior to coagulation. The object of the addition of bile is to inhibit the growth of the other organisms from the throat without interfering with the growth of the diphtheria bacillus.


At this time we had under observation a series of diphtheria bacillus carriers — some of them healthy, others convalescent carriers, some who harbored virulent and others non-virulent organisms — and from these persons throat cultures were taken daily over a period of several weeks. The cultures were made on several different kinds of blood serum media and were examined with great care. All of the cultures found negative at 34 hours and abnost one-half of the positive ones were reexamined at 48 hours. The results are presented in Tables I, II and III.

TABLE I

Results op Cultures Ex.\mined after Incubation for 24 and 48 Houns


Cultures


Examined after 1 incubation for 24 hours


Re-examination at 48

hours of cultures negative at 24 hours


Re-examination at 48

hours of cultures positive at 24 hours



Number


Per cent


Number


Per cent


Number


Per cent


Total


759



549



93



Positive . . .


. 210


27.66


52


9.47


92


93.92


Negative . .


. 549


72.33


497


90.52


1


1.07


TABLE II

Analysis of PosiTn-E Results


Total jiositives

Positive at 24 hours. . . . Positive only at 48 hour


262 210


80.15 19. S4


TABLE III

Virulence of Diphtherw Bacilli in Cultures PosiTm: Only at 48 Hou-RS


Medium


Virulent strains


Nonvirulent Total strains



24

1 3 3


20 ! 44



1



2


Drigalski and Bierast (pis serum -| bile). Drigalski and Bierast (beef serum -| bi'le).


3 3


Total


31


21





Prom Table I it is seen that of the 759 cultures from these carriers, 210, or 27.66 per cent, were positive at the first examination made after approximately 24 hours' incubation. Five


Maecii, 1922]


111


hundred and forty-nine cultures were negative at the first examination and of these 52, or 9.47 per cent, were positive when examined after an additional incubation of 24 hours. Re-examination of 93 originally positive cultures showed that 92 were still positive after 48 hours in the incubator.

An analysis of the positive results (Table II) shows that of the 262 cultures in which diphtheria bacilli were found, 210, or 80.15 per cent, were positive at the iirst examination, and that 52, or 19.84 per cent, were found positive only at the second examination which was made 24 hours later.

From Table III it is seen that among the 52 cultures found negative at the first examination but positive at the second, 31 contained virulent and 21 contained non-virulent strains of diphtheria bacilli. These 52 cultures were made on four different kinds of serum media, on all of which the diphtheria bacillus grows admirably when pure and, as a rule, also when it is present in mixed culture from the throat.

These results speak for themselves and require little comment. From them it is evident that practically one-fifth of the total number of positive cultures were regarded as negative when examined at 24 hours, but were shown to contain diphtheria bacilli when examined a day later. In all instances the original slides were saved for re-examination in case the culture should prove positive at 48 hours. Careful search showed the original report to have been justified in almost every instance, but whenever this re-examination revealed any diphtheria bacilli in the 24 hour slides, the earlier diagnosis was changed and such cases are omitted from this series. It is perhaps unnecessary to add that tubes which showed little or no growth at 24 hours are not included, even though subsequent examination may have revealed the presence of diphtheria bacilli. The practice of examining throat cultures both at 24 and at 48 hours has been continued since these figures were collected and the additional experience of several years has shown the advantage of this procedure. Since introducing it in this laboratory we have isolated typical Elebs-Loeffler organisms from 75 or more of these " delayed positives," thereby confirming the diagnosis originally based on morphology alone. The difference in morphology at 48 hours is practically negligible, being, when it occurs at all, in the nature of an increase in the number and size of the Babes-Ernst granules, thereby facilitating recognition of the organisms when the Neisser or similar contrast stains are used. In general, the development of involution forms seems to be less marked in mixed cultures from the throat than in pure cultures.

In our experience the " delayed positive " culture has been found only among carriers, healthy or convalescent, and has been quite independent of the virulence of the strain of organisms present in the throat. In cases of clinical diphtheria we have practically always found the organisms abundant and their recognition easy in throat cultures incubated for 24 hours, so that no difficulty exists from the source we have mentioned in the bacteriological diagnosis of actual cases of the disease.

The explanation of the " delayed positive " culture is far from clear. Whether the diphtheria bacilli present in cul


tures from the throat of carriers may have a more prolonged period of lag than those from patients in the acute stage of the disease we are not prepared to say. In many instances this is obviously not the case, as the cultures may show at the first examination an abundant growth consisting almost exclusively of diphtheria bacilli. The organisms present in the " delayed positive " cultures, moreover, show a normal rate of growth when isolated in a pure state. We can only suggest that this peculiar retardation may possibly be due to the inoculation of the culture with a very small number of viable diphtheria bacilU and a very large number of other organisms which temporarily overgrow or possibly inhibit the former. Almost invariably, when Klebs-Loeffler bacilli were found to be sparse at 24 hours, they were abundant at 48 hours ; if originally numerous, they were much more plentiful on the following day. In only one instance was a culture which was positive at 24 hours found negative at 48 hours. When once the diphtheria bacillus starts to grow, it apparently is able to hold its own against most of the other bacteria encountered in throat cultures with the exception of the hay bacillus and certain similar organisms. Many cultures have been examined after periods of growth longer than two days, but no evident advantage was found in this procedure.

Similar observations have been made by other workers. Knebel * collected some interesting statistics at the Hygienic Institute in Frankfurt ; the results which form the basis of his report have been condensed and are shown in Table IV.

TABLE W

Results op Examination of Throat Cultures after Incubation for One Day and Two Days

(From Knebel)


Source of cultures


Result after

incubation for

one day


Result after incubation for an additional day



Negative


Doubtful


Negative


Doubtful


Positive


Fresh diplitlieria suspects.


139 576


63 56


43

151

470


17 51


12 16


Fresh diphtheria suspects.


1 55




From Knebel's figures shown in Table IV, it is seen that of the throat cultures from 118 cases — 62 fresh suspects and 56 convalescents — all of which were recorded as doubtful at the first examination, 28 were definitely positive, 66 were definitely negative and only 24 were still considered doubtful when examined a day later.

Table IV also shows that of 159 throat cultures from diphtheria suspects regarded as negative at the first examination, only one was found positive after incubation for another day and in tliis isolated instance both the history and the physical examination of the patient suggested strongly that they were


Knebel, Max : Beitriige zur bakteriologischen Diagnose und Statistik der Diphtherie. Inaugural Dissertation, Giessen, 1912.


112


[No. 37:^


dealing with a convalescent rather than a fresh case of diphtheria. Further information is available concerning four of the seven cases -which were reported as doubtful at the second examination ; the organisms from two of these were shown not to be diphtheria bacilli ; attempts to isolate the organisms seen


A comparison of our results with those reported by Knebel is presented in Table V.

TABLE V

COMPAEISON OF OUB RESULTS WITH ThOSE REPORTED BY KnEBEL


in the culture from another were unsuccessful; the pure culture obtained from the fourth proved to be a\'irulent on animal inociilation.


1 Cultures negative at Source of cultures 24-hour


Cultures positive

when examined

at 4S hours


Conditions were quite different, however, with regard to the 576 negative cultures from convalescents; 55 or 9.54 per cent, proved to be positive when examined next day and these findings were confirmed by further study including virulence tests on the organisms obtained in pure culture. The originally



tion


Number


Per cent


Diphtheria convalescents (Knebel)...

Bacillus carriers, convalescent or healthy (Marshall and Guthrie).

Total


576 549


55 53


9.54 9.47


negative cultures from 51 cases which were moved to the


1125


107


9.51


doubtful column as the result of the second examination.




Knebel dismisses with the comment that a doubtful result is of much less importance in a convalescent than in a diphtheria suspect and that, when dealing with convalescents, one cannot go wrong provided prophylactic restrictions are not relaxed on the basis of a doubtful answer.

Neisser,* who was director of the Frankfurt Institute speaks favorably of Knebel's report and recommends that (1) all cultures showing a poor growth after incubation for one day and (2) all throat cultures from convalescents should be re-examined on the following day.

. Seidel f also lays stress on the desirability of examining all throat cultures from convalescents both at 20 and at 44 hours. He cites an instance in which failure to re-examine a culture which was negative at 20 hours resulted in the discharge of a convalescent child who promptly infected her three Uttle sisters.


From Table V it is seen that our figures agree very closely with those reported by Knebel, although his observations were made entirely upon patients convalescent from diphtheria, whereas ours were made upon bacillus carriers, some of them convalescent but others healthy carriers.

From these results it seems evident that, when dealing with throat cultures from diphtheria carriers, the customary laboratory practice of making single or repeated examinations within the first 24 hours may not yield all of the information available and may lead to erroneous conclusions. The error from this source alone, as judged from the results in 1125 cases, is about 9.5 per cent. If one is interested in determining the duration of the carrier state, in ascertaining the actual condition of affairs, rather than in merely securing some arbitrary number of successive negative cultures required by health regulations prior to discharge of a patient, further investigation is essential.

It is to be recommended, therefore, that in the study of diphtheria bacillus carriers, either healthy or convalescent, all cultures which are negative up to 24 hours be re-examined after another day in the incubator.


Neisser. M.: Bakteriologie der Diphtherie. Centralbl. f. Bakteriol., 1913. Section 1, LVII, Supplement, p. 1. Neisser. M., and Gins, H. A.: Ueber Diphtherie, in Kolle and Wassermann's Handbuch der Pathogenen Mikroorganismen, Ed. 2, Jena. Gustav Fischer. 1913. V, 931.

t Seidel, Otto: Zur Behandlung der Diphtherie, Munchen. med. Wchnschr., 1915, LXII, 1209.


THE HYDROGEN-ION CONCENTRATION OF TISSUE GROWTH

IN FITRO

By M. R. Lewis and Lloyd D. Felton (From the Carnegie Laboratory oj Embryology and the Department oj Pathology, The Johns Hopkins Medical School, Baltimore)


Introduction Before undertaking a study of the effect of bacteria upon growth of tissue culture, it seemed necessary to determine in what way media of different H-ion concentrations influence the activity of tissue cultures, and whether there is a parallelism between what we know of the determining factor of H-ion concentration on the life process of micro-organisms and on cultures of embryonic tissue. Numerous observers have shown that there is both an optimum H-ion concentration for bacterial growth and a limiting or final concentration at which


gro\\'th activity is at rest, both conditions being influenced by the constituents of the medium. It was our purpose, therefore, to determine the optimum and the limiting or final H-ion concentration for tissue cultures, and to show any possible similarity of metabolism between pure type embryonic connective-tissue cells and cellular organisms (bacteria) as gro^Ti ill vitro. At the present the comparison must of necessity be crude, both because of the different conditions under which the two types of living cells exist and because of our meager knowledge concerning the elements necessary for life and propagation of cells.


March, 1922]


113


Technique

Explants of chick embryos in Locke-Lewis solution furnished a satisfactory means of following the hydrogen-ion concentration during the growth of tissues, because of the ease with which a medium of a given H-ion concentration can be prepared and its fluidity at all ages of growth, in contradistinction to plasma. After some experimentation, a colorimetric method was devised (Felton, 1921) by which it was possible to test out the small hanging drop of the tissue culture. This method is not only very simple but also apparently quite exact. A drop of indicator of approximately the same size as the hanging drop was placed directly upon the growth on the cover-slip, shaken or stirred about and, with an abrupt blow, shaken off into a white glass plate. An exact reading of the drop wa.s then obtained by combining a drop of indicator with a drop of a known buffer solution until the same color as that of the hanging drop was obtained.

Tissue cultures were prepared in the usual manner (Lewis and Lewis, 1915). Locke-Lewis solution (85 c. c. of NaCl 0.9 per cent plus KCl 0.042 per cent plus CaCl, 0.025 per cent plus NaHCOg 0.02 per cent plus 15 c. c. chicken bouillon plus 0.25 per cent dextrose) has a H-ion concentration between 6.6 and 7, usually 6.8. When a mediimi of a given H-ion concentration was to be prepared, the sodium bicarbonate and dextrose were omitted and sodium hydroxide or hydrochloric acid was added until the solution, when tested after boiling for 15 minutes, gave the color corresponding to that of the buffer solution of tlie required H-ion concentration. To this was then added sufficient dextrose to make 0.85 per cent. Solutions of H-ion concentrations varying from 4.4 to 9.2, with an increment of 0.2, were used. Explants were made from connective tissue from chick embryos ranging from 5 to 14 days' incubation, but unless otherwise stated the results refer to tissue from embryos of 7 to 9 days' incubation.

The buffer solutions used in these experiments were furnished by Doctor W. Mansfield Clark. The indicators were thymol blue, methyl red, brom thymol blue, brom cresol purple, phenol red, cresol red, and phenolphthalein.

Vaeiabilitt of the H-ion Concentration of Locke-Lewis Solution on Different Kinds of Cover-Slips A few series of cultures sufficed to show that care must be taken in selecting the cover-slips for these experiments. With cover-slips made from certain kinds of glass the control hanging drops, after incubation, varied greatly and many of them became so alkaline that the results of any experiments with different media would have been obscured. All coverslips were placed in dilute sulphuric acid for one or more days before preparing them for use. Covers of three unknown makes, which had been in the laboratory a number of years, were the most satisfactory. When a drop of a solution of a known H-ion concentration was incubated upon these it remained almost unchanged for a number of days, so that after a period of three weeks in the incubator the H-ion concentra


tion was approximately the same as that of the original solution (6.8 to 7.0) . A number of other kinds of cover-slips were tested but proved to be unsatisfactory for our purpose, as the hanging drop sometimes changed within a few days from pH 6.8 to pH 8 or 8.6. It is clear, therefore, that the selection of cover-slips suitable for the purpose is of great importance before undertaking any experiments.

An attempt was made to render the unreliable cover-slips more satisfactory by coating them with celloidin or with paraffin. Those covered with a thin layer of celloidin were favorable for growth; the cells grew out as extensively and lived as long on such covers as on the usual ones, but the hydrogenion concentration of the control drops still varied greatly, due no doubt to the dialysis of the H-ion from the glass. It is possible that an impermeable membrane would produce almost ideal conditions. The alkalinity of these drops did not increase to such a marked extent as on the uncoated cover-slips, but the variations in the results with the individual cover-slips was so great as to render them undesirable for these experiments. On the cover-slips coated with paraffin the drops remained much more constant, but rounded up to such extent that such covers were useless for the purpose of tissue cultures. The limited number of reliable cover-slips obtainable at this time accounts for the small number of esjperiments given in the charts.

Eange of Hydrogen-ion Concentration of Media in which Chick Tissue Exhibited Growth

Pieces of chick embryos were explanted into media having a H-ion concentration varying from 4 to 9.2, with an increment of 0.2. In solutions having H-ion concentrations of 4.0, 4.4, 4.8, 5.0 and 5.2 no growth was obtained, except in one series in which a few migrating cells wandered out from explants of a 68-hour embryo in a solution of pH 5.0. Many explants, from embryos of all ages, were made in solutions of pH 5.5, but none of these grew except in the case of a chick blastoderm of 24 hours' incubation. In general, the younger the embryonic tissue the greater the percentage of growth in media of low pH value.

Approximately one hundred cultures were explanted into solution with H-ion concentrations of 6.0, 7.0, 8.0 and 9.0. The percentage of growth which occurred in these cultures was respectively 71, 93, 89 and 81. In the normal LockeLewis solution, which has a H-ion concentration usually about 6.8, over two hundred cultures were explanted. Of these, 90 per cent grew. The greatest nmnber of cultures grew in media of pH 7 and in the normal solution, and these cultures also exhibited the most extensive growth. It was frequently impossible to differentiate the series of cultures explanted into media having a H-ion concentration of 7.0, 7.2, and 7.4. What growth took place in solutions of pH 6.0 was extensive and healthy, but tissue from embryos of over 11 days' incubation seldom grew in this medium. The growth in solutions of pH 9.0 was frequently small, whereas that in solutions of pH 8.0 was more extensive and lived longer.


114


[No. 373


Htdkogen-ion Concenteation of Culttxees Geown in

Media of Vaeting Hydrogen-ion Concen TEATIONS (pH 6, 7, 8. and 9)

Table 1 shows the results obtained when cultures explanted into solutions of different hydrogen-ion concentrations (pH 6.0. 7.0, 8.0 and 9.0) were tested by means of the drop colorimetric method described above. Wliile the individual cultures varied greatly, there were no marked differences by which cultures belonging to one series could be distinguished from those of another. Cultures explanted into acid media (pH 6.0) were on the whole somewhat more acid than those in normal Locke-Lewis solution, wliile those explanted into alkaline media (pH 9.0) were a little more alkaline than those in neutral solutions.

The lack of any marked differences between the series of cultures was probably due largely to the fact that the explanted piece acted as a buffer, so that shortly after explantation the medium became changed to one more nearly neutral. This was especially so in the alkaline solutions. Cultures explanted into media, which, after explantation, had a H-ion concentration of 8.2 and 9.0, became within 3 to 4 hours pH 6.8 to 7.2 and pH 7.6 to 8.0, respectively. Not enough cultures were examined to justify any definite conclusions, but from the table it is evident that, regardless of the initial hydrogen-ion concentration of the medium, cultures. which contained healthy and extensive growth tended to be neutral, those which failed to grow had usually become slightly acid, and those which had exhibited extensive growth and then degenerated were slightly alkaline. As will be shown later, however, this last point depends upon the amount of dextrose present in the medium.

It is difficult to understand what factors caused our results to differ from those obtained by Eous (1913), who studied cultures of chick embryo tissue explanted into plasma that had been colored blue with litmus. The differences in regard to healthy cultures may be explained on the ground that diffusion takes place much less readily in plasma than in LockeLewis solution ; therefore, whatever acid was produced by the explant may have been held in the immediate vicinity of the explant and thus have become condensed to a sufficient strength to turn the blue litmus pink. This, however, does not account for the fact that in Rous's experiments cultures that failed to grow remained alkaline, while in ours such cultures were usually more acid than those exhibiting good growth. The varied results may be explained upon the basis of different media.

Htdeogex-ion Conuenteation op Cultures Geoavn in Locke-Lewis Solution The hydrogen-ion concentration of the solution used depended upon that of each preparation of bouillon. It was usually about 6.8, but in a few instances was 6.6, and in others 7.0. The amount of dextrose added to the solution varied, as it was measured, not weighed; supposedly about 0.25 per cent to 0.5 per cent dextrose was used. Two hundred and thirty-six cultures were tested in this solution (Chart 1).


Of these, 19 failed to grow, 107 exhibited extensive growth, and 110 were tested after the growth had degenerated.

The age at which the culture was tested had little bearing upon the hydrogen-ion concentration. The condition of the culture, on the other hand, appeared to have a marked influence, as it was found that cultures that had failed to grow were usually slightly acid. The greater number of those tested when the growth was extensive and healthy were neutral, with a range from pH 6.6 to 7.4 ; whereas, after degeneration had taken place, most of the cultures were slightly alkaline, ranging from pH 6.0 to 7.8.

Influence of Dexteose in the Medium upon the Htdro GEN-ION CoXCENTEATION OF THE CULTURES

Tissues grew extensively in media containing from 0.25 per cent to 2 per cent dextrose. When dextrose was omitted from the solution, the cultures often exhibited an extensive cell proliferation withia 24 to 48 hours, but soon became full of vacuoles, and degeneration took place within a few days (Lewis, 1921). When a large quantity (4 to 5 per cent) dextrose was added, the amount of growth was quite variable ; sometimes large, again small, and in some instances consisting of only a few migrating cells. The cells of this growth, however, seldom contained any vacuoles, even after many da3's. The growths in media containing a large amount of dextrose usually lived longer (1 to 2 weeks) than those in media without dextrose (3 to 5 days), but seldom as long as those in cultures containing a smaller amount of dextrose (2 to 4 weeks).

It was difficult to determine the exact amoimt of dextrose most favorable for the cultures. It varied between 0.5 per cent and 1 per cent, depending upon the series; in some experiments, culture in a medium containing 0.5 per cent dextrose exliibited larger growth and lived longer than those in a solution to which 1 per cent dextrose had been added, wliile in other series the results were reversed. From an examination of the numerous cultures (over 500) explanted into media containing different percentages of dextrose, it was found that 0.25 per cent was seldom sufficient sugar to maintain a healthy growth for many days. The cells began to develop vacuoles within 4 to 5 days and died shortly aftei-wards.

Of the cultures explanted into media without dextrose, 71 were tested at death. Of these 19 had a hydrogen-ion concentration of 7, 34 were pH 7.2, 15 were pH 7.4 and 3 were pH 7.6. The longest period of life was 9 days; the greater number of the cultures died between the third and fifth day. Most of the solutions used as media had a hydrogen-ion concentration of 6.4 and 6.6 ; a few were pH 6.8 or pH 7.0. When dextrose was not added to the medium the cultures did not become acid during growth and degeneration. Those failing to grow, however, were sUghtly acid.

As shown in Table I. Chart I, and Table II, cultures containing 0.25 per cent to 0.5 per cent dextrose behaved in somewhat the same manner. On the other hand, cultures in media containing 1 per cent or more dextrose usually had become acid at death. The range of hvdrogen-ion concentra


THE JOHNS HOPKINS HOSPITAL BULLETIN. MARCH, 1922


f(k~



Fig. 1. — A T-day culture ol cuimeetive ti.s^ue from an 8-day cliK-k tnil)r\u. 'Hit' dark area is the explanted piece and the distance from this to the cells at the top of the page may be used as an axis to indicate the extent of the outgrowth of cells wliich IS meant b.v extensive growth. X 43.


i


Maech, 1932]


115


tion varied greatly in the different cultures. Usually, cultures that contained the most dextrose ivere the most acid. Those in 5 per cent dextrose (the largest amount of sugar used in these experiments) were frequently pH 5.8 to pH 6.0. The length of life of the growth in cultures containing more than

TABLE I Hydbogen-ion Concentration of


The cultures after



pH 01 5 were pH 0.4' ?, were pH


3 "

1 was


pH


10 were pH . S


were pH 6.4 " 6.0

6.3! S " " 6.S

0.4 1 was

6.0

6.S


were pH 6.2 " 6.4


pHS


pH9


4 were pH 0. 6 '• " 7. 4 " "7.


1 was pH ' 3 were " ' 3 "


6 were pH G. 3 " "6.6 3 " " 0.8 1 was " 7.


were pH 0. 0.;


1 was 1 " 3 were


1 was pH 6.0 5 were " 6. 3 " " 7.


1 was pH 6.4


0.8


1 was pH6.6 3 were " 6.S 19 " "7.


3 were [ 3 "

8 "

6 •'


6.4 6.6 6.8


1 was pH 6.

1 " " 6.3

1 " " 6.4

7 were " 6.6

11 " " 6.8

11 " " 7.3

7 " " 7.4

3 " " 7.0

1 was " 7.8

3 were pH 0.4

6 • "6.6

7 " " 6.8


3 " " 7.0 5 " " 7.8

1 was pH 0.6

2 were " 6.8 1 was " 7.

5 were " 7.3


The hydrogen-ion concentration of cultures explanted into media having different hydrogen-ion concentrations (pH 6.0, 7.0, 8.0, and 9.0). The cultures which failed to grow were usually tested after 48 hours, but in some instances not until after three or four days. The age at which the cultures exhibiting extensive growth were tested varied from 24 hours to 14 days.

The final hydrogen-ion concentration was obtained by testing cultures just after the cells had died or while one or two still remained alive. This occun-ed between four days and four weeks, usually at the end of 15 days.

1 per cent dextrose seemed to be dependent upon the rapidity with which the formation of acid took place. When death took place, the cells appeared quite different from those in 0.25 per cent to 0.5 per cent sugar, as vacuolization did not occur nor did the cells round up ; instead they became coagulated, retaining their size and shape somewhat as skeleton forms. The cultures that died after a few days of growth had


already become acid (pH 6.0), while those that still exhibited good growth when tested, even after a much longer period of time (10-14 days), had not yet done so.

The final hydrogen-ion concentration of the tissue cultures depended more upon the amount of dextrose added to the

TABLE II


Without dextrose pH O.S.


Cultures in media without dextrose


With 1% dextrose pH 6.8


Cultures in media with 1% de.xtrose


No. of cultures


pH'


" 7.6 Average final pH^7.35


pH6.

" 6.


Average final pH=6.39


The final hydrogen-ion concentration of cultures explanted into media to which no dextrose was added and into media containing 1 per cent dextrose.



Chart I. — The hydrogen-ion concentration of 236 cultures explanted into normal Locke-Lewis solution.

(broken line) represents the hydrogen-ion concentration of the cultures which failed to grow; (smooth line)

that of cultures which were tested when the growth was ex-tensive; o — o — o — o (dotted line) that of cultures which grew well but were not tested until they had degenerated.

Locke-Lewis solution than upon the original hydrogen-ion concentration of the medium. This is shown in Table II.

Discussion From the work reported in this paper it would seem that the optimum H-ion concentration for growth in vitro of embryonic connective tissue cells of a 9-day cliick lies between 6.8


116


[No. 373


and 7.0. The question immediately arises, Why is the optimum H-ion concentration not 7.4, as it is in the blood of an adult chicken? Although not sufficient cultures were tried at a given pH to make it possible to decide this question, the wide range in which tissue cultures grew would make it appear plausible that growth can occur as well at pH 7.4 (same as blood) as at pH 6.8 to 7.0. However, it should be borne in mind tliat we are deaUng with embryonic tissue, more capable, perhaps, of adapting itself to the experimental environment than are the adult cells. This may account for the wide range of H-ion concentrations in which the cells grow. As stated above, the younger the embryonic tissue the higher the percentage of growth in more acid media. Aggazzotti (1913) has shown that there is a gradual neutralization of the yolk as the embryo develops, changing from a pH 4.6 to a pH 6.4 from the first to the twenty-third day of its development. Granting this to be true, a 9-day chick embryo is developing in a more acid medium than an older one and in a less acid medium than a yoimger one. It seems reasonable that the optimum pH for a 9-day chick might be lower than for the adult animal and that this optimum would be the same as that of the yolk-sac at each respective stage of development.

The carbohydrate (dextrose) metabolism of embryonic connective tissue is seemingly very similar to that of bacteria. In a medium without dextrose, the pH increases as it does with most bacteria grown on ordinary dextrose-free medium, while with 0.25 per cent to 0.5 per cent of dextrose the H-ion level remains the same or swings a little to the acid or basic side from the original fluid. The two factors that influence tMs difference are the size of the drop of medium (buifer) and the condition and size of the explant. However, in a medium of 1 per cent dextrose, the entire buffer effect is used up by the acid produced from the dextrose, and the H-ion concentration is always more acid than the original solution. These results


are strikingly parallel to those found in the study of bacteria under similar conditions.

SUMMABT

Tissue cultures of chick embryos explanted into LockeLewis solution of a H-ion concentration between 5.5 and 9.0 exhibited growth. The medium most favorable for growth was one having a hydrogen-ion concentration about 6.8 to 7.0. The addition of dextrose to the medium was necessary for the healthy growth of cells over a period of time longer than three days. Wliile all cultures in solutions containing up to 5 per cent dextrose exhibited growth, those in solutions containing between 0.5 per cent and 1 per cent had the greatest proliferation of cells and remained healthy for tlie longest period of time. Cultures that failed to grow were usually sUghtly acid, while cultures exliibiting extensive growth, when tested, were as a rule nearly neutral.

The final hydrogen-ion concentrations of the cultures depended upon the amount of dextrose in the medium. Those in solutions to which no dextrose had been added were pH 7.0 to 7.6, while those to which '2 to 5 per cent dextrose had been added were often pH 5.6 to 6.4.

BIBLIOGRAPHY

Aggazzotti, A.: Influenza dell' aria varefatta sail' ontogenesi. Arch, f. Entwcklngsmechn. d. Organ., 1913, XXXVII, 1-2S.

Felton, L. D.: A colorimetric method of determining the hydrogenion concentration of small amounts of fluid. Jour. Biol. Chem., 1921, XLVI, 299.

Lewis, M. R.: The formation of vacuoles in the cells of tissue cultures owing to the lack of dextrose in the media. Anat. Rec, 1921, XXI, 71.

Lewis, M. R., and W. H.: Mitochondria (and other cytoplasmic structures) in tissue cultures. Amer. Jour. Anat., 1915, XVII, 339.

Rous, P.: The growth of tissue in acid media. Jour. Exper. Med., 1913, XVIII, 183.


THE RELATION OF H-ION CONCENTRATION TO SPECIFIC PRECIPITATION

By V. E. Mason

(From the Division of Clinical Pathology oj the Medical Clinic, The Johns Hopkijis University and Hospital)


The purpose of this article is to present the results of a series of experiments designed to determine the relation of specific precipitation to the H-ion concentration of the solution in which the antigen-antibody reaction takes place.

The earlier experiments of Michaelis and Davidson ' were made with combinations of sheep serum as antigen and immune rabbit serum as antibody. The H-ion concentration of the medium in which the precipitation reaction occurred was varied by the use of solutions of sodium acetate and acetic acid. Since non-specific protein precipitation occurred, their results were subject to an error dependent on the difficulty of

^ MichaeUs and Davidsohn, Die Abhilngigkeit spezifischer Filllungsreaktionen von der Wasserstoffionenkonzentration. Biochem. Ztschr., Berlin, 1912, XLVII, 59.


determining tlie amount of such precipitation, as may be readily observed in their protocols. They found that specific precipitation occurred between [H] 3 X 10"^ and [H] 6 x 10"* when the antigen was employed in small amounts. If the dilution of antigen was less, however, precipitation occurred in the H-ion range of primary and secondary phosphate solutions, viz., from pH 5 to pH 9. They did not attempt to define the exact range of [H] in which specific precipitation occurred.

In the series of experiments recorded below crystallized egg albimien was employed as antigen. A precipitating serum was produced in rabbits by three daily intravenous injections of 0.01 gm. of antigen in physiological salt solution. At the end of about three weeks the titer of the immune sonmi


March, 1933]


iir


was usually greater than 1 : 100.000. The hydrogen-ion concentration of the mixtures of antibody and antigen was altered by the addition of various amounts of M/1 NaOH and M/1 HsPO^. Since neither of these is a protein precipitant, confusion due to non-specific precipitation was avoided. Furthermore, wide variations of the hydrogen-ion concentration were possible. The NaCl concentration was kept constant by the use of 0.85 per cent NaCl as diluent for antigen.

Procedure

Three parallel rows of 20 small test-tubes were set up and into each tube 0.1 c. c. of immune rabbit serum was introduced. The precipitin titer of the serum was greater than 1 : 100,000 in each experiment. 0.2 c. c. of a mixture of M/1 NaOH and M/1 HaPO^ was added to each tube in such a fashion that tube 1 of each series contained a solution whose pH was about 4 and tube 20 of each series contained a solution of about pH 10. The pH of the solutions in the intermediate tubes of each series ascended from 4 to 10 according to the typical curve. Antigen was next added as follows : To each tube of series 1, 0.1 c. c. of a 1 : 16..500 solution of egg albumen. To each tube of series 2, 0.1 c. c. of a 1 : 8350 solution. To each tube of series, 3, 0.1 c. c. of a 1 : 1650 solution. The tubes were shaken and placed in a water-bath at 37° for 30 minutes. Certain controls were prejjared as follows :

1. pH solutions plus solutions of egg albumen in the same amounts as used in the experiment.

3. pH solutions plus normal rabbit serum in the same amounts as used in the experiment.

3. pH solutions plus normal rabbit serum plus antigen in the same amounts as used in the experiment.

At the end of 30 minutes the results were read and the relative amount of precipitation noted. The pH of the tubes to right and left of the tube in which precipitation was just


visible was determined by colorimetric methods, the dyes recommended by Clark and Lubs being employed.

Kesults

A series of experiments similar to those outUned above were performed and the results were fairly constant. Although there was occasionally difficulty in detecting minimum amounts of precipitation, the end-point was sharp unless the pH of the solution in adjoining tubes diifered by less than 0.5. In general, precipitation was marked in the tubes which contained solutions whose pH ranged from 9.5 to 4.5 inclusive. The degree of dilution of the antigen was without appreciable effect unless this was greater than the precipitin-titer of the serum or low enough to be affected by the phenomenon of inhibition. Furthermore, although accurate methods for measuring the amounts of precipitation were not employed, there was apparently no more precipitate present in the tubes whose pH was near the isoelectric point of serum globuhn (5.4) or of crystalline egg albumen (4.8).

In a further series of experiments similar to those outlined above, antigen and antibody were allowed to come in contact, with consequent precipitation, before the introduction of acidalkali mixtures. The precipitate dissolved rapidly in the tubes which contained a mixture more alkaline than pH 9.5 or more acid than pH 4.5. In the tubes in which the H-ion concentration was between lO"-^ and 10"^-^ inclusive the precipitate persisted, apparently unaltered.

Conclusions Specific precipitation with the solutions employed above occurred between pH 4.5 and pH 9.5 inclusive. Moreover, specific precipitates permitted to fonn in a neutral medium were dissolved if the pH was reduced to less than 4.5 or increased to greater than 9.5.


NOTES ON NEW BOOKS


Ephraim McDowell (1771-1S30), "Father oj Ovariotomy" and Founder oj Abdominal Surgery, with an appendix on Jane Todd Crawford. By August Schachner, M.D., F.A. C.S., Louisville, Kentucky. (7. B. Lippincott, 1921.)

The discovery of vaccination against smallpox, the discovery of anesthesia, and the Listerian doctrine of antisepsis, were perhaps more dramatic in their presentation and reception by the world, but they were surely not more than coordinates in importance with McDowell's heroic effort in opening up the whole realm of abdominal siu'gery, which is the theme of an admirable life of Epliraim McDowell by Avigust Schachner. of Louis^•ille, Kentucky.

In this dehghtfully written work we have all the data for a complete history of one of our great medical pioneers, if not, indeed, the greatest. It has taken years of patient research to collect all the facts of the life of this great doctor frontiersman, and of his heroic patient, Jane Crawford, so that Schachner's work is conspicuous for its untiring industri', and stands out as a model for future historians.

Our author is quite clear and is fully justified in pointing out that McDowell was not merely the first ovariotomist of the world, who thus


not only maugurated a wholly new procedure, in which lor a couple of generations he easdy outstripped all competitors in his low death rate, but that he by this act of opening the peritoneum revealed the whole nascent realm of abdominal surgery.

Just one hundred and twelve years have passed since in December, 1809, Jane Crawford laid herself, a willing sacrifice, upon the table in McDowell's house, and endured the operation of cutting out a large ovarian tumor, confessedly an " experiment, repeating the Psalms as the doctor proceeded with his bold work. All precedents and the advice of all the eminent teachers of the time were against the innovation and, had it failed, what opprobrium would have fallen upon our progenitor's head; but since this rivulet started in the mountains of Kentucky, all the world has added its tributaries until it has become a mighty stream, destined still to enlarge and to flow on as long as man lives on earth and is subject to disease.

Europe at first disbelieved, then opposed, and then adopted the innovation, and last of all tried to rob the backwoodsman of his credit by faking an earlier operator. But to-day, McDowell's reputation is unassailable, and all rejoice to join with Schachner in placing laurels upon his worthy brow.


118


[No. 373


A further dramatic offset to the mis en scene is found in the objection to the operation raised by McDowell's nephew associate who lent only grudging aid in the rash procedure.

Alas, that we have so few such classics in our American medical literature as this excellent book. Mav it reach many of our doctors.

H. A. K.

The Oxford Medicine. By Various Authors. Edited by Henrt A.

Christian and Sm J.^mes Mackenzie. (Oxford University Press,

London and Neto York, 1921.) An inherently unfortunate feature of a system of medicine is this that for better or for worse it seems necessary to include all the diseases in the domain of the internist. The treatment of certain of the subjects, therefore, which are already covered by classical and altogether satisfactorj- monographs is foredoomed to inferiority. In the fourth volume of the O.xford Medicine one finds several sections which are excellent in themselves, but still suffer in comparison with previous well-known and readilj- accessible treatises. One may mention especially the chapters on the spleen, on the muscles, on the bones, and on sepsis. On the other hand, many of the articles are well worth while. Allbutt writes on gout in the older clinical style, leaving the chemical side to his associates; Longcope gives a brief but thorough summan.' of Hodgkin's Disease; Joslin on diabetes is always interesting, and Rountree summarizes the newer points of view in diabetes insipidus. The infectious diseases are well handled, but in a conventional way; the article on pneumonia by Irons is particularly valuable as a summary' of the bacteriological side of this malady. The sections on industrial medicine are of interest, especially the introductory article on the physiolog>' and pathology of work by Cecil and Katherine Drinker. On the whole the volume seems a useful one.

A. L. B.


The Hospital Bvilletin contains details of hospital and dispensarj' practice, abstracts of papers read and other proceedings of tlie Medical Society of the Hospital, reports of lectures, and other matters of general interest in connection with the work of the Hospital. It is is-siied monthly. Volume XXXIII is in progress. The subscription price is $4.00 per year.

(Foreign postage, 50 cents.) Price of cloth-bound volumes, $5.00 each.

A complete index to Vols. I-XVI of the Bulletin has been issued. Price 50 cents, bound in cloth.

NEW PTJBIICATIONS


The following monograph is for sale by The Johns Hopkins Press. Baltimore, Md.:

Relation of Tonsillar and Nasopharyngeal Infections to General and Systemic Disorders. By S. J. Crowe, S. Shelton Watkins and Alma S. Rothholtz. 63 pages. Price, S125.


PUBLICATIONS


The following twelve monographs :


Benzol as a Leucotoxin. By Laurence Selling, M. D. 60 pages. Price, $1.00.

Primary Carcinoma of the Liver. By M. C. Winternitz, M. D. 42 pages. Price 75 cents.

The Statistical Experience Data of The Johns Hopkins Hospital, Baltimore, Md., 1892-1911. By Fkederick L. Hoffman, LL.D., F.S.S. 161 pages. Price, $2.00.

Venous Thrombosis During Myocardial Insufficiency. By Frank J. Sladen, M. D., and Milton C. Winternitz, M. D. Price, 75 cents.

The Origin and Development of the Lymphatic System. By Florence E. Sabin. 94 pages. Price, $2.00.


Leukaemia of the Fowl: Spontaneous and Experimental. By Harry C. Schmeisser, M. D. Price, $2.00.

are now on sale by The John's Hopkins Press, Baltimore. Other monographs will appear from time to time.


The Structure of the Normal Fibers of Purkinje in the Adult Human Heart and Their Pathological Alteration in Syphilitic Myocarditis. By 0. Van Der Stricht and T. Wingate Todd. Price, $2.00.

The Operative Story of Goitre. The Author's Operation. By William S. Halsted, M. D. Price, $3.50.

Study of Arterio-A'enous Fistula with an Analysis of 447 Cases. By Curle L. Callander, M. D. Price, $2.50.

Ligations of the Left Subclavian Artery in its First Portion. By William S. Halsted. Price, $2.00.

The Patholo,gy of the Pneumonia in the United States Army Camps During the Winter of 1917-18. By William G. MacCallcm. Price, $1.50.

Pathological Anatomy of Pneumonia Associated with Influenza. By William G. MacCallum. Price, $1.50. (This monograph will be on sale within a short time.')


CONTENTS

  • Immunologital Reactions of Bence-Jones Proteins. II. — Differences Between Bence-Jones Proteins from Various Sources. (Illustrated.) By S. Bayne-Jones and D. Wright Wilson .
  • Study on Experimental Rickets. XIX. The Prevention of Rickets in the Rat by Means of Radiation with the Mercury Vapor Quartz Lamp. By G. F. Powers, E. A. Park, P. G. Shu'I-ey, E. V. McCoLLUM and Nina Sm.Aioxns
  • A Graphic Method for the Calculation of Diabetic Diets in the Proper Ketogenic-Antiketogenic Ratio. By R. R. Hax.xo.v, M. D., and Wii. S. McCan.n, M. D.
  • The Alveolar and Blood Gas Changes Following Pneumectomy. (Illustrated.) By George J. Hever and W. D. W. Andrus .
  • Posterior Resection of the Rectum and Rectosigmoid (Kraske or Modified) Under Regional Anesthesia. By Gaston L. Lahat, M. D., Paris
  • The Use of the Bone Graft in the Treatment of Pott's Disease. By William S. B.\er, M. D
  • A Suggested Modification of the Wright Opsonic Technique Based Upon the Differential White Blood Count. By Howard B. Cro.ss 142
  • The Dissemination of Bacteria in the Upper Air Passages. II. — The Circulation of Bacteria in the Mouth. By Arthur L. Bloohfield 14.1
  • Bacterial Nutrition Growth of a Hemophilic Bacillus on Media Containing Only an Autoclave-Stable Substance as an Accessory Factor. By T. M. Rivers, M. D 149
  • On the Presence of Nucleic Acid in Bacteria. (A Preliminary Report.) By Ale.xander J. Schaffer, Caspar Folkoff and F. Bavxe-Jones 151
  • The Role of Situation in Psychopathological Conditions. (Abstract.) By Esther Lori.xg Richariis 152
  • Notes on New Books 153

Books Received 153


IMMUNOLOGICAL REACTIONS OF BENCE-JONES PROTEINS II.-DIFFERENCES BETWEEN BENCE-JONES PROTEINS FROM VARIOUS SOURCES

Bj S. Bayne-Jones and D. Wright Wilson (From the Departments of Pathology and Bacteriology and of Physiological Chemistry, The Johns Hopkins University.)


Since 1847, when Bence-Jones^ described the peculiar substance now known as Bence-Jones protein, many preparations of it from different sources have been studied chemicallj'. It has been well established that this substance is a protein with peculiar properties which render it unique among proteins. Furthermore, as the various preparations of this protein have been found to possess in general similar properties, and as the analyses of the specimens from two cases of Bence-Jones. proteinuria made by Hoplvins and Savory ^ agreed within the limits of their experimental error, there has been a


tendency to assume that all preparations of Bence-Jones protein are identical in structure and composition.

The usual determinations of the chemical constituents of proteins, however, have not afforded sufficient data on which to base an opinion as to the identity of those substances. As is well known, immunological reactions have indicated differences between proteins which were apparently alike. In some cases, as for instance in the comparison of the proteins of the eggs of the hen and duck made by Dale and Dakin,^ there has been found some correlation between the structure of the proteins


]l.'l)


[Xo. Mi


and their s])t'cilic c-Iiaracteristics as antigens. As yet, no innniinoldgiial coniiiarisous of varions preparations of the socalled "Bence-.Iones protein" have been made.

Some aspects of Bence-.Iones proteinuria suggest that the opinion that the substance has an invariable coml)osition may not be jiistilied. Bence-Jones proteinuria has been found in association with multiple myelonut, leukemia, carcinomatosis of bone, diseases of the kidney and otiier obscure conditions. Although the origin of Bence Jones protein in the body is unknown, it is conceivable that the characteristics of the substance excreted in the urine might vary as the result of different pathological states. It is possible also that like the erythrocytes of man, and like bacterial proteins — those of the Pncmnococci for example — the specimens of Bence-Joues protein excreted by different human individuals might have specific differences. The specimens of the protein are not all alike physically. There is certainly a great difference in their tendency to crystallize, or even to precipitate spontaneously; and varying temperatures of coagulation liave been reported. Finally, the method of isolation of the protein from the urine might alter its ])Iiysical. cliemical and immunological characteristics.

The purity of the preparations has undoubtedly affected the results of immunological studies on the broad relationship of Bence-Jones protein to other proteins of iuiman origin, particularly the proteins of human serum. Until recently, it was held that an animal immunized to Bence-Jones protein formed an antibody which i)recipitated liuman serum iiroteins. Massini * and Hektoen ° liave shown that by dilution and absorption of the antibodies in sudi sera it is possible with them to show a difference between Bence-Jones protein and human serum ])roteins. In a recent paper," we have described tlie results of an immunological comparison of Bence-Jones ])i'oteins with human serum. In this, we showed tliat a crystalline Bence-Jones protein acts as a single antigen, causing the production of strictly specific antibodies wiiicli do not affect the proteins of human scrum. On the otlier hand, non-crystalline precipitated preparations of Bence-Jones protein were found to contain traces of human serum, which were responsible for the production of antibodies to human serum when these preparations were injected into animals. Aside from the effects attrii)ntable to coutamination witli traces of other proteins in the i)recipitated pre])arations, we think that the procedures, which will be described below, did not alter the fundamental antigenic (pmlities of the Bence-Jones proteins used in these experiments.

In a jtreliminary note,' we rejiorted tliat we had found immunological differences between various specimens of Bence-Jones protein. In tliis i)aper, we sliall present in detail our evidence for the opinion that Bence-Jones i>rotein is not a single substance, but that a group of similar, but not identical, proteins have been included in this designation.


List of Si-ecimexs of Benck-Joxes I'koteix The various preparations of Bence-Jones protein which ere used in this investigation were obtained as follows:


No.



Palknt


Diagnosis


Isolated by:


Method of isolation.


1.


H. M. R.


Bence-Jon es


D. W. Wilson.


Precipitated with




proteinuria


Reported by


spdium sulphate




without de

Waaters s.


and acetic acid.




monstrable



washed and dried




lesions in



with alcohol and




bones. No



ether.




evidence of






myeloma.




2.


H. M. R.


Do.


Do.


Coagulated by heating at 60° C in slightly acid solution, centrifuged and dried with alcohol and ether.


3.


H. M. R.


Do.



ITrine.


4.


H. M. R.


Do.


Do.


Crystallization.


5.


J. E. L.


Multiple m.veloma?


A. Taylor".


Precipitated like No. 1.


6.


F.


Multiple


Guthrie and


C ag u 1 ated at




myeloma.


Boggs 10.


60° C. in solution made acid with acetic acid, filtered, dried o v er H,SO..


7.


S.


Multiple


Guthrie and


Coagulated, like




myeloma.


Boggs i».


No. 6.


8.


J. E. D.


Carcin o m a


D. W. Wilson.


Precipitated like




with meta

Reported by


No-. 1.




stases to


Walters 8.





bones.




9.


J. E. D.


Do.



Urine.


10.


R. L.


Multiple myeloma.


Do.


Precipitated like No. 1.


11.


R. L. !


Do.



Urine.


12.


?


Multiple myeloma.


Rosenbloom n


Coagulated at 60° C.


The 12 preparations of Bence-Jones protein listed in Table I were derived from 5 patients affected with various diseases. Of tliese preparations, one (No. 4) crystallized spontaneously in the urine and was purified as completely as possible by recrystallization. In the comparison with human serum, which we have described, this protein acted as a single antigen. All the otlier preparations listed above contained traces of human .serum proteins.

The immunological studies of these preparations wei-e made by using precipitin, complement-tixation and anaphylactic reactions.

I'rkcu'itin Ke.vctioxs The antisera to the Bence-Jones proteins used for the precipitin and complement fixation reactions were prepared as follows :


April, 1922]


121


Rabbit No. Ill — was immunized to the crystalline Bence-Jones protein No. 1 by 6 intravenous injections of a 1% solution of No. 4 at intervals of 4 to 6 days. The first dose was 2 cc, the last, 20 cc. Ten days after the last injection, when the rabbit's serum caused a precipitate in a 1 to 1,000,000 dilution of a 1% solution of No. 4, the animal was bled.

Rabbit No. 153 — was injected intravenously at intervals of 4 to 6 days with amounts of a 0.5% solution of protein No. 12, increasing from 3 cc. to 20 cc. Six days after the fifth injection, the rabbit was bled. Precipitin titer: 1-100,000 (dilution of antigen).

Rabbit No. 189 — was immunized to Bence-Jones protein No. 7. After 8 injections of amounts of 2 to 6 cc. of a 2% solution of this protein at intervals of 3 to 5 days, the precipitin titer of the serum was 1-2000 (antigen dilution.).

Rabbit No. 195 — was immunized to Bence-Jones protein No. 6. At intervals of 6 days, 1 cc, 5 cc, and 7 cc. of a 3% solution of this protein were injected intravenously. Five days after the last injection, when the animal was bled, the precipitin titer of the serum was 1-8000 (antigen dilution).

The precii)itin tests were made in small clean sterile tubes with clear solutions of the antigens and clear sterile serum. Precautions were taken to eliminate false results due to the growth of bacteria in the mixtures. The BenceJones proteins were dissolved in salt solution with the aid of a small quantity of NaOH. The resulting solutions were neutralized with HCl until they were only very faintly alkaline to litmus. In the tests, the dilutions were made with normal saline. The pH of all dilutions above 1-100 was approximately 7. The various dilutions of the antigens were layered upon the undiluted antiserums, and the first reading made by noting the presence or absence of precipitate at the plane of junction of the two fluids after they had been in apposition at room temperature for 1 hour. The fluids were then mixed, the tubes placed in the incubator at 37° C. for 24 hours, and a second note made of the sediment in the bottom of the tubes. Controls were made by mixing equal quantities of each component, serum or antigen, with salt solution and by incubating these mixtures with the tests. When precipitation occurred in the controls, the corresponding tests were discarded or repeated. The results of the precipitin tests are summarized in the following tables (Tables II, III, IV, V), in which the amount of precipitate is indicated by plus ( + ) signs.


Precipitin reax;tions of antiserura to crystalline Bence-Jones protein vs. Bence-Jones proteins.


Serum 144 Anti— No. 4



Dilution of antigen






Precipitation






g


i






o


= s


g protein


^








""


^


^


'- 1 "


-


No. 1


+ +


+ ++ +


+ + +


+ +


+





2


+


+ +


+ + +


+ + + +


+ + +


+ +


+


3 Urine


+


+ +


+ +


±






4



+


+ + +


+ ++ +


+ + +


+ +


+


5


+ +

6


+ +


-f








7


+

8


+


+


-f


+


',




9 Urine



+


+


±





10



±








11 Urine



+








12



+ TABLE III.

Precipitin reactions of antisenim to No. 6 vs. Bence-Jones proteins.

Serum 195 Antl— No. 6


Precipitation


Dilution


of antigen





with







Bence-Jones protein


4%


1-10


1-100


1-1000


1-8000


No. 1 2 3 Urine


+








4 5


+








6


+ +


+ + + +


+ + +


+ +


+


7


+ +


+


+





S



+






10


+


±







122


[Xo. 374


Precipitia reactions of antiserum to No. 7 vs. Bence-Jon.es proteins.

Serum 189 Anti— No. 7


Precipitation


Dilution


7f antigen





with







Bence-Jones protein


4%


MO


1-100


1-1000



1-2000


No. 1



+ +


+



2


+


+






3 Urine



+ +


+





4


+








5



±


+


+ +


+


6


+ ++4


+ +


+


+



7


+ ++ +


+ + + +


+ + +


+ +


+


8



+


+ +


+ 4

+


10


+ +


+ +


+


+




Precipitin reactions of antiserum to No. 12 vs. Bence-Jones proteins.

Serum 153 Anti— No. 12



Dilution


of antigen




Precipitation

with

Bence-Jones

protein


4%


110


1-100


1-1000


1-10,000


No. 1


+








2 3 Urine


+


±






4 5


+


+


+ + +


+ + +


+


6


+ +


+ +


+ + + +


+ + +


+ +


7



+


+ +


+ + +


+


8





+


±



9 Urine


+


+ +


+ + +


+ +



10





+ +


+ + +


+


11 Urine


+


+


+ +


+ + + +


+


12



+


+ +


+ + +


+


The results of the precipitin reactions presented in Tables II, III, IV and V may be summarized as follows:

(a) The highly potent antisei'um to the crystalline Bence-Jones protein (Xo. 4), having a titer of 1-1,000,000


for its homologous antigen, precipitated all the preparations of Bence-Jones protein. All, however, were by no means afifected to the same degree. This antiserum showed a definite affinity for the preparations derived from the patient H. M. R., precipitating Nos. 2 and 4 to the endtiter, No. 1 in a dilution of 1-10,000, and a 1-1000 dilution of No. 3, which was this patient's urine. Proteins Nos. 8 and were precipitated by this antiserum in dilutions up to 1-1000, while Nos. 5, 6, 7, 10, 11 and 12 were not precipitated when diluted above 1-50. As a consequence of its high titer, this antiserum demonstrated the group or class relationship of the various preparations, and at the same time indicated the sub-grouping among them. It is to be recalled that the preparation used to immunize the rabbit which produced this serum was free from human serum proteins. This antiserum did not precipitate human serum. The precipitin reactions with it, therefore, are strictly specific for BenceJones proteins.

(ft) The antiserum to Bence-Jones protein No. C, having a titer of 1-8000, showed no affinity for the isolated protein from patient H. M. R. (Nos. 1, 2 and 4). As preparation No. G contained a trace of serum proteins, its antiserum also precipitated human serum. To this is to be attributed the precipitate produced when it was added to No. 3, the urine from H. M. R., which contained some serum proteins. While this antiserum slightly afifected the other preparations of Ifence-Jones proteins, it did not precipitate any one of these in a dilution above 1-100.

(c) The antiserum to preparation No. 7 also included a precipitin for human serum proteins, as its antigen contained traces of these proteins. It caused precipitation with all the preparations of Bence-Jones proteins. The preparations from the patient H. M. R., however, (Nos. 1, 2 and 4) were precipitated only in concentrated solutions. On the other hand, proteins Nos. 5, 6, 8 and 10 were precipitated by this serum to its end-titer.

(d) As Bence-Jones protein No. 12 contained traces of human serum proteins, its antiserum precipitated human serum. This antiserum did not precipitate preparations Nos. 2 and 4, the coagulated and crystalline protein from patient H. M. R. Preparations Nos. 1 and 3 from the same patient, containing traces of serum proteins, were precipitated in concentrated solutions by the antiserum to No. 12. The other preparations were precipitated to almost the end-titer by this serum.

While this series of comparisons is not large, it indicates that there are aniigenic differences between these


April, 1922]


123


preparations of Beuce-Jones proteins. Two distinct groups are recognizable, namely :

Groui' I. — Crystalline Bence-.Tones protein No. i, and other preparations, Nos. 1, 2 and 3 from patient H. M. K.

Group II. — Beuce-Jones proteins Xos. 5, 6, 7, S, 10 and 12.

lu addition, tliere are certain less sharply defined differences between the proteins listed in Gi-oup II. For e.xaniple, proteins Xos. 5 and 6 seem to be representatives of different groups.

An attempt was made to define these groups more sharply by applying the method of the absorption of precipitins. The conditions which at present limit the applicability of this method were discussed in our previous paper in relation to the removal of the antibody to human serum proteins from some of these antisera. The method was found to be unsatisfactoiy with the sera and solutions of proteins used in this investigation.

CoMPLEJiEXT Fixation Keactions

Tests were made to determine the ability of the antisera to the crystalline Bence-Jones protein No. 4 and to protein No. 12 to fix complement in the presence of the other Bence-Jones proteins. The sera were diluted with equal quantities of normal salt solution and heated to 56° C. for half an hour. To 0.25 cc. of each serum was added 0.25 cc. of a 1-20 dilution of a ifc solution of each Bence-Jones protein, or a 1-20 dilution of urine containing Bence-Jones protein. These dilutions were found by preliminary titrations to be beyond the limit of their anticomplementary action, except in the case of No. 2. They were, however, just within the range of the effective concentration for the occurrence of precipitation. These sterile mixtures were allowed to stand for 10 hours at 20° C. to permit precipitation to occur. At the end of that time, 0.25 cc. of a 1-10 dilution of fresh guinea-pig serum was added to each tube, and the tubes incubated in the water-bath at 37° C. for 1/2 hour. After this incubation, 0.25 cc. of antisheep red-corpuscle serum, containing 3 units of amboceptor, and 0.25 cc. of 2.5% suspension of sheep corpuscles were added, and the tubes returned to the water-bath. Readings of the presence or ab.sence of hemolysis were made at the end of 1 hour, when all the controls showed the expected results. In the following table, in which the results of these tests are collected, the degree of complement fixation is indicated by plus signs ( + ).


TABLK VI. Jomplement Fixation Reactions.



Bence-Jones


Dilution of



Serum


antigen


antigen


Results of fixation tests


Serum 144


No. 1


1-40


+ +


Anti— No. 4


2


1-20


anticomplementary


Dil. 1-2


3


1-20


+ + -f +



4


1-20


+ + + +



5


1-20


+ +



6


1-20


+



7


1-20


0?



8


1-20


+ +



9


1-20


+ +



10


1-20


-V



11


1-20


+



12


1-20


+


Serum 153


No. 1


1-20



Antl— No. 12


2


1-20


anticomplementary


Dil. 1-2.


3


1-20




4


1-20




5


1-20




6


1-20


+ +



7


1-20


+ +



8


1-20


+



9


1-20


-f



10


1-20


+



11


1-20


+



12


1-20


+ +++


The complement fixation reactions show the general group relationship between the Bence-Jones proteins, and in addition they confirm the results of the precipitin reactions in demonstrating a great antigenic difference between Bence-Jones protein No. 12 and the preparations from patient H. M. R., represented by the crystalline protein No. 4.

Anaphylactic Reactions

The anaphylactic responses of guinea-pigs and of smooth-muscle preparations from these animals were used to continue the immunological analysis of the BenceJones proteins. Young females, weighing about 150 grams, were sensitized by an intravenous injection of 1 cc. of a 1.5% solution of some of the proteins. This injection was not followed by any toxic symptoms. After an interval of 18 to 21 days the second intoxicating injection was given intravenously, or uterine horns were excised from some of the sensitized virgin guinea-pigs and used to obtain graphic records of the anaphylactic contractions according to the method of Schultz and Dale.'^ Sensitization was easily effected by a single in


124


[Xo. 374


jection of the proteins, except No. 4. For the experiments with this protein it was necessary' passively to sensitize the gninea-pigs by an intraperitoneal injection of 1 cc. of serum 144, whidi contained precipitin specific for l>rotein No. 4. Eighteen hours after this injection the animals were found to be hypersensitive to protein No. 4. The data of the experiments and the i-esults of the anaphylactic reactions are summarized in Table VII and Figures 1, 2 and 3.

In interpreting the data of these anaphylactic reactions, it is to be borne in mind that all the preparations of


The anaphylactic reactions confirm the results of the experiments with precipitins and complement fixation. They show clearly that the protein from H. M. R., represented typically by the crystalline preparation Xo. 4, is distinct from the others, and that Bence-.Iones proteins Xos. 5 and 12, while having some similarity, are probably representatives of other groups.

Summary and Comment

Twelve preparations of Bence-.Jones protein obtained by various procedures from the urines of 5 patients, who


TABLE VII. Anaphylactic Reactions.


Guinea Pig


Sensitizing dose

of

Bence-Jones protein


In


terval


Intoxicating dose

of

Bence-Jones protein


RESULT


230


No.



1 cc. 1.5% sol.


19


days


No. 1, 2 CC. 4% sol.


Typical shock, death in 1 min.


231


No.



Ice. 1.5% sol.


19


days


Xo. 4, 1.5 CC. 4% sol.


Slight reaction.


232


No.



Ice. 1.5% sol.


19


days


No. 5, 1.5 cc. 4% sol.


Shock. Death.


233


No.



1 cc. 1.5% sol.


19


days


Xo. 8, 1.5 cc. 4% sol.


-Shock. Death.


234


No.



1 cc. 1.5% sol.


19


days


Xo. 10, 1.5 cc. 4% sol.


Shock. Death.


194


No.


5,


1 cc. 1.5% sol.


19


days


Nos. 1. 4, 6, 7, 8, 10, 12,

1 cc. each. No. 5. See Figs. 1 and 2.


Excised uterus.

No contraction caused by Nos.

1, 4, 6, 7, 8, 10, 12. Strong contraction caused by

No. 5.


293


Nt7.


5.


1 cc. 1.5% sol.


19


days


Xo. 5, 0.23 cc. 4% sol.


Severe Shock.


294


No.


5,


1 cc. 1.5% sol.


19


days


Xo. 4. 1.5 cc. 4% sol.


No reaction.


296


N(7.


5,


1 cc. 1.5% sol.


19


days


Xo. 1, 1.5 cc. 4% sol.


No reaction.


270


No.


12,


1 cc. 1.5% sol.


19


days


No. 12, 0.75 cc. 4% sol.


Shock. Death.


271


No.


12,


1 cc. 1.5% sol.


19


days


No. 5, 1 cc. 4% sol.


Shock. Death.


272


No.


12


1 cc. 1.5% sol.


19


days


Xo. 4, 1 cc. 4% sol.


No reaction.


273


No.


12


1 cc. 1.5% sol.


19


days


No. 1, 1 cc. 4% sol.


No reaction.


274


No.


12


Ice. 1.5% sol.


19


days


No. 1, 1 cc. 4% sol.


No reaction.


5


Passively sensitized to


18


hours


1 cc. 4% solutions of Nos.


No contraction caused by Nos.





No. 4




5, 6, 7 and 4. See Fig. 3.


5, 6 7. Strong contraction followed by desensitization caused by No. 4.


Bence-Jones proteins, except No. 4, contained traces of human serum proteins. As we were unable to measure the amounts of serum proteins in the various preparations, we are unable to correlate the results of these reactions with that factor. The solutions of some of the preparations may have contained sufficient serum proteins to sensitize the animals to those proteins as well as to the Bence-Jones protein component of the mixture, while they may or may not have contained enough serum proteins to cause shock attributable to serum proteins in the intoxicating dose. The error due to the common effect of the presence of serum proteins would undoubtedly confuse the result, and would tend to emphasize the similarity of the preparations of Bence-Jones protein. Nevertheless, the differences exhibited by some of the proteins used in these reactions are so considerable that their significance in most instances is quite clear.


had Bence-Jones proteinuria associated with several different diseases were compared immunologically. Precipitin and complement fixation tests and anaphylactic reactions were studied with these preparations and the antisera obtained by immunizing rabbits to some of them. One of the proteins. Xo. 4, was crystalline and acted in this, as in a j)revious investigation," as a single antigen, free from serum proteins. The other preparations precipitated from urine by various means, contained traces of human serum proteins. As these preparations contained at least two antigens, one of which was not precisely measurable, some difficulties and confusion attended their use. Xevertheless, the results obtained in experiments \\ith the crystalline protein were unequivocal, and most of the reactions with some of the other preparations were sufficiently clear for the purposes of this investigation.


THE JOHNS HOPKINS HOSPITAL BULLETIN. APRIL. 1922


PLATE -XVII


'-i




Fig. 1.— Uterus of guinea pig actively sensitized to Bence-Jo-nes protein No. 5. Guinea pig 194. At A: 0.5 cc. i% Bence-Jones protein No. 4 At B: 0.5 cc. 4% Bence-Jones protein No. 1 At C: 0.5 cc. 4% Bence-Jones protein No. 12 At D: 0.5 cc. 47r Benc^Jones protein No. 5

Fig. 2. — Uterus o-f guinea pig 194 activel.v sensitized to BenceJones protein No. 5.

At A: 0.5 cc. i% Bence-Jones protein No. 10

At B: 0.5 cc. 4% Bence-Jones protein No. 8

At C: 0.5 cc. i</c Bence-Jones protein No. C

At D: 0.5 cc. 4% Bence-Jones protein No. 7 At E: 0.3 cc. human serum

At F: 2 cc. 4% Bence-Jones protein No. 5

Fig. 3. — Uterus of guinea pig No. 5.

Guinea pig passively sensitized to Bence-Jones protein No. 4 by intraperitoneal injection of 1 cc. of serum 144. Reaction tested IS hours later.

At T: 1 cc. 4% Bence-Jcnes protein 5

At R: bath changed.

At F: 1 cc. 4% .Bence-Jones protein No-. G

At S: 1 cc. 4% Bence-Jones protein No. 7

At 4: 1 cc. 4% Bence-Jones protein No. 4


April, 1922]


125


Differences and similai-ities were demonstrated among these proteins. The methods used in isolating the proteins had no appreciable effect upon their immunological relationship, nor was there any obvious correlation between the types of the proteins and the diseases affecting the patients who excreted them. Of course, more specimens of Bence-Jones protein from various different pathological, and perhaps normal sources must be examined in this manner before it can be asserted positively that neither the disease with which the proteinuria is associated nor the method of isolation of the specimen determines the basic antigenic character of the protein. It is suggested, however, that such differences as exist may be related to the fundamental impress which an organism, bacterium or animal, places upon most of the proteins which are formed in its body.

From the immunological reactions of the preparations used by us, we are able to draw the following conclusions :

Conclusions. 1. Under the term "Bence-Jones protein" have been grouped a number of proteins which are similar but not identical.


2. Certainly two, and possibly three groups of BenceJones proteins are recognizable by immunological tests.


BIBLIOGRAPHY


J. Physiol.. Cambridge, Biochem. J., Cambridge, Leipzig, 1911,


1. Bence- Jones, H.: Philosophical Transaction, Royal Scciety, London, 1848, CXXXVIII, 55.

2. Hopkins, F. G. and Savory, H.

1911, XLII, 189-250.

3. Dakin, H. D. and Dale, H. H.: 1919, XIII, 248-257.

4. Masslnl, R.: Deutsch. Archiv f. klin. Med., CIV, 29-43.

5. Hektoen, L.: J. Am. Med. Assn., Chicago, 1921. LXXVI, 929.

6. Bayne-Jones, S. and Wilson, D. W. : Johns Hopkins Hosp. Bull., 1922, XXIII, 37-43.

7. Bayne-Jones, S. antl Wilson, D. W.: Proc. Soc. Exp. Biol, and Med., 1921, XVIII, 220-222.

8. Walters, W.: J. Am. Med. Assn., Chicago, 1921, LXXVI, 641-645.

9. Taylor, A. E. and Miller, C. W.: J. Biol. Chem., Baltimore, 1916, XXV, 281-295.

10. Hoggs, T. R. and Guthrie, C. G.: Am. J. Med. Sciences, Philadelphia, 1912, CKLIV, 803-814.

11. Rosenbloo-m: J. Arch. Int. Med., Chicago, 1912, IX, 256-257.

12. Dale, H. H.: J. Pharmacol, and Bxper. Therap., Baltimore,

1912, IV, 167-223.


STUDY ON EXPERIMENTAL RICKETS. XIX.

THE PREVENTION OF RICKETS IN THE RAT BY MEANS OF RADIATION WITH THE MERCURY VAPOR QUARTZ LAMP

By G. F. Powers and E. A. Park

(Dept. of Pediatrics, Yale University, New Haven, Conn.)

P. G. Shipley (Dept. of Pediatrics, Johns Hopkins University, Baltimore, Md.)

E. V. McCOLLUM AND NiNA SiMMONDS (School of Hygiene, Johns Hopkins University, Baltimore, Md.)


In a previous article ^ we showed that when rats were placed on a rickets-producing diet (diet 3143) they did not develop rickets if they were exposed to direct sunlight.


1 The Prevention of the Development of Rickets in Rats by Sunlight. XI. P. G. Shipley, Dept. of Pediatrics, Johns Hopkins University, Baltimore, Md., E. A. Park, G. F. Powers, Dept. of Pediatrics, Yale University, New Haven, Conn., E. V. McCollum and Nina Simmonds, Schoc?l of Hygiene, Johns Hopkins University, Baltimore, Md. Proc. Soc. for Exper. Biol, and Med.. Oct. 19, 1921.

STUDIES OX EXPERIMENTAL RICKETS. XIV. The Prevention of the Development of Rickets in Rats by Sunlight. P. G. Shipley, Dept. of Pediatrics, Johns Hopkins University, Baltimore, Md., E. A. Park, G. P. Powers, Dept. of Pediatrics. Yale University, New Haven, Conn., E. V. MicCollum and Nina Simimonds, School of Hygiene, Johns Hopkins University, Baltimore, Md. Jour. Am. Med. Assn., Jan. 21, 1922, Vol. IS, pp. 159165.


In the resume of the literature on the subject of liglit in relation to rickets which is introductory to that article, attention was called to the fact that radiation other than sunlight had been used to prevent or cure rickets in human beings. Buchholz used the "Gliihlicht" which he stated was poor in chemically active rays; Huldschiiisky, Putzig, Karger, Riedel, Erlacher and Mengert reported the use of the mercury vapor quartz lamp, and Winkler the use of the X-ray.

All the evidence as to the preventive and curative effects of the radiations from the mercury vapor quartz lamp in the rickets of human beings has been furnished by the X-raj'. In order to determine the protective action of these radiations in experimental rickets in rats and also to examine the bones themselves we perfonned the following experiments :


12G


[No. 374


Nineteeu rats, mostly mixed black and white aud about seveu weeks old, were placed on diet 3143 which, as previous experieuce has shown, produces rickets comparable iu every respect to the rickets mauifestiug itself in human beings. The ratiou has the following composition:

Per cent

Wheat 33.0

Maize 33.0

Gelatin 15.0

Wlieat Gluten 15.0

NaCl 1.0

CaCOa 3.0

It contains nearly twice the optimal content of calcium aud is decidely below the optimum in its content of phosphorus and of fat-soluble A. Otherwise it is well constituted. Nine rats were kept as control animals under ordinary laboratory' conditions in a room completely screened with windows of ordiuaiy glass. Ten rats were exposed to the radiations from a Hanovia mercurj- vapor quartz lamp (Alpine type).

One of the control animals (16Y) was found paralyzed thirty-eight days after being placed on the diet (age about eighty-eight days) and was killed. We have previously pointed out that the development of paralysis of the posterior extremities not infrequently occurs in rats fed on diet 3143. Another control animal (2CY) was killed fifty-eight days after being placed on the diet (age about one hundred and eight days) ; and the other seven animals were killed sixty-four days after being placed on the diet (age about one hundred and fourteen days).

The rayed animals were exposed to the radiations from a mercury vapor quartz lamp for varying periods of time for sixty-four days aud were then killed. The animals were exposed to the radiations for two minutes ou the first day; the period was gradually increased during the succeeding seven days to one hour daily ; for the succeeding fifty-six days of the experimental period the animals wei'e rayed for two to six hours daily, the length of time being determined largely by the availability of the lamp. The cage used was constructed so that animals could not be shielded iu any way from the radiations. The floor of the cage was placed three feet from the quartz tube.*

The rayed animals never showed conjunctivitis, but

» STUDIES ON EKPERIMEINTAb RICKETS. VIII. The Production of Rickets by Diets Low in Phosphorus and PatSoluble A. McCollum, E. V., Simnionds, Nina. Shipley, P. G., Park, E. A. Jour, of Biol. Chem., Vol. XLVII. Ko. 3, August, 1021.


We have found since the conclusion of these experiments that, before our use of the Alpine Lamp was begun, it had depreciated about eighty-five per cent (estimated) in its output of ultra-vioiet rays. This estimate was purely a guess made by a representative of the Hanovia Company, but of its essential truth we have no doubt. Prolonged exposure to the radiations of the lamp has not caused pigmentation of the skin in children.


early in the experiment spots of pigmentation on the ears of some of them were observed; this was especially markcvl iu the albinos. This pigmentation was not present at the time the animals were killed. The albinos among the rayed animals showed slight yellow tinting of the hair ou their backs.

As the experiments progressed, certain differences in the behavior of the two groups of animals were ob.served. The rayed animals were extremely active, the contrast between the two groups becoming more and more marked and reaching its maximum in the second third of the experimental period. Whenever any one came near the cage in which these animals were, a constant scurrying to and fro was almost always the striking feature observed. Not only were the control animals markedly less active, but their gait was distinctly waddling in character after the third or fourth week of the experiment. The disability of the control animals was well illustrated by the fact that toward the end of the experiments seven of the animals got out of their cage and were all recovered with relatively little difficulty.

The rayed animals always seemed to be hungry and, al though the food given to each group was not measured, it was obvious that the amount consumed by the animals receiving mercury lamp radiations greatly exceeded that consumed by those in the control group. Increased appetite, then, was a striking characteristic of the rayed animals.

One of the rayed animals gave birth to six young on the thirty-seventh day of the experiments.

The autopsies revealed umny differences in the animals. As shown in the following table, the measurements corro borate the observations made as to the better physical development of the animals exposed to the radiations from the mercun' vaiior quartz lamp. P" "m

' ' '^ Increase in

Conlrol RaJialed Radiated over

Animals (8) -Animals (9) Controls

Average Weight 140 grams. 157 grams. 12

Average Nose-Tail Length 31.6 cm. 34.5 cm. 9

Average Nose-Rump Length 15 cm. 16.1 cm. 7

Average Tail Length 16.6 cm. 18.4 cm. lO

The hair of the rayed animals was noticeably thicker and coarser than that of the control animals. The hair ou the backs of the albino rats exposed to ultra-violet rays was tinted a slight lemon yellow. There \Aas no enlargement of the epiphy.seal juucfions iu the rayed aiii mals. On opening the bodies of the rayed aiiimal'< the most striking feature was the great amount of tal ileposited in the subcutaneous tissue and iu the i)eritoneal cavity. In some animals this deposition was treiuen dous. It occurred in some of the control rats but was never great. The muscular development of the rayed rats was better than that of the control animals. There were no deformities of the thorax aud no gross evidences of rickets in the long bones of the rayed auinuils.

The control rat (IGY). autopsied thirty-eight days after the beginning of the experiment, showed the tyi>ical pic


THE JOHNS HOPKINS HOSPITAL BULLETIN, APRIL. 1922








Fig. 3


Fig. 4


April, 1922]


127


ture of advanced rickets. The iucisoi- tcetli were brittle, tlie molar teeth were loosened ; there was eiilargemeut of the knees and wrists; the costo-choudral junctions were enlarged and bent inwards, and there were numerous fractures of the ribs. The tibiae and femora on section showed the typical gross picture of rickets.

The other control animals, autopsied about four weeks later, showeil less marked but equally definite signs of rickets. The teeth were only slightly more easily fractured than normal. There was moderate enlargement of the epiphyses of the long bones of the extremities. The costo-choudral junctions were enlarged but there was little deformity of the chest and no well marked fractures of the ribs. The bones of the e.xtremities cut with diminished resistance and in the femora of some there were gross evidences of rickets on section. The viscera showed no pathological chang&s. The amount of subcutaneous and peritoneal fat was much le.ss than in the rayed animals. The muscles of the control aninuils were relatively poorly developed.

Microscopic examinations of the bones were confirmatory of the gross findings. In .sections of the long bones of the rayed animals there were no evidences of rickets. The Hue of demarcation between the cartilage and shaft was sharp and clear cut. The deposition of calcium in the provisional zone of cartilage was heavy and complete. The trabeculae showed only the thin nuirgin of osteoid which is seen in healthy, growing animals and which must be regarded as physiological.

The sections of the long bones of the control animals without exception showed rickets, but in some specimens the rachitic process was moderate in severity. Tlie trabeculae in all sections wei'e surrounded by broad osteoid borders and the calcification of the provisional zone of cartilage was fragmentary and exceedingly irregular. The degree of rickets usimllj- found in rats on this diet is more severe than is shown in some of the si)ecimens from these animals. This is probably explained by the fact that the animals were fifty days old when placed on the diet and were in some instances one liundred and fourteen days old when killed. During the period covered by the experiments, therefore, activity in growth was declining and with it there was a decline in the activity of the rachitic process.

Discussions

From this experiment it is possible to say tluit rats fed on the rickets-i)roduciug diet (diet '.MA'^) are protected from timt disease by exposure to radiations from a merCU17 vapor quartz lamp. It is possible to say further that radiations from the mercury vapor quartz lamp affect not the skeleton alone but indeed the whole organism. While the development of a normal skeleton in the rayed animals is a "striking, visible and measurable" efl'ect of the radiations on a single tissue, growth, good muscular development, storage of fat, improvement in the condition of the hair, stimulation of sexual development and


reproductive power are evidences that the radiations have a favorable influence upon the animals as a whole. ISo far as we are able to discern, the action upon rats fed the rickets-producing diet (diet 3113) of the radiations of a mercury vapor quartz lamp in securing an efficient utilization of the substances which are directly or indirectly concerned with ossification and calcification and in promoting general bodily vigor is in no way ditterent in respect to these matters from the action of cod-liver oil and of sunlight.

Summary

1. The object of the experiments was to determine whether or not radiations from a mercury vapor quartz lamp prevent the development of rickets in the rat.

2. A diet was employed which at room light regularly gives rise to a disease identical in its es.sential features with rickets as seen in the human being. The diet was high in calcium, low in phosphorus and was insufficiently supplied with fat-soluble A. In other respects it w'as well constituted.

3. Nineteen rats were placed on the diet. Ten were exposed to radiations from a Hanovia "Alpine mercui-j' vapor quartz lamp for varying periods of time daily over a period of sixty-four days. Nine rats were kept under conditions of ordinary room light as control animals.

1. One of the control animals was killed after thirtyeight days; another after fifty-eight days and the remaining seven after sixty-four days. All of these animals showed gross and microscopic evidences of rickets.

5. The ten rats exposed to the radiations from the mercury vapor quartz lamp were killed after sixty-four days. These aninuils were free from rickets both grossly and histologically.

6. The beneficial effects of the radiations from the mercui-j- vapor quartz lamp were not limited to the skeleton, since the condition of the rayed aninuils underwent a general improvement.

7. The effects of the radiations of the mercury vapor quartz lamp on the growth and calcification of the skeleton of the rat and on the animal as a whole seem to be similar to, if not identical with, those brought about by direct sunlight ami by cod-liver oil.

DESCRIPTION OF PLATES Figs. 1 and 2. Mdcrophotographs of sections of femora of rats fed on diet 314 3 and exposed to radiations from a mercury vapor quartz lamp. Note sharp demarcation between shaft and cartilage at metaphysis (a), the heavy calcification of provisional zone of cartilage (b) . and the wide bony trabeculae without osteoid borders (d).

Figs. 3 and 4. Microphotographs of sections of femora of rats fed on diet 3143 and kept at room light. Note wide irregular metaphysis. Fig. 3. a — Proliferative cartilage.

a — Islands of cartilage with (bFig.3) deposition of

calcium on the diaphyseal side, c — Osteoid trabeculae. Fig. 2. b — Cartilage is irregular and uncalcified.


128


[No. 374


A GRAPHIC METHOD FOR THE CALCULATION OF DIABETIC DIETS IN THE PROPER KETOGENIC-ANTIKETOGENIC RATIO

By R. R. Hannox, M.l)., and Wm. S. McCann, M.D.

{From the Chemical Division, Medical Clinic, Johns Hopkins Hospital, Baltimore, Md.)


The study of the balance between ketogenic and antiketogenic substances in the metabolism, having been put on a quantitative basis by the admirable work of Shaffer/' -• ^- almost immediately found its application in the treatment of diabetes mellitus. Shaffer found that the ketogenic substances were the fatty acids, and certain of the amino acids of protein. The antiketogenic substances were glucose, the glucose yielding amino acids of protein and the glycerol of the fat, which is known to be capable of yielding glucose.* It was found that the complete oxidation of ketogenic substances did not take place unless there were present at least one molecule of antiketogenic substance for each molecule of the ketogenic. Shaffer arrived at these conclusions on the basis of in vitro experiments, metabolic observations on starving subjects and diabetic patients, including studies of the respiratory exchange.

Woodyatt '^ has applied Shaffer's data to the calculation of diets for diabetic individuals in whom it is desirable to preserve the proper balauce between ketogenic and antiketogenic substances. He has converted Shaffer's figures for the molecular proportions into terms of grams of the various foodstuff's. The antiketogenic substances are reduced to the term available glucose or G, which eciuals the sum of glucose available from free carbohydrate, the carbohydrate moiety of the protein molecule, and that which may arise from glycerol of the fats. Thus G=0.5S P+0.1 F+C, where r=grams of protein, F=grams of fat, and C=grams of carbohydrate.

In calculating the ketogenic substances, expressed as available fatty acids or FA, Woodyatt has not followed Shaffer's figures closely. For in.stance, 42% of the weight of protein is made up of amino acids which do not yield glucose. Of these only three, leucine, phenylalanine, and tyrosine, are known to be ketogenic, making up only 23% of the total weight of protein. Woodyatt calculates FA=0.46 P+0.9 F. In view of the unknown behavior of the remaining amino acids of protein it is believed that Woodyatt's estimate is on the safe side.

Woodyatt estimates that the equimolecular ratio of ketogenic to antiketogenic substance obtains when FA 1.5 0.46 P -f 0.9 F


G 1 0.58 P -h 0.1 F + C

This on being simplified gives the expression F=2 C+0.546C P.

In discussing the objects of dietary adjustment in diabetes, Woodyatt has called attention to a fundamental


conception which has been overlooked by practitioners who have followed the methods of treatment of diabetes largely in vogue in the past few years. This is the fact that the tolerance of the patient for glucose cannot be estimated simply from the amount of free carbohydrate in the diet, but that it must be estimated in terms of available glucose from all sources. That since the metabolism goes on uninterruptedlj- during starvation considerable amounts of glucose maj' be arising in the organism especially if the protein metabolism is high. He showed iu an illustrative case, a patient with severe diabetes in whom the protein metabolism was presumably high (urinary nitrogen figures not given), that the giving of a diet low in protein with considerable fat and free carbohydrate was well borne. This was presumably' due to a sparing of body protein by fat, in keeping with the results of Newburgh and Marsh," though unfortunately exact calculations cannot be nuide, as there were no data regarding urinary nitrogen or heat production of the patient, except a rough calculation of the requirements based on body weight.

However, the conceptions of diabetes set forth above seemed so rational that it was determined to Introduce it iu the practice of the medical clinic of the The Johns Hopkins Hospital. Since its introduction very satisfactory results have been obtained. To facilitate the work of preparing the diet prescriptions one of us (R. H.) has prepared the graphic chart. Fig. 1, which has been so useful that we have been led to make it available for general use. All points on the graph bear the relationship between protein, fat and carbohydrate expressed in Woodyatt's equations previously given. The chart is given much additional value by reason of the fact that it provides for the calculation of this relation.ship over a wide range of levels of protein metabolism. In the plan outlined by Woodyatt " the total caloric I'equirement of the patient was estimated on the basis of body weight, and an arbitrary figure for protein was used, 1 gram protein per kilogram of body weight. It seemed more rational to estimate the measure of total metabolism iu terms of calories per square meter of body surface area, and to consider the protein metabolism in terms of percentage of the total. Thus, on the graph, diagonal lines represent the total calories. Radiating lines from the intersection of the axes represent the percentage of total calories in the form of protein. The ordinates represent grams of protein, the abscissae the grams of carbohydrate which are required to maintain ketogeuic-antiketogenic balance for any given level of protein metabolism. Thus


THE JOHNS HOPKINS HOSPITAL BULLETIN. APRIL. 1922


PLATE XVIII


/v?


/0%



10 20


B


X 40 50 60 70 ao 90 100 HO 120 130 140 150 160 170 160 190 CARBOHYDR/iTE IN GRAMS.


April, 1922]


129


if the patient requires 2000 calories per dieru, and if it is desired to give lO^o of the calories in the form of protein, the point of intersection of the 2000 caloric line and the 10% line locates on tJie ordinate 49 gms. of protein, and on the abscissa 68 gms. carbohydrate.


Total calories — (calories fro m protein-fcaibohydrate) _ 9.3


gi-ams fat.


To avoid this latter calculation a second graph has been superimposed upon the fli-st. A vertical line AB, parallel to the ordinate axis, is intersected at various points by the radiating percentage lines. For each intersecting point a second ordinate scale gives the ratio of fat to carbohydrate ¥/C This is the factor by which the weight of carbohydrate must be multiplied to give the requisite number of grams of fat.

Thus the 10% line intersects the line AB at a point which indicates that F/C is 2.39. Then the number of grams of fat required =2.39X68=162.5. The prescribed diet would then be protein 49 gm., carbohydrate 68 gm., fat 162.5 gm. This diet would furnish 1920 calories, and would yield a maximum of 113 grams of glucose in the metabolism.

If the minimal protein metabolism for a particular diabetic jiatient were known (urine N. figures), and if his caloric requirement were properly estimated or measured, it would then be possible to find the minimum carbohydrate and the maximum fat allowable if the patient is to be maintained at the minimal level of nitrogen balance. For instance, a man 30 years old, 170 cm. in height, weighing 60 kg., requires at least 1612 calories per diem. After a period of observation on a diet furnishing 5% of the calories from protein, it is found that his protein metabolism, as shown by the urinary nitrogen excretion, has reached a minimum of 50 grams per diem. Reference to the chart shows that this constitutes 12.6% of his total metabolism. If a diet is to be given which will just cover this minimal protein requirement (50 gm. protein), the quantity of carbohydrate which is prescribed will be 51 gms., and of fat 2.53X51 gins.=129 gms.

It will be found interesting to make the following calculation of two diets each furnishing 2000 calories from the graph. In one case the protein forms 40% of the total energy value. In the other case protein furnishes only 10% of the total energj\


40% Trotein Cals. 10% Protein Cals.


2000 calorie diets. Available

P F C Glucose

195 125 9 134.6

49 165 68 112.9


It will be seen that a patient, whose tolerance might be just sufficient to permit the taking of 2000 calories with the smaller amount of protein, would probably


develop hyperglycemia or glycosuria if the same number of calories were given with the higher percentage of protein. The diet containing less protein permits the use of much greater amounts of free carbohydrate.

It is of interest also in this connection to consider the diet of the Eskimo, which has been studied by Krogh and Krogh,' who found that about 447© of the total energy was derived from protein. The graphic chart shows at a glance that in such a dietary very small amounts of free carbohydrate serve to prevent ketogenesis.

It is a matter of common observation, to which attention has been called by Allen and DuBois,* that the level of protein metabolism is almost invariably high in patients with severe diabetes and acidosis. One might well ask, therefore, whether an increase in amount of pi'otein metabolized may not be one of the mechanisms by which the diabetic organism protects itself against undue ketogenesis.

At a recent meeting of the American Society of Biological Chemists, Shaffer made a further report of his studies on antiketogenesis which indicated that at times as much as two molecules of fatty acid can be metabolized completely in the presence of one molecule of antiketogenic substance. Since the 2: 1 ratio does not hold in all cases, it has seemed much better to adhere to the formulae constructed on the basis of the 1 : 1 ratio, as this gives a small margin of safety. Tlie data regarding the molecular proportions of ketogenic and antiketogenic substances in the metabolism of our patients will be given in a subsequent communication.

REFERENCES

1 Shaffer. P. A.: Antiketo'genesis I. An in vitro analogy. Journ. Biol. Chem., 1921, XLVIl, 433.

"Idem: Antiketogenesis II. The ketogenic-antiketogenic balance in man. .Journ. Biol. Chem., 1921, XL.VII, 449.

'Idem: Antiketogenesis III. Calculation of the Ketogenic Balance from the Respiratory Quotients. .Journ. Biol. Chem., 1921. XLIX, 143.

Cramer: Miinchener Med. Wochenschr., 1902. XLIX, 944. ' Woodyatt, R. T.: Object and Method of Diet Adjustment in Diabetes. Arch. Int. Med., 1921, XXVIII, 125.

6 Newburg, L. H. and Marsh, P. L.: The Use of a High Fat Diet in the Treatment of Diabetes Mellitus. Arch. Int. Med., 1921, XXVII, 699. Reference No. 6.

'Krogh, A., and Krogh, M.; Diet and Metabolism of Eskimos. Medelelser oni Gronland Kommissionen for Ledelsen af de Geologiske og Gecgrafiske Undersogelser I Gronland, Copenhagen, 1914.

8 Alleni, F. M. and DuBois, E. F.: Clinical Calorimetry. 17th Paper. Metabolism and Treatment in Diabetes. Arch. Int. Med., 1916, XVII, 1010.


130


[No. 374


THE ALVEOLAR AND BLOOD GAS CHANGES FOLLOWING PNEUMECTOMY

By (lEoRGE J. Heier and W. 1). W. Aniirus. (From the Himterian Laboratory of Experimental Surgery.)


In some pieviotis experiments ' it was found that very shortly after the removal of one Inng the remaining lung would begin to enlarge, and by this enlargement eventually would fill the entire thoracic cavity. As the lung enlarged, it displaced the heart so that this organ later came into contact with the lateral thoracic wall, and by necropsy examinations it was repeatedly demonstrated that when the heart was in contact with the thoracic wall the enlarged lung completely filled the thorax. A method was, therefore, at hand of following the enlargement of the lung by observing the displacement of the heart with the fluoroscope, and as a result of many examinations it was found that the lung which remained after a total pneumectomy completely filled the thorax in from four to six weeks. The capacity of the lung to undergo this compensatory enlargement seemed very great. Even when not only the entire lung upon one side but the largest of the three lobes upon the other side were removed, the two remaining lobes of one lung, with the exception of the space obliterated by the retraction of the thoracic wall and the elevation of the diaphragm, would enlarge to fill the entire thorax. As to the exact nature of this enlargement w-e have no knowledge at present, but some work to determine this point is being carried on in the Hunterian Laboratory.

This anatomical change in the remaining lung we have naturally considered a compensatory enlai'genient — an attempt on the part of the organism to replace the loss of lung tissue. As has been stated, the enlargement begins early, but it requires from four to six weeks to reach its maximum. Yet during this period of four to six weeks the animals apparently do not suffer from the lack of lung substance. They are lively and active; they are not dyspueic. AVe have thought, therefore, that there must be some immediate functional compensatory mechanism whereby the respiratory needs of the animal are probably assured, until the comparatively late anatomical changes in the remaining lung shall have taken place. Tlie purpose of the present work was to discover whether or not there was such an immediate compensatory mechanism.

In such a mechanism probably the circulation and


respiration both jilay a jiart, the circulation by an increase in the rapidity of blood flow through the remaining lung, the respiration by changes in the alveolar air and blood gases. It is only with the latter — the changes in the alveolar air and blood gases — that this report concerns itself.

Methods. — In animals, the subjects of these experiments, determinations of (1) the alveolar carbon dioxide content, (2) the alveolar oxygen content, (:>) the carbon dioxide content and capacity of the blood plasma, (4( the oxygen content and capacity of the whole blood, (5) the hemoglobin content and red cell count, were made before operation to serve as controls for subsequent similar determinations. The alveolar air was collected by the Plesh - method, the animals rebreathing a certain amount of air from a hag until equilibrium was established between it and the air in the lungs. Samples from the bag were then analyzed in a Hablane apparatus for carbon dioxide and oxygen. The collection and analysis of the blood were made by the method of Van Slyke,^' * all necessary precautions being taken to eliminate contact of the blood with the air or loss of blood gases by diffusion. All samples both of air and blood were obtained after the dogs had been absolutely at rest for at least ten minutes. The observations on the alveolar air and blood gases were made immediately after operation, 24 hours after operation, three and five days after operation, and thereafter at intervals of several days to a week. Controls were made upon animals subjected to operations other than pneumectomy, in order to eliminate the possible effects of operations themselves upon our findings.

Summary of Results. — [a] Effect an Alveolar Carbon Dioaide. The increased ventilation of the lungs due to anesthesia causes a fall in the alveolar carbon dioxide tension. After operation, however, the alveolar carbon dioxide tension rises, and within 24 hours is about equal to or above the normal value as determined before operation. It continues to rise until about the eleventh day, then gradually falls, reaching normal by about the twenty-fifth day (Curve 1).


April, 1922]


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(6) The Effect on Alveolar O.ryyen. Anesthesia causes a rise of the alveolar oxygen of approximately 2 per cent above the normal value as determined before operation. Following operation the alveolar oxygen shows a marked fall, averaging Si^ per cent, until about the eleventh day, after which it again tends to rise to its normal level. In two animals it reached the preoperative value in twentyeight days ; in another it had not reached its former level in sixty-six days. The second rise in the alveolar oxygen value occurs coiucideutly with the fall in alveolar carbon dioxide tension (Curve 2i.


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(c) The Effect on Blood Carbon Dioxide Content and Capacity. The anesthesia causes an average fall in the carbon dioxide content of the blood of from 8 to 10 volumes per cent. For a period of ten days after operation there occurs a rise of from 41/2 to 13 volumes per cent (average, 91/2) above the normal value as determined before operation : then a gradual fall to about normal by the twenty-fifth day (Curve 3).



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(d) The Effect on the Blood Oxygen Content. Ane.sthesia causes a slight rise of about 2 volumes per cent. This is followed within 24 hours after operation by a marked fall of approximately 4i^ volumes per cent, in oue case by a fall of 11 volumes per cent, or nearly half the total oxygen capacity of the blood. This fall in the blood oxygen content continues approximately up to the eleventh day after operation. In two cases it again rose to normal by the twenty-fifth and the thirtieth days; in one animal it remained at a low level until the twentythird day (Curve 4).

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CHART 4 — VENOUS BLOOD « 0, CONTENT O O'. CAPACITY

(e) Effect on Blood Oxygen Capacity. The effect of anesthesia is in our experience variable, there being either a rise or fall in the blood oxygen capacity. Following operation there is an average rise in the oxygen capacity of the blood of 3i/4 volumes per cent, this increased value being niaintaine<] during the period of our observations


132


[Xo. 374


(sixty-six days.) In one animal dying from distemper twenty-eight days after operation, there was a progressive fall in the oxygen capacity (Curve 4|.

(/) The Effect on Oxygen Unsaturutioii, Percentage. Anesthesia causes a rise in the oxygen unsaturatiou I)ercentage in two cases, a fall in tiiree cases. Within 24 hours of operation there is a very marked rise of from 9 to 30 per cent, with an average rise of 19.6 per cent. This rise in the oxygen unsaturatiou percentage continues up to the eighth day, at which time the average rise is 23.4 per cent. Subsequently, there is a fall but not quite to the previous normal value as determined before operation (Curve 5).



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(//) Ohseri-atiom on the Blood Count. The day following operation there is an average rise in the red cell count of 666,000. On the third day after operation this rise averages 800,000 above normal, one aninml showing an increase of 1,200,000 red cells. This rise in the red cell count corresponds to an increase in hemoglobin of from 15 to 20 per cent. So far as our ob.servations go (extending to the sixty-sixth day in one case) this increase in the red cell count is maintained, two dogs observed on the fifth-third and sixty-sixth days having blood counts of 7,000,000 or more (Curves 6 and 7i.

Briefly, then, the effects of pneumectomy are a rise in alveolar carbon dioxide and a fall in alveolar oxygen, these alveolar air changes being associated with a tempoary rise in the carbon dioxide content and capacity of the blood, a marked fall in the oxygen content, and a marked rise in the percentage of oxygen unsaturatiou. Concomitantly, there is a marked rise in the hemoglobin content of the blood and therefore in its oxygen-carninf



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.„.



^^








t;:

.




.


.


....



...^.

.


7 ') 11 13 15 17 19 21 23 25 27 29 31 Days alter operation

CHART 7 — HEMOGLOBIN (Calculated)


capacity, which may be interpreted as a compensatoiT mechanism. As has been noted, the changes in the alveolar carbon dioxide, alveolar oxygen, carbon dioxide content and capacity of the blood, and percentage of oxygen unsaturatiou, are only temporary; and approximately within thirty days normal relations are again established. The increased hemoglobin and oxygen-carrj-ing capacity of the blood, however, persisted during our period of observations (sixty-six days). They serve at least in part to supply the respiratory needs of the animal perhaps until complete anatomical changes in the remaining lung have taken place.


April, 1922]


133


PROTOCOL. OF EXPERIMENTS



Date


Day


Alveolae Air


Venous Blood


Dog


c

<D


a o

i B


a


a o


1

s s


o

o" u


a <v

a

8 o"



a o

5 g 1 "

Bf

d"


.2 ^

XJ 0)

o -JJ

11


a

o O

U

m


T,


11/ 1/21


—1


5.34


40.6


15.72


119.5


39.8


46.4


14.64


19.03


23.1


103.4




11/11/21


+9


5.04


38.3


11.23


85.3


41.4


48.1


16.47


20.95


21.4


112.8




11/18/21


16


5.10


38.7


10.43


79.2


52.3


58.6


15.13


20.95


23.0


112.8




11/25/21


23


5.39


40.9


11.22


85.2


57.8


62.5


14.82


24.41


39.3


131.9




12/ 2/21


31


5.48


41.6


10.23


77.7


51.9


55.7


17.18


24.12


28.7


130.4




12/15/21


44


5.38


40.8


11.71


89.0


49.4


53.2


14.50


23.70


38.8


127.5


7,192,000



1/ 6/22


66


5.21


39.6


13.26


100.8


42.4


48.1


14.87


23.54


36.8


127.2


7,360,000







T/


11/12/21


—2


4.76


36.1


12.08


91.8


41.6


62.5


13.66


19.00


28.6


102.7




11/14/21



3.45


26.1


15.60


118.5


35.0


38.7


15.36


19.08


19.5


103.1




11/15/21


+1


3.51


26.6


13.50


102.6


35.0


42.4


9.70


15.95


39.2


86.2




11/17/21


3


4.74


36.0


12.95


98.3


44.5


48.4


10.25


21.14


51.5


114.3




11/19/21


5


5.08


38.6


12.39


94.16


46.4


52.0


7.90


16.40


57.9


88.7




11/22/21


8






50.2


57.6


5.46


17.29


56.8


93.5




11/25/21


11


5.61


42.5


10.00


76.0


43.6


48.5


9.75


16.72


41.7


90.4




11/30/21


16


5.07


38.5


11.83


89.9


43.3


50.5


7.80


16.38


52.5


88.5




12/ 5/21


23


4.80


36.4


11.99


91.1


42.3


47.5


6.44


14.53


55.7


78.5



T,


11/14/21


—1


5.07


38.5


12.80


97.3


47.3


55.9


15.81


23.12


31.7


125.0




11/15/21



3.60


27.4


16.01


121.7


38.8


49.2


17.74


22.42


20.7


121.2




11/16/21


1


4.80


36.5


12.20


92.7


57.7


72.0


9.26


23.24


60.15


125.6




11/18/21


3


5.39


40.9


11.00


83.6


52.0


61.6


12.95


21.95


41.0


118.6




11/21/21


6


5.29


40.2


11.10


84.3


59.6


67.2


11.91


24.45


51.3


132.1




11/22/21


7


6.24


47.4


11.08


84.1


59.2


79.0


14.23


25.59


44.4


138.2




11/26/21


11


6.12


46.5


8.38


63.7


60.1


67.5


18.34


24.89


26.3


134.8




12/ 1/21


18


5.80


44.1


12.00


91.2


52.5


58.5


14.29


22.86


37.2


123.9




12/ 8/21


25


4.97


37.7


12.28


93.3


50.0


59.5


15.02


22.97


34.6


124.2




12/15/21


32


5.62


43.3


11.32


87.16


51.6


56.5


16.42


23.69


31.9


128.0




1/ 3/22


53


5.12


38.9


12.37


94.0


47.1


53.8


14.21


24.26


40.6


131.1


7,000,000








Died of distemper on the 28th day after operation


134


[No. 374


PROTOCOL OP EXPERIMENTS — Continued.



Date


Day


A-LVEOLAR Air


Venous Blood


Dog




o


a

S3


d _o

a

o'


a

a o U

o" o


oi

O

d" o


1 o o O


1

6


a d"


.s s

II

ii 1^


a o

oi


T.


11/26/21


—1


5.25


39.9


14.05


106.8


52.0 56.5


16.91


23.72


28.8


128.2


5,296,000



11/27/21



4.20


31.9


16.08


112.9


49.5


55.2


19.02


22.44


15.2


121.3




11/28/21


1


5.23


39.6


11.31


86.0


52.4 59.0


15.86


25.52


37.9


137.9


5,872,000



11/30/21


3


5.42


41.2


12.22


92.9


61.3 65.3


14.29


25.21


43.4


136.3


6,480,000



12/ 5/21


8






51.0


61.0


16.22


27.61


41.3


149.2


6.096,000


Ts


12/ 3/21


—1


5.02


38.2


11.14


84.7


48.1


59.5


19.16


24.27


21.1


131.1


5,392,000



12/ 4/21



4.94


37.5


12.94


98.3


33.8 40.0


20.11


25.51


26.7


137.4


5.700,000



12/ 5/21


1


5.23


39.7


9.60


73.0


44.3 52.3


13.10


26.25


50.1


141.9


5,970,000



12/ 7/21


3


5.35


40.7


9.29


70.6


52.4


60.0


8.42


25.69


67.2


138.8


5,816,000



12/10/21 12/13/21


6


4.89


37.1


13.36


101.5


49.5 1 59.5


12.87


23.61


45 5


127.4


4,888,000



9


5.26


39.5


10.89


52.8


46.2 1 53.8


10.26


22.06


50.3


119.6


5,328,000



12/16/21


12


5.37


41.0


10.95


83.2


51.9 55.7


10.12


25.21


59.8


136.2


5,912.000



1/ 3/22


28


4.96


38.1


12.00


93.2


47.1 52.8


12.38


25.35


46.1


137.0


5,872,000






-^",0


12/11/21


—1


4.87


37.0


12.42


94.4


45.8 55.3


18.00


25.39


29.1


137.2


4,816,000



12/12/21



5.22


39.7


13.36


101.5


51.5


55.7


18.39


28.95


36.1


156.4


4,896,000



12/12/21


6 hi-s.P.O.


5.52


41.9


10.95


83.2


51.9


59.5


15.53


24.99


37.9


135.1


4,944,000



12/13/21


1


5.30


40.3


11.44


86.9


46.5


55.7


13.99


25.76


45.7


142.7


5,560,000


BIBLIOGRAPHY

1. Heuer, G. J., and Dunn, G. R.: Experimental pneumectomy. Bull. Johns Hopkins Hosp., 1920, XXXI, 31-42.

2. Plesh, J.: Hamodynamische Studien. Ztsclir. f. exper. Path, u. Therap., Berl., 1909, VI, 380-618.


3. Van Slyke, D. D.: Studies of acidosis. II. A method for the determination of carbon dioxide and carbonates in solution. J. Biol. Chem., Bait., 1917, XXX, 347-368.

4. Van Slyke, D. D.: Gasometric determination of the oxygen and hemoglobin of blood. J. Biol. Chem., N. Y., 1918, XXXIII. 127-132.


POSTERIOR RESECTION OF THE RECTUM AND RECTOSIGMOID (KRASKE OR MODIFIED) UNDER REGIONAL ANESTHESIA*

Bj- Gaston L. Labat, M.D., l'ari.s {Special Lecturer on Anesthesia, The Mayo Foundation, Rochester Minnesota.)


Apart from early diagiicsis and the choice of methods iu dealing with cancer of the rectum and rectosigmoid, one of the most interesting problems connected with the surgery of that portion of the large intestine is the choice


Presented before The Johns Hopkins Hospital Medical Society, Baltimore, October, 1921.


of the method of anesthesia, especially if tlie operability is raised, as has been the teutlency for the past few years. Among tlie patients suffering from the disease are those with chronic conditions of the urinary, respiratory, or circulatory systems, which make them poor operative risks under ordinary circumstances. Obesity is apt to


April, 1922]


13a


increase the mortality, or at least to cast a heavy shadow ou the operative jjrognosis. The colostomy itself, in the case of very obese persons, is attended by considerable risk.

Nephritis is one of the important causes of operative mortality. It is usually of an acute type, superimposed on a chronic process. Many patients die some weeks after the operation from nephritis, cardiovascular disease, and so forth, which they had at the time of the operation. Infection (general peritonitis, pelvic cellulitis), obstruction and exhaustion are, according to the present study, the most frequent causes of death.

Although the anesthetic agent used for long and traumatizing operations such as the Kraske or any other type of posterior resection of the rectum is seldom mentioned in the literature, the presumption is that it has almost always been general anesthesia by ether inhalation. The morbid influence of ether on lungs, kidneys, and arteries with pathologic lesions is too well known to be deserving of more than mere simple mention in this paper. The fatal blow given by the lipoid solvent to the progressively exhausted heart laboring under fatty degeneration needs no discussion. In apparently normal ])ersons, the unfavorable influence of ether on the kidneys, lungs, and gastrointestinal tract is most apparent in the early days following operation; and the patient recovers generally without any complication attributed to the auesthetie. Albuminuria, glycosuria, lipemia, glycemia and acidosis, all clear up within a relatively short time. But occasioiuilly a condition of exhaustion exists at the time of the operation, a condition chiefly due to anesthesia or the inability of the patient to adapt himself to ether inhalation. Some patients recover from this condition ; but others And their power of resistance gradually decreasing. The natural reaction to infection is thus greatly diminished. Cellular life is weakened in the central nervous system which loses control over the vital functions of the economy. The kidneys at times find themselves helpless to deal with the abundant waste material coming directly from the operative wound, and the material which overflows or leaks from the defective liver.

Siich disturbances may be avoided or considerably lessened by the use of regional anesthesia, which does not, as a rule, aft'ect the general condition of the patient. But it must be clearly understood that the block method is not meant to improve chronic conditions which are present at the time of the operation, nor to prevent the development of acute disease started just before the operation. Regional anesthesia does not increase the resistance of the jiatient, but it reduces to a minimum an unfavorable oi)ei'ative prognosis by not atfecting the vital functions of the main organs of the body. Regional anesthesia does not prevent pulmonary embolism, but it takes away the danger of pulmonaiy complications, since it does not interfere with the respiratory organs. Edema


of the lungs, pulmonary congestion, or pneumonia will not set in as a postoperative complication if the usual elementary precautious are taken. Since regional anesthesia does not affect the gastrointestinal tract, there is no postoperative nausea and vomiting, no acute distension of the bowels with consequent paresis, and no strain on the abdominal cavity and its contents immediately after the operation. Postoperative paralytic ileus is prevented more effectually by the normal i)eiistaltic movements of undisturbed bowels. Tendency to bleed is also greatly reduced and convalescence rendered shorter. In considering the advantages and the bright postoperative clinical picture shown by the majority of patients who were considered very poor surgical risks before the operation, regional anesthesia has unquestionably strong claims against ether anesthesia or any other form of iidialation narcosis, which occasionally results in serious complications. Regional anesthesia is the method preferred for posterior resection of the carcinomatous rectum and rectosigmoid, whatever be the condition of the patient. In this type of case, it can be said without error that all patients are amenable to the method; but it may be well to emphasize a few points which are occasionally, if not always, overlooked, especially if regional anesthesia is only applied to i)articularly poor surgical risks.

In trying to increa.se the percentage of operability, the question of local conditions of the disease and of metastasis is generally solved by exploration at the time colostomy is performed. How far the radical cure is indicated, and the type of operation best suited to the particular case, are matters I shall not discuss, although the choice of operation is a significant factor in the final settlement of the prognosis quoud vitam in cases of carcinomatous rectum and rectosigmoid. After the operation has been decided on, two questions remain to be answered: Will the patient stand the operation? Will he stand the anesthesia needed for the operation added to the operation itself? The answei-s depend on the condition of the main organs and their surgical resistance, measured in terms of their progressive alteration from the time of the colostomy, the coefficient risk being added to the operative prognosis by the personal equation of the surgeon. The risk due to the type of anesthesia has too long been overlooked ; and it is now time to consider the opportunity of using regional anesthesia exclusively for the Kraske or any other types of posterior resection of the rectum and rectosigmoid.

The preconceived idea of the absolute safety of regional anesthesia is apt to influence the operability favorably, although it cannot be expected to alter the pathologic conditions existing at the time of exploration. All patients who seem to be good operative risks quoad the lesion may otherwise be veiy poor surgical risks ; and, in raising the operability, one should be cautioned against those cases that are on the borderline. In considering


13C


[Xo. 374


the advisability of a radical cure, the actual resistance of the patieut after the colostomy should be the chief guide. Some patients continue to decline after the colostomy, and can scarcely pull through that first-stage operation, especially when it has been performed under ether anesthesia. I'rogressive dehydration cannot be avoided, and from fifteen to twenty days after, which is the usual time for the second stage oi)eration, these patients are weaker than ever. Whatever anesthesia is then used, they will seldom come out of that condition of pogressive exhaustion which silently cai'ries them across the borderline into a state of coma which starts during the operation and ends with life on the second or third day. In those desperate cases regional anesthesia only permits the surgeon to risk the colostomy, and the prognosis of the second-stage posterior resection is greatly improved.

A study of thirty-three cases in which operation was performed in the Mayo Clinic from October, 1920 to June, 1921, inclusively, brings out jjoints which tend to prove the innocuousness of the method of regional anesthesia:

1. Previous diseases do not seem to influence the operative risk, provided no acute process prevails at the time of the operation. Patients having a previous history of diphtheria, pneumonia, grippe, pleurisy, scarlatina, measles, chicken pox, acute Bright's disease, and so forth, whether contracted recently or several years before, have made uneventful recoveries from operations performed by means of regional anesthesia.

2. Chronic lesions of the heart, with or without compensation, are not conditions which contra-indicate the use of the regional method, provided the resistance of the patient seems fair. Patients with enlarged hearts (1.5 to 2 cm. to the left) with extrasystoles, associated with a systolic blood pressure of 1G5 mm., diastolic 96 mm. and deficient kidneys; patients presenting mitral incompetence and aortic stenosis, with a systolic blood pressure of HO mm., diastolic 90 mm., and pulse 116, have left the hospital in from ten to twenty-two days following the posterior resection of the anus, anal canal, rectum, and lower sigmoid.

3. Pulmonary tuberculosis does not seem to influence the operative prognosis. An operation was performed under paravertebral and sacral block on a woman, aged thirty-six years, who gave a history as follows: She had had four pregnancies, two of which were extra-uterine, one rupturing. One pregnancy ended in a miscarriage at three months. She had had "anemia" twelve years before; pneumonia, in childhood, and eight and two years before; typhoid fever with peritonitis twenty two years before; and active tuberculosis ten years before. Her sputum was positive May 4, 1921, when a left inguinal colostomy was performed under regional anesthesia. One month later the anal canal, entire rectum, and 25 cm. of the sigmoid were excised. The growth was in the rectosigmoid, extending down into the rectum. A loop of the


sigmoid and one of the small intestine had dropped down and become adherent to the growth. The small intestine was separated, but the peritoneal coat, where it was adherent, was removed. A loop of the sigmoid about 10 cm. above the, growth, which had become adherent to the growth, was removed with it. The gland.s ami fat which were involved and adherent to the promontory and sacrum were removed. The upper end of the sigmoid was turned in twice and fastened to the peritoneal wound. The posterior wall of the uterus was drawn into the cavity to help fill the large peritoneal defect.

The extent and type of the operation performed in this case is a real test of the method of regional anesthesia. No particular care was taken to avoid or lessen the usual manipulations generally adopted under ether anesthesia. The patient made an uneventful recovery and left the hospital on the twentieth day after the operation.

5. Diabetes, at least mild and chronic, easily controlled by dietary measures, has to all ai)pearances no influence on the operative prognosis if regional anesthesia is used. A woman, aged seventy-two years, was operated on for carcinoma of the rectum April 1, 1921, while sufl:ering from mild chronic diabetes. Her urine output was 800 c.c. in twenty-four houi-s. The urinalysis showed specific gravity 1.016 ; reaction, acid ; sugar, a trace; and pus cells, 1 to 6. The patient left the hospital twenty days after the operation with a clean postoperative record.

6. Operation may also be safely performed in renal conditions of a chronic type. A woman, aged forty-seven years, had an average urine output of 500 c.c. in twentyfour hours, the urinalysis showing specific gravity 1.029; reaction, acid; albumin 1; pus cells 3; and a combined phenolsulphouephthalein return of 35 per cent. She gave a previous histoi-y of having had diphtheria, scarlatina, pneumonia, grippe, and pleurisy, and at the time of the operation was surtering from mitral incompetence and aortic stenosis. A posterior excision was made of the anus, anal canal, rectum, and lower sigmoid. She passed an average of 650 c.c. of urine every day following the operation. She recovered uneventfully, and left the hospital ten days after the operation.

Preparation of Patients for Regional Anesthesia

For the one-stage Kraske operation, the patieut receives a preliminary hypodermic injection of morphin 1/6 gr. and scopolamin 1/300 gr., one hour before anesthesia is begun, and a second dose of the same strength immediately after the completion of the anesthesia.

For the colostomy in the two-stage operation, one hypodermic injection of the narcotics is, as a rule, sufficient to dull the mentality of the patieut and dismiss any apprehension due to consciousness at the time of the operation, provided it is given one hour before the anesthesia, and the colostomy is performed ten minutes after the completion of the anesthesia. In very nervous


April, 1922]


137


patients, if the first dose does not seem to liave produced the desired effect, a second dose is given at the time of the anesthesia.

The patient is prepared for the second stage Kraske in the same manner as for the one stage. Individual cases sliould be considered in administering two doses of the combined narcotics. If during thp manipulations for inducing anesthesia, the attitude of the patient reveals suflicient psychic control there is no reason for giving the second dose; it should be reserved for alleviating postoperative pain.

The use of morphin and scopolamin in such weak doses is not meant to produce, and in fact does not produce, a twilight sleep nor a semiwaking condition during which the surgeon loses control of his patient, but simplj- to enable the patient to feel more comfortable in that rather awkward position he must assume during the operation.

Anesthesia for the Colostomy

For the left rectus colostomy, two procedures are available, the abdominal field block, and the paravertebral block.

The abdominal field block is the procedure iisually employed because it is easily and quickly accomplished, and requires no special long and delicate training. It affords almost complete relaxation of the abdominal muscles and perfect anesthesia of the parietal peritoneum within the blocked area. If gentleness is used, the pelvic organs, as well as the liver, may be explored, provided the incision is long enough to allow the hand to steal into the abdominal cavity without force. This is most easily done after the lips of the wound have been clamped and raised. In the majority of cases, there is no sharp pain during gentle exploration, but only an abdominal sensation referred to the epigastrium, such as that due to intestinal colic. The sensation ceases with the exploration, which ordinarily is of very short duration. Some patients require just enough ether or gas to make them lose consciousness during this stage of the operation. If the excitation period is reached, exploration becomes impossible, owing to lack of relaxation which can only be obtained by bringing the patient to the surgical stage of general anesthesia; and in such case he loses the benefit of the regional anesthesia. It is, therefoi-e, wise to insist on the necessity of a judicious use of ether or gas during exploration.

The average patient, well prepared by the preliminary narcotics, and familiar with the requirements of regional anesthesia, cooperates willingly and, if gentleness is used, the colostomy is painlessly performed. If the patient is obese, or if the mesocolon is short, a few^ whiffs of a general anesthetic contributes greatly to establish favorable conditions. This combined method is safer than any general anesthetic alone.

The patient lies in the recumbent dorsal position, and the operative field is prepared as for any surgical opera


tion ; one coat of tincture of iodin is sufficient for the [)urposes of regional anesthesia. Intradermal wheals are raised with a fine needle along the left costal margin, from the xiphisternum to the tip of the eleventh rib, and from that point to the iliac crest, (Fig. 1). A fine needle of convenient length (8 to 10 cm.) is then passed through each of these wheals in succession and deep injections made within the muscle laj'ers of the abdominal wall, followed by subcutaneous injections which join all the wheals, except the first to the last. All the injections along the costal margin are made in a plane perpendicular to the surface of the skin and passing through the wheals; those from the costal margin to the iliac crest are made in a similar manner. Two walls of anesthesia, meeting at about the level of the tip of the eleventh rib, are thus created, cutting off the nerve supply of half the abdominal wall. The anesthetic is a 0.5 per cent solution of neocain * or procain, containing 15 minims of adrenalin solution 1:1000 per 100 c.c. The quantity injected varies from 100 to 200 c.c, according to the weight of the patient.

The paravertebral block is more difficult and, in order to give it successfully, fairly good training is required; but it affords a wider field of anesthesia and greater facility in handling the bowel, in case the mesocolon is short. It is customary to inject from the eighth dorsal to the third lumbar nerve on the left side with from 5 to G c.c. of a 1 per cent, neocain or procain solution at each nerve. Paravertebral block thus performed does not abolish the abdominal sensation present during exploration, but it lessens it to a certain extent. In the majority of cases, the colostomy is performed painlessly without the aid of ether or gas. If regional anesthesia is to be induced by the average surgeon, the abdominal field block, according to my experience, should be given preference.

Anesthesia for the Kraske or Any Modified Posterior Kesection

One hour after the preliminary hypodermic injection of morphin and scopolamin has been given, and twenty to thirty minutes before the hour fixed for the operation, the patient is gently wheeled into the operating room or into the special room which, in a few institutions, is specially equipped for inducing regional anesthesia. He is placed flat on his stomach and a cushion slipped under his hips to raise the sacral region and render the landmarks more accessible. The operative field is prepared in the usual manner, exposing the whole lumbosacral region, from the first lumbar vertebra to the tip of the coccyx. One hundred and fifty cubic centimeters of a 1 per cent, procain solution containing 15 minims of adrenalin solution 1 :1000 is placed in a cup at hand, and the special regional anesthesia syringe with one set

Neocain' is a French product made by Corbiere and Lionnet, Paris. France, put up in sterile capsules of 0.5 gm. each. It is readily soluble in saline solution and of very low toxicity.


138


[No. 374


of needles of different dimensions tested as to efficiency before starting tlie manipulations.

The best results are obtained by combining the caudal, transsacral, and paravertebral block of the last three lumbar nerves (Fig. 2), starting with the caudal block, then injecting the sacral nerves, and finishing with the paravertebral block. Whatever be the level of the grow-th, one and the same procedure should be applied, although anorectal and rectal lesions require a smaller anesthetic field. It is always preferable to prepare the patient in such a way that no sub.sequent injections will be necessai7, if the surgical manipulations are cari-ied beyond the contemplated area. For instance, an error of diagnosis may be followed by the excision of many centimeters of bowel through the posterior route, in order to reach the growth situated high in tlie sigmoid colon. When the lesion lies in the rectosigmoid juncture, the procedure described here gives absolute anesthesia throughout the operation. It is, therefore, advisable, I insist, not to modify the procedure according to the height of the lesion, but to use it integrally in all cases. 1. Caudal or epidural bZocA-.— The sacral hiatus is defined by a depression which is felt in the middle line, at about the juncture of the coccyx with the sacrum, and bounded by the sacral cornua on each side and the fourth sacral spinous process on the middle line a little higher up. These three prominences, palpable in the majority of cases, form the angles of a triangular surface at the middle point of which the needle is introduced with ease and success. The spinal puncture needle, with its stylet in and its bevel turned upward, is introduced at this point, in a direction making an angle of about 45 degrees with the surface of the skin. After piercing the sacrococcygeal membrane which, like a screen stretched across the sacral hiatus, closes the lower extremity of the sacral canal, the point of the needle strikes the anterior wall of the canal. It is then withdra^^^l 1 or 2 mm. and the hub of the needle swung downward toward the gluteal cleft, reducing the angle of 45 degrees to about 20 degrees. The needle is advanced gently and gradually into the sacral canal, always keeping along the middle line, until about 6 cm. of its length have disappeared. If the point of the needle impinges on the posterior wall of the canal and is stopped a short distance from its point of entrance, it is ordinarily released by the application of pressure on the sacrococcygeal membrane with the left forefinger placed on the needle at the site of puncture. If this device is not successful the needle has to be withdrawn and reintroduced a little higher. When the needle has been correctly introduced, its stylet is withdrawn and time is allowed to make sure that no blood or cerebrospinal fluid comes out. In such case the needle is drawn back a few millimeters until the flow ceases and the syringe, filled with a 1 per cent, solution, is connected with the needle. It is a verj' good practice to aspirate before injecting fluid, so as to feel quite certain that


no intraspinal nor intravenous injection is actually made. Abnormal extension of the dural sac below the second sacral vertebra is exceptional, but its existence cannot be denied. Injection should be very slow, owing to possible after-efl'ects due to rapid absorption by the structures contained in the sacral canal. A total amount of 30 c.c. of the anesthetic solution thus injected into the sacral canal establishes favorable conditions for the transsacral block.

2. Transsacral ilock. — This procedure consists in injecting the sacral nerves individuallj* hj introducing the needle through the posterior sacral foramina, which can be reached with some precision only by taking accurate superficial landmarks. These ai-e the posterior superior spine of the ilium and the sacral cornua. A wheal is raised about 1 cm. medial to and below the said iliac spine and another wheal is placed just above the sacral cornu on the same side. The distance between them is divided into three parts by two other intermediate wheals. The second, third, fourth, and fifth sacral foramina are thus easily and accuratelj' defined. The first sacral foramen is found by raising a wheal about 2.5 cm. above that which nuirks the second sacral foramen, following the same general direction. The thickness of the soft tissues overlying the sacrum being much greater in the upper portion of the bone, needles of different lengths are used according to the height of the puncture. It is most convenient to use a needle 5 cm. long for the last three foramina, one 8 cm. long for injecting into the second sacral foramen, and one 10 cm. long for the first foramen which is vei-y deeply situated (Figs. 3 and 4). It is customary to begin with the second foramen, which seems to be easier to locate than the first and which helps considerably in locating the others. The needle is introduced perpendicularly to the surface of the skin and gently advanced toward the posterior aspect of the sacrum until its point touches the bone. A little practice ordinarily gives an almost accurate tactile sense of the rich fibrous structures overlying the foi-amen and spreading out, so to speak, in its immediate neighborhood. The direction of the needle is slightly changed if the foramen is not reached by a first puncture; but gentleness should be used in approaching the bone so as not to hurt the patient, traumatize the region by repeated punctures, and damage the point of the needle, which bends to a hook and tears through the tissues while it is withdrawn. This faulty techuic of most beginners can be improved rapidly by a short training on the cadaver, especially if every effort is made to visualize the framework through the soft overlying structures. After losing contact of the posterior aspect of the sacrum, the needle passes through the posterior sacral foramen and, according to the foramen, is introduced more or less deeply, because of the uneven thickness of the bone. For the same reason the quantity of solution injected varies with the foramen. It is customary to inject 6 c.c. of the solution in the first


THE JOHNS HOPKINS HOSPITAL BULLETIN, APRIL. 1922


PLATE XIX fl.

4



April, 1922]


139


foramen and to reduce by 1 c.c. each time the quantity injected into each subsequent foramen. Starting from the second foramen injection is therefore made of 5, 4, 3, and 2 c.c. respectively, in descending order of foramen, thus injecting 40 c.c. of the solution, which, added to the 30 c.c. used for the caudal block, makes a total of 70 c.c. for the sacral block.

3. Paravertehral block.- — After inducing the sacral block, as described, bilateral injections of the last three lumbar nerves are made as follows: Wheals are raised 3.5 to 4 cm. from the middle line of the spine, according to the weight of the patient, opposite the upper edge of the spinous process of the third, fourth, and fifth lumbar vertebne (Fig. 2). A needle 12 cm. long is introduced through each of these wheals in succession and advanced in a direction perpendicular to the skin surface until contact is taken with the transverse process of the vertebra. The needle is then withdrawn a little, for the purpose of changing its direction, and reintroduced toward the spine, making an angle of about 20 degrees with the middle plane on the body. The needle passes above the transverse process and is stopped at 2.5 to 3 cm. after it has passed the bone, and 8 to 10 c.c. of the solution is injected while the needle is slightly moved to and fro. Efforts should be made not to hit the nerves, but to deposit the solution in their immediate vicinity. The same rule applies to the sacral block. In order to reach the fifth lumbar nerve the needle is passed below the transverse process of the fifth lumbar vertebra. The aspiration test for blood should be renewed several times during each injection, thus making sure that the point of the needle does not lie within the lumen of a blood vessel. Not more than the exact quantity of the solution should be taken in the syringe each time, so as not to exceed the total amount of 150 c.c. of the 1 per cent, solution prepared beforehand. The injection of greater quantities is likely to produce toxic symptoms. Procain is ten times less toxic than cocain, but still its toxicity must be remembered, and likewise the possible presence of impurities in the commercial drug. Toxic symptoms are exceptional ; but if they should appear, a subcutaneous injection of spartein sulphate 0.05 gm., caffeiu 0.25 gm., and strychnin sulphate 0.001 gm. improves the condition. No such mishap is expected from the method itself which is the safest now known; but attention should be called to the fact that it may occur through faulty technic, and the anesthetist should know what to do in such cases.

After making the last injection, the .sensibility of the anus and perineum is tested by clamping the region. Complete relaxation of the anal sphincter is a proof that the entire rectum, bladder, and prostate have been anesthetized, although there is no way of testing beforehand the sensibility of the parietal peritoneum in connection with the mesosigmoid, and the pelvic organs in women.


Conclusions

1. The choice of the method of anethesia is one of the most interesting problems connected with the surgery of the rectum and rectosigmoid.

2. Most of the postoperative complications may be attributed to inhalation narcosis, especially ether, and can be avoided or considerably lessened by the use of regional anesthesia.

3. Regional anesthesia does not increase the resistance of the patient, but leaves the vital functions of the body in the same condition.

4. The anesthesia does not prevent pulmonary embolism, but excludes the danger of pulmonary postoperative complications, provided no acute condition exists at the time of the operation.

5. The anesthesia has no ill-effects on the gastrointestinal tract; thus the possibility of paralytic ileus and the tendency toward hemorrhage are reduced.

C. All patients are amenable to the method, but one should be cautioned against borderline cases.

7. Previous conditions, such as chronic lesions of the heart, with or without compensation, high blood pressure, pulmonary tuberculosis, diabetes, at least mild and chronic, easily controlled by dietary measures, and chronic renal conditions do not seem to interfere with the operative prognosis.

8. The use of morphin and scopolamin controls the psychic state of the patient and greatly contributes in establishing favorable conditions during the operation; but the stage of twilight sleep must not be reached.

9. With the abdominal field block procedure, colostomy is performed painlessly, provided the patient is not too obese and the mesocolon is not too short. Exploration is possible in the majority of cases, if gentleness is used.

10. The sacral block, consisting of the caudal or epidural and trans-sacral block, added to the paravertebral block of the last three lumbar nerves on both sides, constitutes the method of choice for the posterior resection of the carcinomatous rectum and rectosigmoid.

11. The administration of the anesthesia is not diflBcult, but it requires practice and patience, irrespective of the gentleness which must always be used in handling conscious patients.

12. If the anesthetic does not give complete anesthesia throughout the operation, the administration of a first stage ether anesthesia during the deep manipulations constitutes a combined method much safer than general narcosis alone.


140


[No. 374


liEGENDS

Fig. 1. — Abdominal fieJd block for Colostomy, resulting in anesthesia of left half of abdominal wall.

Fig. 2. — Regional anesthesia for posterior resection of rectum and rectosigmoid.

-)- Site of puncture for caudal or epidural block.

1, 4, 3, 2, 5. Sites of puncture for transsacral block.

6, 7, 8. Sites of puncture for paravertebral lumbar block.


Fig. 3. — Oblique lcm;gitudinal section of the pelvis showing the right sacral foramina, the entire sacrum being preserved. This is intended to show the variable thickness of the soft tissues overlying the sacrum and the different directions of the needle.

Fig. 4.— Oblique longitudinal section of the pelvis passing through the right sacral foramina, showing the progressively decreasing thickness of the sax;rum from top to bottom, a condition which commands the use of variable quantities of anesthetic solution according to the site of injection.


THE USE OF THE BONE GRAFT IN THE TREATMENT OF POTT'S DISEASE.

By William S. Baer, M.D. (Associate Professor of Clinical Orthopedic Surgery, The Johns Hopkins University.)


The ten years which have now elapsed since Albee first called attention to the use of the bone graft as an aid in the cure of tuberculosis of the spine, furnish a period of time sufficient to allow us to draw some conclusions as to the value of the procedure. With this aim in view I have collected the cases of tubei-culosis of the spine treated by this method by myself or my associate, Dr. Bennett, and our assistants.* The cases to be reported are fifty in number and in each one the bone graft operation was done pi'ior to December 31, 1919. Thus two years at least, and in many cases more, have elapsed since the operation, so that a careful analytical study of the series will throw some light upon the efficacy of the operation and the indications for its employment.

We have arbitrarily^ divided our cases into three groups, according to the results obtained.

First: Good results. In this class there are

a. Ko symptoms or signs of active tuberculosis in the

vertebral column. I). No support is necessai-y.

c. There is no kyphosis present or no increase in the preoperative kyphosis.

Second : Fair results. In this class there are a. No symptoms or signs of active tuberculosis. 6. No brace or support has to be used at the pre.sent

time. c. A post-operative increase of the kyphos has

occurred.

Third: Poor results. Patients in whom the process is still active ; those in whom support is still necessary, or those who have died. Again, the cases have naturally divided themselves into three groups, according to age at the time of operation. a. Up to the age of six years, the period of infancy. h. From six to sixteen years of age, the period of greatest growth.


I am greatly indebted to Dr. Wlnthrop Phelps for his work In correlating these cases.


c. From sixteen years upwards, — the period when growth for the most part has ceased.

This means that in Class a — up to six years of age — our results have been as follows: Good — 7. % Fair — 21.5% Poor — 71.5%

In Class 6 — six years to sixteen years : Good — 33-1/3% Poor — 66-2/3%

In Class c — sixteen years and upward : Good — 90% Poor — 10%

These figures go to show that if the operative procedure is instituted before the age of six years the ultimate result is apt to be poor, while between the age of six to sixteen years it is somewhat better. If, however, we include in class a under good results tlie cases in which all symptoms have ceased, but where the kyphos has inci'eased, the relative comparison is a good result in class a of 28.5% against a good result in class 6 of 33-1/3%, or, almost the same. According to these figures, then, operative procedures in the ages of infancy and of growth, have been successful in less than one-third of the cases, even if we are so lenient as to include those cases in which the kyphosis has been increased.

On the other hand, in class c, of the patients operated upon after the age of sixteen, ninety jjer cent have given a good result. This would tend to show that in adult cases in which growth of the body has ceased, and in which the human body has concentrated its efl'orts on repair, the addition of a bone graft has given excellent results. The rationale of this improvement may be that the bone graft acts as an intra-corporeal support so that traumatism is better resisted ; or that the blood supply between the various vertebral bodies is increased and changed by the new medium placed along the spinous processes.


April, 1922]


141


Localization' of the diseaJic:

For the cervical region tlie poor results were 100%.

For the iniddorsal and lower dorsal region there

were good results in 39% and poor results in 61%.

For the dorso-lumbar and lumbar region there were

good results in 73% and poor results in 23%.

Those regions of the spine in which the vertebral body is the largest and hence relatively more resistant to necrosis have given better results. Contrary to what would naturally be supposed, the amount of lessened mobility of the spine, owing to anatomical conditions, as in the mid-dorsal and dorsal region, does not seem to have had any favorable influence on the progress of the case toward recovery.

The effect of the presence of a kyphosis on the ultinwte result of the operation: In the cases in which there was no kyphosis at the time of operation the result of the operation was 100% recovery. In those in which there was a slight kyphosis at the time of the operation, the result was a cure in 50% of the cases. In the cases in which there was a moderate kyphosis there was a cure in 50% of the cases. In those in which there was a marked kyphosis there was a cure in only 25% and a failure in 75%. In other words, the greater the kyphosis at the time of operation the less the chances of a cure. There are several factors which probably afl'ord an explanation of these results.

Tuberculosis starting after the age of growth is far less likely to produce a kyphosis than when the human body is in the growing stage. Most of the patients who have little or no kyphosis are adults. As we have already mentioned, in adult ca.ses a cure is effected in 90%.

When a kyphosis is large, the bone graft is iisually weakened in making it conform to the curve of the kyphosis ; indeed, under these circumstances it has often been broken. The graft may break at the time of the operative procedure, or it has been known to hold for as long as two years after it has been embedded and then break. It has further been noted that when a graft has broken, the acute symptoms, which had been overcome by the operation, have reappeared almost immediately. It behooves us, then, in operating, to employ as large and stable a graft as can be obtained.

The numher of spines fixed at the time of operation: This has varied from four to eight spinous processes. It has always been our aim to take in the spinous processes of the affected vertebrae and, when possible, at least the spinous processes of two normal vertebrae above and below the affected ones. In marked kyphoses, composed as they are of many diseased vertebrae, this is not always possible, and when accomplished, it is often done at the expense of breaking the graft. Kit when it is broken, mobility occurs. The fixation is tested in various ways : ( 1 ) by a lateral picture of the spinal column sometime after the graft has been embedded; (2) by a leadline tracing of the patient in an upright position and in a


forward bending posture. The results of our cases have shown that the fixation has been complete in 75% of our cases.

Duration of the disca.se before operation: This plays a very definite role as regards the successful outcome of the case. In general, the longer the duration of the disease before the operation, the more likelihood of recovery. In the ca.ses in which the disea-'se had existed less than six months previous to the operation good results were obtained in 37.5% and poor results in 62.5%.

When the disease had existed one year: Good results — 55% Poor results — 45%

When the disease had existed two years: Good results — 60% PcTOr results — 40%

AVhen the disease had existed over two years: Good results — 78% Poor results — 22%

The real cure of any case of tuberculosis of the spine depends on the amalgamation or fusion of the aft'ected vertebrae and of the aft'ected vertebrae to the normal vertebrae above and below. This takes time. There is no known method which will afford a short cut by which this may be accomplished. Calv6 states that in tuberculosis actual ossification that assures repair does not begin until about three years after the onset of the disease. Hence, the cases which have been in existence two years are farther on the road to repair than those of more recent date. The bone graft does not bring about directly consolidation of the vertebral bodies, but only of the spinous processes. But, I believe that it helps indirectly by the formation of a new blood supply and that by its stabilizing effect it alleviates the acute process of the disease.

Duration, of post-operative fixation: The after-treatment of Pott's cases recommended by Albee was as follows : The patient is kept in the recumbent position without any plaster jacket or other support for six, eight or ten weeks; he is then allowed to move around without any support, on the assumption that the graft becomes stronger and that too more quickly if the normal physiological activity of the body be permitted. Our experience does not support this view; on the contrary, we believe that proper support must be given in all cases after the operation. In adult cases the period of fixation after operation is much less than in those of infancy and during the years of growth. In the adult case our average was six months, whereas, in infancy and during the period of growth, it was from two and one-half to thi-ee years. If this support were not applied, acute exacerbations of the disease soon appeared. It was also found in this growing period that, if a proper support were not w^orn after the operation, the kyphosis invariably increased, the graft itself was not capable of preventing further deformity; being viable, it was subject to the same


142


[No. 374


influences and the same laws of strain that had brought about the original kyphos, and instead of preventing a further deformity as growth went on, it bent, becoming more rounded and indeed even acute in outline. In oi'der to prevent the occurrence of a deformity a proper brace or support had to be worn over a long period of time, at least through most of the period of growth.

Influences o-n abscesses: In general the implantation of a graft has had a beneficial action on the active symptoms of the disease and on the abscess formation. In several cases in which an abscess was present before operation, the rapidity of its disappearance was hastened by the implantation and consequent fixation. On the other hand, absces.ses often reappeared when the graft became broken or fixation was not good.

Mortality: Although we have been fortunate enough to encounter no immediate fatality following the operation, the ultimate mortality, properly attributable to it, was eight per cent. The gravity of the operation should make one vei-y careful in the selection of his cases. Children, in infancy and in the growing period, afl'ected with tuberculosis of the spine, and in many cases having other foci of tuberculosis, are not good risks. An extensive procedure of this nature is such a severe drain on their vitality that they are made more liable to a spread of the tuberculosis and to secondary infections, such as influenza, which ultimately prove fatal. The adult, however, is a far better risk and far better able to undergo it.

Conclusions In drawing conclusions, therefore, from this series of cases, one can hardly agree with the statement of Dr.


Albee that "Pott's disease must now be regarded as a distinctly surgical affection, the operative treatment a sine qua non, to be given precedence over all other therapeutic measures. Mechanical treatment must now occupy a minor position, to be employed only as a post-operative adjunct, or with patients who refuse surgical intervention or are not good surgical risks." My own feeling is tliat in Pott's disease operative procedures should be used only in selected cases, and simply as one of the aids in making a cure, but not as the chief means. Pott's disease is a pathological condition which must necessarily run a very chronic course and its cure is accomplished only when amalgamation of the vertebral bodies is assured. This necessarily takes time. Mechanical means, therefore, particularly in infancy and in the growing child, must be the main method for stabilization of the vertebri¥, and when the graft is used it is to be regarded only as an aid and must not be relied upon alone. In infancy the operation is questionable owing to its magnitude and to the tendency which it has to weaken the system and thus favor the incidence of general tuberculosis and other infectious diseases. In this class of cases the ultimate, if not the immediate, mortality is high. In the growing period, from six to sixteen years of age, in carefully selected cases the bone graft may be used, but must be considered only as an aid, and proper mechanical treatment must be kept up until the period of growth is over or amalgamation of the vertebrse has taken place. In adults the bone graft is indicated in the majority of cases, but even here mechanical support should be used for at least six months.



BOOKS RECEIVED


Greek Medicine in Rome. The Fitzpatrick Lectures on the History of Medicine Delivered at the Royal College of Physicians of London in 1909-1910. With ether historical essays. The Right Hon. Sir T. Clifford Allbutt, K. C. B., M. A., M. D., F. R. C. P., F. R. S., Hon. F. R. C. P. I., Hon. M D., Hon.LL.D., Hon. D. C. U. Hon. D. Sc, etc. 1921. 8°. 633 pages. Macmlllan & Co., London.

Life and Times of Amhroise Parr. l.'iVi-l.'i'id. With a New Translation of his Apolog>' and an Account of his Journeys in Divers Places. By Francis R. Packard, M.D. With twentytwo text illustrations, twenty-seven full page plates and two maps of Paris of the 16th and 17th centuries. 1921, 8°. 297 pages. Paul B. Hoeber, New YoTk.

The Oxford Medicine. By Various Authors. Edited by Henry A. Christians, A. M., M. D. & Sir James Mackenzie, M. D.. F. R. C. P., LL. D.. F. R. S. In six volumes. Illustrated. Volume IV. Diseases of Lymphatic Ti,isue. Metabolism, Locomotory Apparatus. Industrial Disease and Difectious Di.'ieases. 1921, 8°. 938 pages. Oxford University Press. American Branch, New York.

History of the Pennsylvania Hospital Unit. (Base Hospital No. 10, U.S.A.) In the Great War. 1921. 8°. 253 pages. Paul B. Hoeber, New York.


Laboratory Handbook for Dietetics. Ph. D. Revised edition. 1921. 8': Company. New York.


By Mary Swartz Rose, 1.56 pages. Macmillan


Woman's Hospital in the ^tate of Netv York. Report of the Scientific Work of the Surgical Staff. Edited by George Gray


Ward, Jr., M. D., F. A. C. S. Volume III, 1920. 8°. 195 pages. C. V. Mosby Company, St. Louis, Mo.

Morris's Human Anatomy. A Complete Systematic Treatise by English and American Authors. Edited by C. M. Jackson, M. S., M. D. Sixth edition, revised and largely rewritten. Eleven hundred and sixty-four illustrations, five hundred and fifteen printed in colors. 1921. 4". 1.507 pages. P. Blakiston's Son & Co., Philadelphia.

American Child Hygiene Association. Transactions of the Eleventh Annual Meeting. 1921. 8°. 440 pages. Press of Franklin Printing Company, Baltimore, Md.

Columbia University in the City of New York. Studies from the Laboratories of the Department of Surgery. Volume III, 1918-1920. 8°.

Connecticut fitate Medical Society. Proceedings of the 129th Annual Convention. Editor Charles Williams Comfort, Jr. 1921. 8 = . 265 pages. Published by the Society.

Ergebnisse der Chirurgie und Orthopiidie. Herausgegeben von Erwin Payr und Hermann Kiittner. Vierzehnter Band. Redigiert von H. Kiittner. Mit 137 Textabbildungen. 1921. 8°. 956 S. Julius Springer, Berlin.

The Rockefeller Institute for Medical Research. Studies. Volume

XXXVIII. 1921. 8°. 580 pages. The Rockefeller Institute for

Medical Research, New York. The Heart. Old and Keiv Views. By H. L. Flint, M. D. With

Illustrationis. 1921. 8°. 177 pages. Paul B. Hoeber, New

York.


Syphilis and Its Treatment. With Especial Reference ta Sypliilis of the Sliin. By Wilfred S Fox, M. A., M. D.. B. C. (Cantab.), M. R. C. P. (London). With fifty-three illustrations, twentytwo in colour on fourteen plates and thirty-one in blacli and white on twen'ty-eight plates. 1921. 8°. 195 pages. Paul B. Hoeber, New York.

Text-Book of Materia Medica for Nurses. Compiled by Lavinia L. Dock. Seventh edition. Revised in accordance with the ninth decennial revision of the U. S. pharmacopseia. 1921. 12°. 315 pages. G. P. Putnam's Sons, New York and London.

Modern Italian Surgery and Old Universities of Italy. By Paolo De Vecchi, M. D. Fareword by George D. Stewart, M. D. With fifteen full page illustrations. 1921. 8°. 249 pages. Paul B. Hoeber, New York.

The Microtomisfs Vade-Mecum. A Handbook of the Methods of Microscopic Anatomy. By Arthur Bolles Lee, Hon. F. R. M. S. Eighth edition. Edited by J. Bronte Gatenby, B. A., B. Sc., D.Phil. (Oxon.), D. Sc. (Lond.), F. R. M. S. With the collaboration of W. M. Payliss, M. A., D. Sc. (Oxon.), F. R. S., F. R. M. S. [and others]. 1921. 8°. 594 pages. P. Blakiston's Son & Co., Philadelphia.

The Diseases of Children. By the Late Sir James Frederic Goodhart, Bart., M. D., LL. D. Aberd., F. R. C. P. Eleventh edition. Edited by George Frederic Still, M. A., M. D., F. R. C. P. With 60 illustrations. 1921. 8°. 942 pages. Paul B. Hoeber, New York.

The Principles and Practice of Medicine. By the Late Sir William Osier, Bart., M. D., F. R. S. and Thomas McCrae, M. D. Ninth thoroughly revised edition. 1921. 8°. 1168 pages. D. Appleton and Company, New York and London.

Diseases of the Skin. By Richard L. Sutton, M. D. With nine hundred and sixty-nine illustrations, and eleven colored plates. Fourth edition, revised and enlarged. 1921. 8°. 1132 pages. C. V. Mosby Company, St. Louis.

Studies in the Palaeopathology of Egypt. By Sir Marc Armand Ruffer, Kt., C. M. G., M. D. Edited by Roy L. Moodie, Ph. D. 1921. 4°. 372 pages The University of Chicago Press, Chicago, Illinois

The Lister Institute of Preventive Medicine. Collected Papers. No. 15, 1918-19: No. 16, 1919-20. Part 1. Bacteriological, Epidemiological, Pathological and Statistical Papers. Part II. Physiological, Zoological and Biochemical Papers. London.

A Guide to Urinary Diseases. By Adolphe Abrahams, O. B. E., M. D. (Cantab.) M. R. C. P., (1-ond.) and A. Clifford Morson, O. B.E., F. R. C. S. (Eng.). 1921. S = . 120 pages, Longmans, Green & Co., New York. Edward Arnold & Co., London.

Atlas for Electro-Diagnosis and Therapeutics. By F. Miramond de Laroquette, M. D. Authorized Translation by Mary Gregson Cheethan. With Foreword by Robert Knox, M. D. 1921. 8°. 180 pages. Paul B. Hoeber, New York.

The Blood Supply to the Heart in its Anatomical and Clinical Aspects. By Louis Gross, M. D., C. M. With an Introduction by Horst Oertel. With twenty-nine full page plates and six


text illustrations. New York.


1921. 4°. 171 pages. Paul B. Hoeber,


A Manual of Selectei Biochemical Methods as Applied to Urine, Blood and Gastric Analysis. By Prank P. Underbill, Ph. D. 1921. 8°. 232 pages. John Wiley & Sons, Inc., New York.

Surgical Diseases of Children. A Modern Treatise on Pediatric Surgery. By Samuel W. Kelley, M. D., LL. D. Second edition, revised and enlarged; illustrated. 1914. 8 = . 789 E. B. Treat & Co., New York.


A Pocket Surgery. By Duncan C. L. Fitzwllliams, C. M. G., M. D., Oh. M., F. R. C. S. (Edin. and Eng.). 1921. 12". 348 pages, Longmans, Green and Co., New York; Edward Arnold, London.

Human Embryology and Morphology. By Arthur Keith, M. D.. P. R.S., LLlD. (Aberdeen), F. R. C. S. (Eag.) Fourth edition, revised and enlarged with nearly 500 illustrations. 1921. 8°. 491 pages. Longmans, Green and Co., New York: Edward Arnold, London.

A Manual of Diseases of the Stomach. By William MacLennan, M. B. With the Assistance of J. SalisbuiT Craig, M. B., Ch. B. 1921. 8°. 392 pages. Longmans, Green and Co., New Y'ork and London.

The Life of Jacob Henle. By Victor Robinson. M. D. 1921. 8°. 117 pages. Medical Life Company, New York.

Clinical Surgical Diagnosis. By P. de Quervain. [Third English edition.] Translated from the seventh edition by J. Snowman, M. D. With 731 illustrations and 7 plates. 1921. 8°. 914 pages. William Wood & Co., New York.

The Oxford Medicine. By various Authors. Edited by Henry A. Christian, A. M., M. D., and Sir James Mackenzie, M. D., F. R. C. P., LL. D., F. R. S. Volume V. Infectious Diseases (Cont'd) and Diseases Due to Animal Parasites. Volume VI. Diseases of the Cmitral Nervous System. Under the Editorial Supervision of Sir James Purves Stewart, K. C. M. G.. C. B., M. D., P. R. C. P., 1921, 4°. Oxford University Press, American Bi-anch, New York.

Oxford Medical Publications. Publishers: Henry Prowde, London; Hodder & Stoughton, London. The following 5 volumes:

The Early Diagnosis of the Acute Abdomen. By Zachary Cope, B. A., M. D., M.S., Lond., F. R. C. S. Bag. 1921. 8°. 223 pages.

The Care of Eye Cases. A Manual for the Nui-se, Practitioner and Student. By Robert Henry Elliott, M. D., B. S. (Ijond.). Sc. D. (Edin.), F. R. C. S. (Eng.). With 135 illustrations. 1921. 8°. 172 pages.

^Heart Disease and Pregnancy. By Sir James Mackenzie. M. D.. P. R. C. P., LL. D., Edinburgh antl Aberdeen. P. R. S.. P. R. C.P.I. 1921. 8°. 138 pages.

Obstetrics and Gynaecology. Edited by John S. Pairbairn, M. A., B. M., B. Ch. (Oxon), P. R. C. P. (Lond.), P. R. C. S. (Eng.) 1921. 4°. 950 pages.

The Anatomy of the Human Orbit and Accessory Organs of Vision. By S. Ernest Whitnall, M. A., M. D., B. CH. (Oxon.), M. R. C. S.. L. R. C. P. (Lond.) Illustrated largely by photographs of actual dissections. 1921. 8°. 428 pages.


CONTENTS

  • The Evolution of Human Races in the Light of the Hormone Theory. By Prof. Sir Arthur Keith 155
  • The Pharmacological Action of Adrenalin on the Sphincter Pylori of the Foetus. By P. G. Shipley, M. D., and K. D. Blackean, M. D. 159
  • Occurrence of Ansemia in Rats on Deficient Diets. (Illustrated.) By William M. Happ 163
  • The Significance of the Influenza Bacilli. By Arthur L. Bloomfield
  • The Intercolumnar Tubercle, an Undescribed Area in the Anterior Wall of the Third Ventricle. (lUustratei.) By Thacy Jackson Putnam ISl
  • Pseudomyxoma Peritonei. By Edward Novak, A. B., M. D.
  • Histamine as an Inflammatory Agent. By William Bloom
  • Treatment of Non-Encapsulated Brain Tumors by Extensive Resection of Contiguous Brain Tissue. By Walter E. Dandy 188
  • Spontaneous Labor Occurring Through an Obliquely Contracted, Kyphotic, Funnel Pelvis. (Illustrated.) By J. Whitridge Williams 190


THE EVOLUTION OF HUMAN RACES IN THE LIGHT OF THE HORMONE THEORY

By Prof. Sir Arthur Keith (Conservator of the Museum and Hunterian Professor, Royal College of Surgeons, London. Eng.)


There is no need for a Herter Lecturer to apologise to Alumni and students of Johns Hopkins LTniversity for choosing a subject which has apparently no direct bearing on medical practice. No LTniversity has perceived more clearly than Johns Hopkins that rational medical practice must be based on a complete knowledge of the normal machinery of the animal and human body and has, therefore, in the past welcomed every enquiry which was likely to broaden the basis of this knowledge. When we set out to discover how the races of mankind have arisen, we have to make a full demand on all we know concerning the factors which regulate the growth of the


Abstract of Lecture I under the Herter Foundation delivered before the Johns Hopkins University on October 5, 1921.


human body — particularly on the action of the glands of internal secretion. Much of what the world knows concerning these glands it has learned from Johns Hopkins University — from Howell, Gushing, Goetsch, Abel, Barker, MacCallum and many others. In these lectures I hope to return, with some degree of interest, the preliminary outfit which the anthropologist borrowed from the clinician and pathologist.

Darwin, Hunter and the Hormone Theory I can best introduce the problems to be discussed in these lectures by carrying my audience to Darwin's home, near the village of Down, situated in the chalk uplands to the south of London, where, in the year 1870, the great observer was applying his machinery of evolution to the


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"Descent of Man." After he had made use of all the natural agencies at his command, the action of selection, the supposed effects of use and disuse, the manner in which one part of the body is correlated with that of another part, he had to declare that none of these could explain how the Negro had come by one set of characteristic features, the Mongol by another set and the European or Caucasian by a third. The missing agency which Darwin was in search of has been discovered accidentally by medical men working on disorders of nutrition and growth in the out-patient departments and wards of public hospitals. It was working under such circumstances that clinicians came to realize that there is placed in the human, as in the animal body, a group of glands, which, through the formation of chemical substances — or Hormones as Starling named them in 1904: — determine the individual and racial characters of every man, woman and child. It is a matter worthy of remark that not only Darwin but also his great predecessor, John Hunter, stood on the verge of this discovery — and yet both were sidetracked. John Hunter successfully transplanted the testes from one fowl to another; he grafted the cock's spur on the hen's leg, and the hen's spur on the cock's leg; his earliest investigation was to show that the human testis which failed to descend was arrested because of imperfect development; no one ever realized more clearly than Hunter did that the growth of all these features of the body, which he named secondary sexual," depends on the presence of healthy sexual glaads. Darwin also realized this to the full, but he concentrated his attention, not on how sexual characters are produced but on how they may be selected and perpetuated once they have come into existence. In the history of discovery we learn as much from the failures of brooding geniuses as from their successes. Both Hunter and Darwin had the misfortune to work ahead of the physiological knowledge of their time.

The Features of Neanderthal Man and of the Acromegalic Contrasted

Soon after 1908, when the Council of the Royal College of Surgeons of England placed me in charge of the Museum which a long line of Conservators had built up round Hunter's glorious nucleus, I had to rearrange the skulls of ancient and modern human races. In one section w^ere two specimens of historical interest ; one was a cast of the Neanderthal calvarium which Shaatfhausen had sent to Huxley in 1860 and on which the latter had based his statement that Neanderthal man was the most primitive, most ape-like and most ancient type of humanity known to him. The other specimen was a remarkable modern skull which led Dr. Barnard Davis, the BTitish craniologist, flatly to contradict Huxley by asserting that Neanderthal characteristics could still be found in the population of Europe. I noted with interest that the pituitary fossa in the Barnard Davis skull was double


the ordinary size, that all the characteristics of a regulated acromegaly were present and that it was these acromegalic traits which gave the apparent resemblance to the Neanderthal type. At the date of wiiich I write there was ample evidence to prove that a disordere<l enlargement of the pituitary was directly related to the appearance of acromegalic traits and that the effects could be best accounted for by regarding the pituitary as a centre in which growth-hormones were elaborated. The query naturally arose in my mind, as it did in that of others : Can the pituitary be concerned in the evolution of human races?

At the time I commenced this investigation an important truth was dawning on the minds of European anthropologists. The late Professor Gustav Schwalbe of Strassburg, strongly backed by Dr. Adloflf of Berlin, led a movement which completely altered the then prevailing conception of Neanderthal man ; Schwalbe held that he must be regarded, not as the Pleistocene ancestor of modern Europeans, but as a separate and extinct species of humanity. With the establishment of Schwalbe's position, we had to alter our mental vista of man's past. All the existing races of mankind are members of a single species but as we go into the far past we find not only separate species of humanity, but separate genera. We have to base our conception of the conditions under which mankind was evolved not on what wc .see in the modern world of man, but on what we can see now' among man's nearest allies — the anthropoid apes. These we find broken up into diverse genera and .species, confined to definite localities. All discoveries of recent years justify the belief that in the remote past mankind was represented by numerous local genera and species and that the machinery of evolution must have been both active and effective. The nature of this machinery is not beyond recall ; we may safely presume that it may be studied now in living groups of anthropoid apes — amongst gorillas, chimpanzees and orangs. We shall find among anthropoid apes the same hormonic mechanism which shaped the ancient and motlern racial types of mankind.

Here, too, before following the straight thread of my lecture, I may mention another line of evidence which has influenced the direction of my investigations. We are apt to think of evolution as a process of the past and that mankind has now come to an evolutionary standstill — so far as racial difi'erentiation is concerned. When we look at the distribution of the chief types of humanity as seen in the modern world it is no longer possible to share in this belief. We cannot explain why the Mongolian type was formerly confined to one region of the earth, the negro to another and the European to a third, or why intermediate types link them together, unless we proceed on the hypothesis that evolution has been and is now at work. In every continent we find human races at all stages of differentiation — from an


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incipient state such as is exemplified by the British Anglo-Saxon and Celt, where the degree of physical differentiation is slight, up to a complete stage, such as represented by Negro and white man where the degree of physical differentiation is unmistakable. The quest we set out on is the discovery of the physiological machinery concerned in the differentiation of mankind into its various physical types.

Acromegaly is a True Disorder of Growth

For the purpose of our search uo condition is so instructive as that known to clinicians as Acromegaly. In a short period of years after the onset of this disorder, a new and distinctive type of being is produced which the clinician recognizes at a glance as acromegalic. All the bodily structures and characters involved in the change are just those which tlie anthropologist knows to be concerned in the differentiation of human races. The texture and frequently the tint of the skin is changed; the growth and distribution of hair alters, the nose, lips, jaws, cranium and thorax undergo characteristic transformations; stature and the proportions of the body and limbs become modified. To study and estimate the degree and nature of these acromegalic changes we must apply the same exact methods as anthropologists employ in the study of human races.

Ten years ago I published the results of a craniological examination of acromegalic skulls ^ and showed the general nature of the changes which had occurred in them. At that time I had not sufficiently appreciated the complexity of the processes involved iu the growth of the normal cranium, for, to provide room for the growing brain, new bone has to be laid down at varying rates along certain sutural lines, while along other lines it is being absorbed. No less elaborate is the growth mechanism of the face, for, as the teeth erupt, additional bone is interpolated along certain sutural lines and deposited on certain areas while a process of absorption is taking place along other lines and areas. Each bone of the cranium and face represents a separate growth element and it is only by studying the changes which occur in each element that we come by a precise knowledge of how the face of a boy of five years of age is transformed into the face of an adult man. The result of study along these lines on additional acromegalic material has been to show that the differences between a uornuil and an acromegalic skull are exactly of the same nature as those seen when we compare the skull of a boy in his thirteenth year with one of an adult num. Beyond any doubt, with the onset of acromegaly a ti'ue growth process is reawakened and that reawakening is a continuation of the normal process which should cease when the adult stage is reached. The disorderly enlargemeut of the pituitary is directly related to the resurrection of the process of growth.


1 An Inquiry into the Nature of the Skeletal changes in Acromegaly. Lancet, April 15, 1911.


Acromegalic Changes Have a Functional Significance

It is only when we realize that the size and shape of the skull are determined by the functional purposes which it has to serve and by the manner in which it serves them, that we really profit by craniological enquiry. In the fii'st place the skull has to serve as a brain carrier, providing a cavity with walls so fashioned that additional accommodation can be easily and economically provided for the growing brain. In the second place, the skull has to serve as a carrier of sense organs — the eye, nose and ear. In the third place, it has to provide the whole of the bony scaffolding concerned in mastication, and which also answers, in a secondary degree, to the needs of speech and respiration and deglutition. In the fourth place it has to serve as a complex mobile lever on which the muscles of the neck act.

Let us look for a moment at the alterations undergone by acromegalic skulls in respect to these four functions. Apparently the brain increases in bulk and the cranial cavity in size; at least the mean cranial capacity of the acromegalic skulls at my disposal is distinctly above the average for normal skulls. As a sense carrier there is no change, save as regards the nose. But as regards its third or masticatory function, very remarkable and instructive changes occur. The area on the side of the skull from which the temporal muscle arises, becomes greatly extended, particularly in a forward direction. In the forward extension the frontal bone undergoes a transformation involving changes along the coronal suture, the throwing out of great supra-orbital bulwarks which serve as fulcra of origin for the temporal muscle. The transformation is usually facilitated by an enormous extension of the frontal air sinuses. The origin for the masseter muscle — the zygomatic arch — is strengthened, elongated and lowered in position. The areas and processes from whicli the pterygoid muscles arise, are also strengthened and extended. The ascending ramus of the mandible, which has to provide an area of in.sertion for those muscles, elongates greatly and sometimes increases iu width as well. All parts of the body of the mandible become the sites of true growth; the alveolar margin, carrying the tooth-sockets, is remodelled and extended. The chin and the lower mandibular border are greatly augmented — strengthening and extending the bony scaffolding on which the floor of the mouth is set. All parts of the upper jaw — with one exception — share in the general enlargement of the masticatory system. The exception is the area cari-ying the alveolar margin and liard palate; these parts, we shall see anon — may not suffer a perceptible degree of change.

Now the parts just named — the chin, the size and form of the mandible, the prominence of the cheek-bones, the size and projection of the ascending nasal process of the


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upper jaw, the degree of developiueut of the supra-orbital ridges — are all of them immediately concerned in giving the face its individual, as well as its racial, characteristics. All of them are linked together to serve in one function — mastication. The pituitary acts not in an anatomical but in a physiological manner; it can somehow stimulate and correlate the growth of all the anatomical elements concerned in a single function. We shall see presently that other hormones, besides those arising in the pituitary, can and do influence the growth of the face. It is sufficient for the meantime to be certain that the characterization of the face — the chief signboard of race — is under the control of a hormone system.

Further evidence in favour of the view that the growth functions of the pituitary are grouped on a physiological — not an anatomical — basis is seen when we study the adaptations undergone by the skull to serve as a lever. To that area of the skull to which the muscles of the neck are attached we nmy give the name of "nuchal platform"; it is fashioned out of cartilage bone, comprising the whole of the cartilaginous part of the occipital bone and the mastoid part of the temporal bone. In acromegaly the nuchal platform has its area greatly enlarged ; all its bounding and intercrossing muscular ridges — comprising the mastoid processes and occipital ridges — are enormously strengthened. The need for some mechanism to correlate the growth of the masticatory bony scaffolding and nuchal platform is made very evident in the skulls of anthropoid apes. The gorilla exerts the strength of its massive body through its huge jaws ; hence in this animal a supreme development of the masticatorj' scaffolding is correlated with an enormous expansion of the nuchal platform. In the chimpanzee, a near relative of the gorilla, the jaws are reduced and so is the nuchal platform. The difference between Neanderthal man and modern man — in respect of jaw and neck — corresponds in degree to that seen between gorilla and chimpanzee. As a boy's jaws gi"ow, his neck enlarges; he continues to take a bigger size in collars as each new tooth erupts. In women the head is poised on a slender neck ; their maxillary and nuchal development represent an adolescent stage of the male. In these instances, we see the pituitary acting on a series of anatomical elements moulding and adapting them to a single physiological end. Nuchal characters also serve to differentiate races. It is a remarkable fact that the most extensive facial and nuchal development is found amongst Europeans.

All Systems of the Body Are Affected

IN Acromegaly

Too much stress cannot be laid on the fact that in acromegaly all the tissue systems of the body are involved. In every case of acromegaly which has received a complete examination, it has been found that not only are


the skeletal and cutaneous tissues affected but so too are the circulatory, the pulmonary, the alimentary-, renal and connective tissue systems. All of them are involved in overgrowth with the exceptions of the sexual system, which undergoes atrophy, and the nervous system, regarding which our evidence is incomplete. Nor can too much emphasis be laid on another fact that in the later stages of the disease — all of these systems suffer degeneration and atrophy. Some years ago the Museum of the Royal College of Surgeons obtained a subject of this disease — a man who had begun to show symptoms of acromegaly at the age of twenty-four and had died twenty-five years after the onset, the disease having been steadily progressive. Preparations, naked-eye and microscopic, were made from all the sj'stems and organs of this individual and are now preserved in the Museum of the College. Evei-y cell of the body of this subject was found to have suffered in the general disorder of growth. For example, the muscles of mastication had not merely undergone a normal degree of hypertrophy; they had undergone a real process of growth — the individual fibres having increased in length, diameter and number. This was also true of all the muscles of the tongue and mouth. The temporal and facial arteries were abnormally large and their muscular coats greatly increased, but, as was the case throughout the whole arterial system, the inner coat was thickened, it encroached on the arterial lumen. From these facts one infers that the pituitary acts upon all the structural elements which are comprised within a functional unit and somehow keeps their growth in unison The Nature of the Disorder Known as Acromegaly So far I have merely put forward the evidence in support of the view that in Acromegaly we have exposed for us part of the growth-machinery concerned in the differentiation of racial characteristics. The pituitary and substances formed within it are essential parts of the machinery. Before we proceed further it is necessary to ask the question: Is Acromegaly the result of a breakdown of one of the growth mechanisms of the normal body? I think this question must be answered in the affirmative. It is a breakdown of the body's chief adaptational mechanism — the mechanism which "makes the back equal to the burden it has to bear." That a mechanism of this nature must be present in the body, becomes apparent, I think, if one will but mark what happens in the various systems of the body when a man or animal is placed under training. The muscles are the structures chiefly concerned but it would be useless for them to undergo a hypertrophy unless they had some means of calling forth a corresponding growth reaction from heart and vessels so as to be supplied with an increased amount of blood, from the lungs, to meet their increased need of oxygen ; from the alimentary system, so as to be supplied with increased energy-material ; from the kidneys, so as to rid them of their increased refuse


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products, from the skeletal ami articular and ligaineutous systems, so that they become fit to withstand the increased stresses produced In* hypertrophied muscles. These effects cannot be produced in a merely mechanical way; we must postulate an organized system of some kind and the one which answers all the circumstances of the case is that of hormones. In Acromegaly we find a disorderly manifestation of all the results which follow increased use of muscles — bones strengthen, particularly their muscular ridges and impressions; the joint-surfaces enlarge, the ligaments are thickened, the heart, arteries and veins hypertrophy, the lungs and chest undergo a true growth; the apparatus of mastication and the alinientar\- canal become hypertrophied, renal substance is increased. If we suppose that this adaptational mechan


ism, which makes hypertrophy answer to increased action, were to be set in motion, not through the normal stimulus of muscular activity but through some condition arising in the pituitary itself, then we can understand why it is that the human body should be subject to such a disorder as Acromegaly.

One other fact supports the theory here put forward regarding the nature of Acromegaly. AVhy should the hands and feet be the parts of the body which suffer first and most in Acromegaly? If my explanation is the true one, then the feet and hands should be the most susceptible, for nothing is more certain than that hard manual labour will produce marked growth responses in the hands and feet.


THE PHARMACOLOGICAL ACTION OF ADRENALIN ON THE SPHINCTER PYLORI OF THE FOETUS

V. G. Shipley, M.D.

AND

K. D. Blackfan, M.D.

(From the Department of Pediatrics, Johns Hopkins University. Baltimore, Md.)


Pirie's f recent suggestion that hypertrophic stenosis of the pylorus in newly born infants may be due to fcetal hyperadrenia makes it necessai-j' to view that condition from an entirely new angle.

The development of hypertrophic stenosis according to I'irie comes about as follows: — (1) Reflex stimulation of the adrenal medulla from some internal or external receptor, e. g., the preputial nerve endings, is followed by (2) the discharge of an increased amount of adrenalin into the blood stream.} (3) This increased adrenal discharge stimulates the splanchnic and causes, according to whether it is constant or intermittent, either increased tone or spasmodic Contraction of the alimentary sphincters (ileocolic and jtyloric I because of the socalled reverse innervation of these muscle groups. Hypertrophy of the sphincter pylori follows because of this spasm or increased tone, and persists into post-f(etal life. The cause of stimulation disappears or is persistent only as some minor defect (phimosis) which is not usually recognized as causally associated with the graver pathological result. It mav, however, be sufficient to maintain


t Pirie. G. R.. Lancet, September 2fl, 1919.

Jin the British Medical Journal of Xovember 26. 1921, (p. 891) Gray and Reynolds have suggested that the hypersecretion of adrenalin may be maternal as well as foetal.


the severity of the morbid condition for which the infant comes under treatment.

Pirie cited cases of babies suffering from hypertrophic stenosis who recovered after circumcision and mentioned the autopsy of a child dead from hypertrophic stenosis at which gross adrenal lesions were found to support this attractive hypothesis. It is obvious that this hypothesis is dependent on the so-called "reversed innervation" of the gastrointestinal sphincters. In other words, the statement must be accepted that while the augmentor fibres supplying the gastrointestinal musculature in general reach the digestive tract from the vagus nerve and the inhibitors from the splanchnic, the muscles of the pyloric and ileocolic sphincters are augmented through the splanchnic and depressed by way of the vagus. Stimulation of the splanchnic nerves (peripheral), according to this theory, results in relaxation of the entire gastrointestinal musculature except the ileocolic and pyloric sphincters which are said to be thrown into a state of increased tonus by the impulse following splanchnic excitation.

Almost nothing is known about the behavior of the intestine during intrauterine life.

It has been generally accepted that peristalsis occurs before birth because lanugo hairs are found in the first meconium. These hairs could only have reached the large intestine by passing through the whole gastrointestinal tract after having been swallowed with amniotic fluid into which the_y had been cast off. The general opinion regarding ante-natal intestinal movement has been that peristalsis is feeble and infrequent until just before the birth of the foetus. Yanese,* indeed, found that in human foetuses he was able to produce peristalsis and local contractions as early as the ninth week of intrauterine life. Before this date, when the myenteric plexuses and the longitudinal muscle coat begin to develop, this author claimed to have been able to produce neither peristaltic movements nor local contractions by direct stimulation. He concluded, therefore, that peristaltic movements are of neurogenic rather than myogenic origin.

In the course of a study of the physiology of the gastro-intestinal tract in the foetus we have observed the action of adrenalin solutions on the musculature of the stomach and intestine, and since the appearance of the hyperadrenia theory of pyloric stenosis, it has seemed worth while to record the reaction of the foetal sphincter pylori to solutions of adrenalin. Solutions of adrenalin were tested on ring and strip preparations of isolated sphincter pylori and on preparations of the pylorus in situ.

Isolated preparations of embi-yonic pyloric muscle show very characteristic regular spontaneous contractions when kept in oxygenated warm Kinger solution. The graph made by such an isolated ring is shown below (Fig. No. 1). Contractions usually start two or three minutes after the preparation is set up and increase steadily and rapidly in strength until a maximum contraction is reached, which may be maintained for two or three hours. WhUe the contractions are usually singularly rhythmic, the rhythm maintained may be multiple (in groups of two or three beats, Fig. No. 2) and occasionally, in chilled or shocked preparations, the contractions are irregular in rate and force. The character of the curve made by these muscular contractions is strikingly uniform — a rapid contraction indicated by a steep, almost vertical ascending limb, and a rapid initial relaxation slowing to its completion, so that the descending limb of the curve blends gently with the horizontal. Ascent or relaxation may be interrupted by secondary' contractions which produce ana- or cata-crotic notches preceding the relaxation of the muscle, or the curve may be topped by a smooth or serrated plateau; double contractions may occur which are followed by a contraction wave of extra height, or by a compensatory pause, a phenomenon which we shall discuss in more detail later.

Now if a solution of adrenalin be instilled into the Ringer bath in which the strip of muscle is working, a pronounced change in the rhythm and force of the contraction takes place which is characteristic and constant for a given dose of the drug. If a small dose of the salt is given (0.0005 mgm. in 50 c.c. of the bath solution), the interval between the contractions is lengthened immediately so that three or four times the usual period may intervene between contractions without any departure of the lever from the base line (Fig. 3b). When the muscle does contract, however, it may do so with somewhat greater force, so that the resulting curve of the contraction wave is very slightly higher than the curve of the contractions immediately preceding the instillation of the adrenalin solution. On the other hand, after a slightly larger dose, the amplitude of the contraction may be diminished, the interval remaining the same, or both amplitude and frequency of contraction may diminish. When the amplitude of the contraction is very much diminished, the return to normal contraction force may be gradual (Fig. 3a). This is maintained until the preparation is washed out or the drug is destroyed by oxidation. This decreased frequency of contraction is more pronounced as larger doses of the drug are given until, following the exhibition of massive amounts (0.3 mgm. in 50 c.c), the muscle may remain completely paralyzed for half an hour or more, even though the specimen has been twice or three times washed. Return of contractility to such a paralyzed muscle is like the onset of contractions in a fresh, shocked, preparation. The muscle contracts at first to a hardly recordable extent and gradually progressively, minute by minute, the contraction attains its full strength (Fig. 4). The action of adrenalin on pyloric strips is quite the same.

The description of the increase in the size of the contraction following the administration of small doses of adrenalin might make one feel that, besides the depressor action of the drug, some pressor action was present also which increased the contraction of the muscle, but a study of the graphs made from untreated preparations shows that an increase in the strength of contraction is seen following any pause in the course of the spontaneous rhythmic muscular movements or lengthened inten^al between contractions. It will be seen, therefore, that this apparent increa.se in contraction is analogous to the heightened response of the cardiac muscle after the removal of the inhibition caused by vagus stimulation.

The above account of the results of splanchnic stimulation holds good not only in the pig foetus, on which most of our studies have been made, but also in all other mammals which we have studied, including man. Since pyloric stenosis occurs only in man, as far as we at present know, an examination of human foetal material is necessary in order to exclude the possibility of special peculiarities. This is especially so since human tissue, at least in one instance, is said to vary from that of other mammals in its response to chemical stimulation (see Barbour's description of the response of the human coronary arterj- to adrenalin). It may be well, therefore, in this connection to give a protocol of one of the experiments on the pylorus of a human foetus.

The child R. M., female, prematurely born in the sixth month, weight 907 gms., admitted in the eleventh hour of extra-uterine life. The child was said to have grown progressively weaker since birth.

Death at 8.30 A. M., the day following admission.

At 10 P. M. the stomach was opened and found filled with a slightly alkaline fluid consisting of the milk feeding given two hours before death. The milk showed no evidence of coagulation.

The pylorus was normal to inspection. It was cut into strips one of which was immediately suspended in 50 c.c. of oxygenated Tyrode's solution at 39° C. and attached to a light lever.

Strip No. 1. Spontaneous contraction of the muscular strip started immediately and continued with experimentally induced variation until 11.20 P. M., when the preparation was discarded. The instillation into the bath of 0.1 c.c. of a 1 : 100,000 solution of adrenalin chloride (P.D.) (a concentration of 1: 50,000,000) was followed by no other change in the contraction than a slight slowing of the contraction rate. The instillation of double the amount of the drug was equally unproductive of appreciable effect. Following the instillation of 1 c.c. of 1 : 100,000 solution of the drug, however, (1 : 5,000,000) the amplitude of the contraction curve was considerably diminished and the exhibition of 1 c.c. of a 1 : 10,000 solution (1 : 500,000 concentration) caused a relaxation of muscular tone and diminution of contraction amplitude to approximately one-fifth of the normal value, a condition of affairs which persisted until, just before removal of the strip from the bath, stimulation with 0.1 mgm. of histamine restored approximately the pre-experimental muscular vigor (Fig. 5).

Strip No. 2. four hours and 20 minutes after death, 2 hours and 50 minutes after removal from the cadaver, gave an equivalent reaction to that given by preparation I.

Strip No. 2, studied at 3.30 P. M. on the day following death under the same conditions described above, gave only feeble contractions which were marked by inhibited adrenaline chloride 1 : 50,000,000 and which entirely ceased under the influence of the same drug 1 : 5,000,000. This strip was kept at refrigerator temperature in Ringer's solution for 31 hours after death.

It will be seen from the above experiment that the re action of the human foetal pylorus differs in no respect from the response to adrenalin given by the pyloric musculature of foetuses of other mammalian species. This response is in the nature of relaxation and inhibition rather than of stimulation and augmentation.

We have been able to confirm the results given by ring


and strip preparations, isolated from the foetal body and maintained in vitro, by experiments in which the pylorus was left in situ and the adrenalin was exhibited to the pylorus with the blood and nerve supply uninterrupted.

Increased secretion of adrenalin il would seem, could hardly result in anything else than a decrease in the tone of the pyloric muscle, and intermittent discharges of adrenalin, instead of inducing periodic pyloric spasm, would rather result in periods of relaxation of the gastrointestinal motor mechanism.

Furthermore, since the action of adrenalin on the muscle of the gastro intestinal tract depends on stimulation of the endings of the splanchnic, reversal of valvular innervation, if it exists in the adult intestine, must be a phenomenon confined to post-foetal life.

It is interesting to note that in the pig foetus, even in those with a C. R. length of only 6 cm., it is possible to see definite peristalsis when the gut is exposed after delivery from the uterus. Foetal peristaltic movements follow the "law of intestine" as laid down for the adult animal, bear the same relation to the Tonus rings and are affected in the same way by stimulation. Contractions of segmentation occur and are more readily seen than in the adult gastrointestinal tract and, in brief, intestinal motor mechanism has the potentiality to carry out the movements described as characteristic for the gastrointestinal tube in post-embryonic life.

Fig. 1. — Spontaneous contractions of an isolated ring of embryonic pyloric muscle (pig).

Fig. 2. — Spontaneous contractions of a ring of pyloric muscle from a pig embryo, to show compound contractions and multiple rhythm in a cooled preparation. Temperature of the bath 36° C.

Fig. 3. — a and 6 show the effects of small doses of adrenalin chloride on the contractions of rings of embryonic pyloric muscle (pig) suspended in 50 c.c. of Ringer-Locke solution. While the contraction rate is much slower after very many doses of adrenalin, the tone of the muscle remains unchanged. Recovery may be sudden or gradual. During the period of recovery the amplitude of the contractions may return to normal or slightly above, while the frequency is still diminished. The immediate effect of the drug is the diminution of both amplitude and frequency of contraction.

Fig. 4. — ^Shows complete cessation of contractions of a ring of pyloric muscle of a pig embryo after 0.3 mgm. of adrenalin chloride was added to a bath of 5 c.c. Ringer-Locke solution.

Fig. 5. — Curve showing the effect of adrenalin chloride on a preparation of the pyloric muscle of a human foetus which contracted spontaneously in 50 c.c. of Ringer-Locke solution. This curve shows that the tone of the muscle was impaired by the drug as well as the frequency and amplitude of the contractions.


1


162 JOHNS HOPKINS HOSPITAL BULLETIN [No. 375

Fig. 1. Spontaneous pontraction of a ring of pyloric muscle. Pig foetus C. R. 115 mm. Ringer-Locke solution.

39° C. 39° C.

Beginning fatigue.

4.05 P.M. 4.10 P.M. 4.15 P.M. 4.25 P.M. 4.30 P.M.


Fig. 2. Compound contractions and multiple rychm of a ring of pyloric muscle induced by cooling. Pig fcEtus C. R. 14 6.1 mm.

k a ill .Liii k kii- L k k k 'hijj.


Fig. 3a. Pylorus of a pig foetus. C. R. 157 mm. in 2 c.cm. of Ringer-Locke solution.


fi 1


Adrenalin chloride— .01 mgra. Rate slowed —Amplitude slightly increased.


Fig. 3b. Effect of Adrenaline Cliloride on pylorus of a pig embryo. C. R. 171 mm. Ringer-Locke. 38° C.

Adrenalin! Chloridi 0.005 mgm. A. C. 0.01 nigm. A. C. 0.0005 nigm.


Effect of adrenaline chloride on the spontaneous contractions of the pylorus. Pig embryo C. R. 155 mm. Ringer-Locke

solution. 20 c.cm.


».Jo^AjvAAJt-^AJJ-U^^AAMAAA/jJiAj'XUiA/L_


A. C. 0.3 mgm. Washed out.


R. M. Pyloric muscle (strip). Human premature — lived 32 hrs. Weight 4410 gms.

A. C. 1.0 c.c— 1100,000.


May, 1922]


163


OCCURRENCE OF ANAEMIA IN RATS ON DEFICIENT DIETS

By William M. Happ

(From the Department of Peiliatrics. The Johns Hopkins University.)


Introduction. Numerous attempts Lave been made to produce aiia?mia in animals by means of diets poor in iron. In these experiments attempts Lave often been made to ascertain wLetLer inorganic iron given by luoutL causes the regeneration of the blood in anaemia. Meyer ' summarized the literature on this subject in I'JOG.

Hall - fed white mice on the following diets :

1. Casein 37. S

Starch 28.3

Fat 30.1

Salts* 3.7

2. Casein 20.0

Butter 3.34

Fat 11.66

Cellulose 0.7

Starch 60.5

Salts 3.7

He found that the total iron content of the ash of the mice kept for three weeks on these diets decreased 40 per cent. The feeding of iron by mouth increased the number of red blood cells and hsemoglobin. The ingredients of these diets were purified foodstuffs and relatively free from iron. The nature of the fat used was not stated. The animals lost rapidly in weight and lived only four weeks on diet No. 1 and six weeks on diet No. 2. No water soluble B was supplied in these diets.

Schmidt ^ was not able to produce ana?mia in tLe first generation of wLite mice fed on a diet poor in iron. The blood of their young, however, showed a reduction of haemoglobin and red blood cells, with poikilocytosis, anisocytosis and polychromasia. When iron was added to the diet, the blood became normal. Enlargement of the spleen was noted in these anaemic mice.

Other autliors, Kunkel,* Cloetta, Abderhalden," MulIcr," Tartakowsky,* and others, fed animals on diets of milk, milk and bread, and milk and rice, to study the regeneration of tLe blood following bleeding and tLe effects of the administration of inorganic iron. These experiments sLowed that a reduction in the amount of iron bound in the tissues (reserve iron) and in the circulating blood (circulating iron) resulted in animals fed on such diets. The depletion could be made up by feeding iron by mouth either in inorganic or organic combination. They showed further that the reserve iron was depleted before the amount of iron circulating in the blood as haemoglobin was diminished. There have

Composition of salt mixture.

K,CO,, 0.97

NaCl 0.7

CaHPOi 1.8

MgCl, 0.18


been few studies other than those regarding the hiemoglobin content of the blood of animals on deficient diets.

It is a fact that the prolonged feeding of milk alone to an infant may be followed by the appearance of a secondary anannia. Because of the work of Bunge and Abderhalden " this ana'inia has been attributed to a depletion of the reserve store of iron with which these infants are born, and a consequent reduction in the circulating iron (ha-moglobin). Again, there are anaemias associated with rickets in infancy in which a peculiar symptom complex is present, associated with various qualitative and ([uantitative cLanges in tlie blood picture. Children with these anaemias usually have an enlargement of the spleen, which is occasionally extreme, and a slight enlargement of tLe liver and lymph nodes, and a secondary an.emia with or without leucocytosis. There is often a lymphocytosis, and immature cells of both red and white blood cell series are found in the circulating blood (normoblasts, megaloblasts, myelocytes, pathological lymphocytes, etc. ) . This clinical picture has received various titles, such as "von Jaksch's anaemia," "anaemia pseudoleuka'mica infantum" and "splenic anaemia of infants." It is in all essential respects a secondary anannia. The frequency of its association with rickets has been emphasized by practically every author on the subject (.see review bj' Evans and Happ).^" The cause of this aniemia has not been established. Anaemia of this type appears to depend upon more factors than does the simple secondary anaemia which results from a diet low in iron, such as a prolonged milk diet.

On account of the frequency of anannia in infants with rickets the question arose as to the existence of anaemia in rats made rachitic by faulty diets. Through the kindness and co-operation of Dr. McCollum, Miss Simmonds and Doctors Shipley and Park, I was able to study the blood of rats on various faulty diets. These authors have found that if growing rats are placed on diets in which certain fats, butter fat, cod-liver oil, etc., are absent, or pi-esent only in small quantity, and in which there is a certain disproportion between the amounts of calcium and phosphorus, lesions of the bones result which are analogous to the lesions of the bones of young children with rickets."

The result of the examination of the blood of rats on various faulty diets is given, togetLer with the diet employed in each instance.

Technic. Klineberger and Carl,'- and later Powdermaker," found that the number of cells in blood obtained from the tails of rats was subject to great variation. For this reason the following procedure was employed in obtaining blood for


164


[Xo. 375


counting. The rat was lightly anesthetized and laid on a board, ventral side up. The hair over the thigh was cut close. The femoral (superficial) vein, which lies just beneath the skin, was exposed. This vein is quite prominent, especially in albino rats. It was cut with sharp scissors. In this way sufficient blood could be obtained with insignificant trauma for a red and white blood cell count, hcemoglobin determination, a cresyl blue preparation for reticulated red blood cell count, and smears for a differential blood count. As a rule, the flow of blood stopped promptly, and if the animal was to be kept, a drop of collodion was placed on the wound. The counts given in this paper, however, are the results of the first examination, unless otherwise stated. No animal was bled previous to the making of the blood count, the results of which are given. The red and white blood cell counts were done in the ordinary manner, Tiirck's and Hayem's solutions being used for diluent, and the blood was counted in a Karl Zeiss hasmocytometer with Neubauer ruling. The haemoglobin determinations were made with the Sahli apparatus. The hsemoglobinometer standard was so prepared that it gave a reading of 95 per cent for blood from the normal human adult. A reading of 95 per cent indicated approximately 14.25 grams of haemoglobin per 100 c.c. of blood. The blood films for differential counts and for the counting of the reticulated red blood cells were made by the following technique: A clean glass pipette of small bore or a white blood cell pipette was rinsed with a saturated solution of sodium oxalate, the fluid withdrawn and the inside of the pipette thoroughly dried. Several drops of blood from the incised thigh vein were drawn up into the pipette and blown in and out several times on a watch-glass. In this manner the blood was kept from clotting. A film was made from this oxalated blood in the usual manner. These films were stained, when dry, with Wilson's modification of Romanowsky's stain, mounted on a slide, a differential count of the white blood cells was made and the nature of the red blood cells noted. Then a drop of the oxalated blood was drawn into the pipette together with a drop of 1 per cent cresyl blue in normal salt solution. The blood and the stain were well mixed by blowing the contents on a watch-glass. A drop of this mixture was then blown on a cover-slip, films were made and stained with Wilson's modification of Romanowsky's stain, dried and mounted. By this method a preparation was obtained which showed beautifully the reticulum in the red blood cells. These preparations were permanent. Films kept for one year in this laboratory still show the reticular stain of the red blood cells. The percentage of reticulated cells was determined by counting a certain number of red blood cells, 500 or 1000, and recording the number of cells showing reticulum.

Cages. In these experiments three kinds of cages were employed. They are designated as

1. Galvanized iron cages. The iron wire was well insulated by galvanized metal.

2. Wood and iron cages. These are standard cages used in Dr. McCollum's laboratory. They consist of a wooden frame with galvanized iron-wire sides.

3. Wooden cages, made entirely of wood and glass. Food Cups. Zinc cups were employed in the first two types

of cages and glass cups in the third.

Drinking Water. The stock rats were given tap-water, the remaining rats were given doubly distilled water. This was first distilled through a copper still and then redistilled through glass. Animals receiving such water were given one drop per week per animal of the following solution:


Iodine 2 grams

KI 5 grams

H^O 500 c.c.

The norvuil blood of the rat. Klineberger and CarP= give the following average counts for the blood of the rat (blood from thigh vein) :

R. B. C. ...9,300,000 P. M. N 16.

Hb 105% (Sahli) P. M. E 3.5

W. B. C 15,200 L. lymph 24.5

S. lymph 53.5

L. M 0.5

T 2.

100.0 These authors found no difference in the blood of the

two sexes. The red blood cells of normal animals showed

some auisocytosis and polychromasia. The leucocytes

were predominantly small lymphocytes. The platelets

tended to group themselves in masses.

Donaldson " gives the following average counts for

albino rats on a "scrap" diet.

R. B. C 7,600,000 — 9,200,000

Hb 85% to 100%

W. B. C 7,200 to 16,000


P. M. N, P. M. E., P. M. B, Lymph.

the


nor


. .44 to 71% . . to 3% . . . .30 to 55

type of the


Neither the source of the blood ha?moglobinometer used is stated.

Gelling and Green/° in a preliminary report of some studies on blood regeneration in the rat on various diets, give the normal red blood count as between 7.5 and 10.5 millions and the haemoglobin percentage as 110 to 140 (Smith-Cohen). They state that the number of blood cells per c.mm. in rats of different ages and sexes falls within these limits.

In discussing the blood of normal rats several factors should be taken into consideration : first, the species of rat used ; second, the diet given as the stock diet ; third, the presence of respiratory infections which are common in laboratory rats and which may modify the total leucocyte count and differential formula; and fourth, the source of the blood. White blood cell counts made on blood from the end of the tail are higher than counts made from venous blood and show a higher polymorphonuclear percentage. I also found that blood from the heart contained a uniformly lower white blood cell count than blood from the veins. The number of red blood cells and the percentage of haemoglobin, however, were the same in blood from both these sources. It was necessary in some of the small rats to draw the blood from the heart, as a free flow could not be obtained from the cut vein. It should be remembered that the white blood cell counts are lower in these cases than if they had been made on blood from the thigh vein.

In our series of experiments the rats used were hybrids of the albino and the black Norway rat.

The diet employed as a stock diet for our animals was as follows:


May, 1922]


165


Whole ground wheat Cracked corn Ground rolled oats Flaxseed meal


3 parts 1 part


This was given ad Ubitiim and in addition the rats received a small quantity of pasteurized milk and tapwater daily.


CHART I. BLOOD COUNTS OF NORMAL RATS AT DIFFERENT AGES.



Blood from


Hb. %


R. B. C.


W. B. c.


Reticulated R. B. C. %


DIFFERENTIAL |


REMARKS



P. M. N.


p. M. E.


Lymph.


LM. T.


u.


Newlyborn* Newlyborn


Heart


75


3,200,000


4,000 4,000


93


24 43


2


30 17


8 5


8 32


Myelocytes 4. Nucl. . Anisocytosis.

R.B.C. 250 in count. 1 Polychromasia cor.

ing 100 W.B.C. f responding to per. Myelocytes 2. Nucl. / centage of Retic.

R.B.C. 280 in count- \ R.B.C. See photo ing 100 W.B.C. j graph.


15 Days


Heart




1,800


19


5



95





No Nucl. R. B. C. Marked leucopenia.


20 Days


Heart Thigh vein


80


4,800,000

'5;i2o;ooo


3,600 4,800 3,600 4,200 9,000


20


2 22




90 76


8 2




No Nucl. R. B. C. Rarity of P. M. N. forms.


26 Days


Thigh vein


85


18


12



76


4


8



1 Mo.


Thigh vein Thigh vein


80 85


7,000,000 6,592,000


6


16 24




76 74


8 2




2 Mos.


Thigh vein


90


7,680,000


8,000


9


16



84






4 Mos.


Thigh vein Thigh vein


110


11,200,000


9,600 8,000


3


24


6


68


2




9 Mos.


115


10,800,000


1


26


4


68


2




10 Mos.


Thigh vein


110


9,856,000


12,000


—1


14



85


1





Under newly born are included rats under 24 hours old. Chart I shows the results obtained in rats of different ages. The blood of a young rat differs from that of the adult, in that in the former the haemoglobin content and the red blood cell counts are relatively lower, the white blood cell count is lower (a relative leucopenia) and the percentage of lymphocytes is relatively higher. Therefore, it is important in studying the blood of a rat of known age to compare it with a normal for its age. The normal count for a young rat would indicate a slight anaemia for an adult rat. We may, therefore, speak of a physiological anaemia in the nursing rat.


The diets which were used in these experiments may be roughly divided into two groups. The first of these included simple diets which had milk in one form or another as a basis. The second group includes the more complex mixtures of various purified foodstuffs. Rats were fed on

1. Pasteurized milk.

2. Pasteurized milk and bread.

3. Evaporated milk (Pet Brand).

4. Condensed milk (Eagle Brand).

5. Dried skimmed milk (Krystallak).

6. Dried skimmed milk with various additions.

A diet of pasteurized milk alone will not cause pathological changes in the blood of rats, and rats may be reared to the third generation on diets of milk and bread without developing antemia.

Neither evaporated milk (Pet Brand) nor condensed milk (Eagle Brand) induced ansemia in rats even when the intake was limited to 8 c.c. and 4 gm., respectively, per diem in order to restrict the growth of the animals.

The attempt to cause anajinia by diets of dried skimmed milk (Krystallak) was a failure. Nor was anaemia produced by diets which contained Krystallak with yeast, butter fat or bread, or any combination of these additions to the skimmed milk diet. When, however, casein was added to a ration of Krystallak and butter fat, an anaemia was produced in two animals.


A. A female rat was started on a diet of

Krystallak 95

Butter fat 5

100 at the age of 25 days, when she weighed 30 grams. She was on the diet five months, during which time she gained in weight to 130 grams. At this time casein was substituted for 15 per cent of Krystallak:

Krystallak 80

Casein 15

Butter fat 5

100 The animal was kept in a galvanized iron cage on this diet for four weeks and lost 30 grams in weight. The rat appeared pale. Blood was taken from a thigh vein:

R. B. C 1,920,000

Hb 20% Leucopenia too marked

W. B. C 1,600 for accurate differential.

Red cells showed slight anisocytosis and poikilocytosis and there were many nucleated red cells. Polychromasia was fairly well marked. Approximately 70 per cent of the white cells were lymphocytes.

Autopsy. The tissues were very pale. The spleen was normal in size. The bones appeared normal and cut with resistance. Lungs normal.

. B. A male rat, which was in cage with the above rat, was put on the same diet at the age of 25 days, when weighing 30 grams. He was on the diet five months, during which time he gained in weight to 1^0 grams. Fifteen per cent of casein was added, and the rat was kept on this diet for 6


166


[No. 37.'.


weeks, on which he lost 20 grams in weight and developed pallor. Blood from thigh vein:

R . B. C. . . .1,472,000 P. M. N 51

Hb 15 — 20% Metamyelocytes 5

W. B. C 5.600 P. M. E 5

Eosinophilemyeloeytes. 1

Lymph 27

T 1

Unclassified 10

100* 67 nucleated red cells were seen in counting 100 W. B. C. There was slight anisocytosis and poikilocytosis. Polychromasia was well marked.

Autopsy. As for above rat.

Microscopic sections: Bone. The cartilage was not calcified. There was a narrow subchondral zone of irregular trabeculae with a great deal of osteoid tissue. The cortex of the shaft was thickened and spongy. The marrow was congested.

The following types of complex diets were also studied to determiue if possible the relations of certain deficiencies and excesses to the production of anaemia.

7. Purified diets (a) without iron, (6) with iron.

8. Diets low in fat-soluble A.

9. Diets low in calcium.

10. Diets low in calcium but with au excessive amount of fat-soluble A.

11. Diets low in water-soluble B.

12. Diets low in phosphorus and fat-soluble A.

13. Diets low in calcium and in fat-soluble A.

li. Other diets which produced changes in the bones. 7. No anwmia resulted in the first generation and only slight anwrnia in the second generation from a [jurified complete ration without added iron.

For this series of animals a purified diet was used (indicated below as purified diet without iron).

Salt mixture.

Washed casein . . 25** NaCl 0.173

Butter fat 10 MgSO, 0.266

Agar 2 K,HPO, 0.954

Wheat germ 10 CaH,(POJ2.. 0.540

Salt mixture 3.5S Ca Lactate. . . 1.300

Washed dextrin, .qs. ad 100 NaH.PO^ .... 0.347


3.580


In the formulas of the differential counts. P. M. N. = polymorphonuclear neutrophile leucocytes.

P. M. E. = Eosinophile.

Lymph. ^Lymphocytes (large and small).

T. = Transitional monocytes.

•* I take pleasure here in thanking Dr. McCollum for outlining and analyzing for me this and the other complex diets which were used.

t The diets of the mother rats were never changed during lactation, that is. if the mother of a litter was on a deficient diet, she continued to receive it while the young were being nursed. The second generation animals received the same food as their parents, unless otherwise stated. Animals of the second generation on a faulty diet may be said, therefore, to have received the deficient food since birth, although it has been shown that the breast milk of a mother on a deficient diet is a better food for her young than the diet she receives is for her.


For the control .series the identical diet (indicated below as purified diet with iron) was used except that ferrous lactate 0.118 grams was added to the salt mixture, bringing the salt mixture to 3.7 in.stead of 3.5S7c.

Both of these groups of rats were kept in wooden cages with paper for bedding and with glass doors and glass food and drinking cups. They did not come in contact with iron.

7a. Purified diet without iron.

A. A mother rat was put on the purified diet without iron at the birth of her young and remained on it while she nursed them.f Blood was examined from thigh vein of a male rat of this litter which had been on the same diet for 2% months. His weight at this time was 250 grams.

R. B. C 12,240,000 P. M. N 18

Hb 1157c Lymph 70

W. B. C 12.000 T 2

Reticulated R. B. C. .1% Unclass 10 (smudge cells)

100 At the age of 2 Vi months a splenectomy was performed under ether anesthesia. The spleen was larger than normal and weighed 0.91 grams. The animal recovered promptly and was kept 2 1/^ months longer on this diet; weight 320 grams (total of 5 months on a diet); blood from thigh vein:

R. B. C 9,920,000 P. M. N 7

Hb 110% P. M. E 6

W. B. C 40,000 Lymph 84

Reticulated R. B. C. . . 3% To 3

100 This animal had a severe respiratory infection.

Microscopic examination of the spleen showed a simple hypertrophy.

Autopsy. Subcutaneous fat abundant. The tissues were of good color. There was no infection about the wound and no hypertrophy of the lymphatic tissue. The bones were hard and normal in appearance.

B. A male rat of the same litter as the preceding rat on the same diet five months, but no splenectomy performed; weight 2 80 grams, blood from thigh vein:

R. B. C 12,300,000 P. M. N 11

Hb 1.15% P. M. E 6

W. B. C 12,800 Lymph 78

Reticulated R. B. C. 3% T 3

LTnclassified 2

100 Autopsy. The spleen was larger than normal, weighing 0.85 grams. Autopsy findings as in preceding rat. 7b. Purified diet with iron.

A. Control rat for the above. The mother of this rat was put on a purified diet with iron at birth of the young. A male rat of her litter was on this diet for 2% mohths. Weight 250 grams. Blood counts at this time from thigh vein gave the following result:

R. B. C 11.872.000 P. M. N 14

Hb 115% P. M. E 2

\V. B. C 15.000 Lymph 76

Reticulated R. B. C. 1-2% T 4

Unclassified 4


100


I


I


May, 1922]


167


A splenectomy was performed under anesthesia; the weight

of the spleen was 0.52 grams. The animal made a good

recovery and was kept on this diet for 2 % months longer,

making a total of 5 months on "a diet. Weight 310 grams. Blood from thigh vein:

R. B. C 9,920,000 P. M. N 29

Hb 115% P. M. E 9

W. B. C 12,400 Lymph 60

rr 2

100 Microscopic examination of the spleen showed it to be normal.

Autopsy. Entirely negative, wound perfectly healed. B. A male rat of the same litter as A. on the same diet for five months. No splenectomy performed; weight 290 grams; blood from thigh vein was counted as follows:

R. B. C 12,640,00 P. M. N 10

Hb 115% P. M. E 1

W. B. C 14,400 Lymph 84

T 5

100

Autopsy. The spleen weighed 0.62 grams. Examination entirely negative.

The blood of two other i-ats of this series was examiued with i)ractically the same results. The blood of rats of the first generation on a purified diet without iron was found to be practically identical with that of rats on the same diet with iron added. The spleen of rats on the former diet, however, was uniformly larger than the spleen of those on the latter diet. Splenectomy had no effect on the blood picture.

^J'Kjht aiKTiiiia resulted in some of the animals in the second generation receiving the purified diet tvithout iron.

A. A female rat, whose mother had been on the purified diet without iron since her birth, at the age of 19 days weighed 21 grams. The blood of the mother was normal after the weaning of the litter. She had been on the deficient diet during lactation. Blood from thigh vein:


P. M. N 32

Lymph 64

T 4


R. B. C 4,112,000

Hb 45%

W. B. C 3,400

Reticulated R. B. C. 15-20%

100 No nucleated red cells were seen. There was slight anisocytosis. no poikilocytosis. but fairly marked polychromasia.

Autopsy. Spleen normal in size, weight 0.77 grams, tissues somewhat pale, bones cut with resistance.

B. A young rat, whose mother had been on a purified diet without iron since her birth, at the age of 4 2 days weighed 29 grams. Blood from thigh vein:


R. B. Hb. . W. B. Retic.


C.


. .6,400,000

60%

4,600

C. . . .15%


P. M. N 40

Lymph 60

T

100


The red cells showed moderate polychromasia, otherwise they were normal.

Autopsy. The spleen was small, weighing 0.1 gram. Liver and tissues fairly good color. Lungs normal. The bones cut with resistance.


C. One of the young of a mother rat which had been on a purified diet without iron since birth, at the age of 24 days, weighed 2 5 grams. Blood from thigh vein:

R. B. C 7,360,000 P. M. N 28

Hb 67% Lymph 72

W. B. C 7,600 T

Retic. R. B. C. ...10%

100 Slight aniso- and poikilocytosis and polychromasia. No nucleated R. B. C. seen. No autopsy.

D. A young rat of the same litter as the preceding but aged 2 months. Weight 110 grams. Blood from thigh vein:

R. B. C not done P. M. N 14

Hb 100% Lymph 86

W. B. C 5,600 T

Retic. R. B. C 8%

100 Slight polychromasia, red cells otherwise normal in appearance.

Autopsy. Tissues and viscera of good color. The spleen was slightly enlarged, the thymus persistent and bones cut with resistance.

E. Mother rat was put on purified diet with iron at birth. Blood of mother normal after weaning litter. One female of this litter at the age of 19 days weighed 32 grams. Blood from thigh vein:

R. B. C 6,144,000 P. M. N 10

Hb 80% Lymph 88

W. B. C 3,400 T 2

Retic. R. B. C 10%

100

There was a slight anisocytosis and polychromasia. No

nucleated red cells were seen.

Autopsy. The spleen was normal in size, weighing 0.1

gram; the thymus somewhat enlarged. The bones were hard

and cut with resistance.

F. Rat, aged 1 month, weight 40 grams. Third generation on purified diet with iron. Blood from thigh vein:

R. B. C 10,240,000

Hb 110%

W. B. C 2,600

The red cells appeared normal and the differential blood picture was essentially normal.

No autopsy.

7a. Diet 2.

Casein 12.5

Butterfat 10.0

Wheat germ 5.0

Agar 2.0

Salt mixture without iron. . 3.7 Dextrin 67.0

100.0 The diet was made of purified food stuffs. It was iron free and had about half the protein content of the last mentioned diet. Two generations of rats were reared on this diet. Four rats of the second generation, weighing 33 to 40 grams, were kept on this diet from the age of 3 days for three months. At the end of this period they weighed 130 to 150 grams, on purified diet with iron. Blood from thigh vein:

R. B. C 10,880,000

Hb 90%

W. B. C 7,000

The smears showed red blood cells which were apparently normal. The differential count was normal.


168


[No. 375


\j. Tissues and viscera of good color. The spleen was enlarged; the bones appeared to be normal.

Microscopic sections of the bones of rats raised on the above purified diets showed practically normal bones. The cartilage was well calcified but the trabeculte were somewhat fewer in number than normal, i. r., the bones were slightly osteoporotic.

8. Tlic hlood of rats on a diet so deficient in fat soluble A as to produce xerophthalmia showed no anwmia.

The diet which was used had the following composition.

Rolled oats 40.0

Flaxseed oil meal 8.3

NaCl 1.0

CaCOa 1.5

Dextrin 4 9.2


100.0 9. The following diets were adequate in all respects except for a deficiency in calcium.

Wheat 20.0

Maize 15.0

Rice 9.5

Rolled oats 9.5

Peas 10.0

Navy beans 10.0

Casein 10.0

While milk powder 5.0

NaCl 1.0

Butter fat 10.0

100.0

Wheat 20.0

Maize 19.5

Rice 9.5

Rolled oats 9.5

Peas 9.5

Navy beans 9.5

Casein 10.0

NaCl 1.0

NaHCOj 1.5

Butter fat 10.0


100.0


The blood of rats fed on these diets was quite normal. 10. This was also true of the hlood of rats which were

kept on the following diet which contained an excessive

amount of calcium and fat.

Wheat 20

Maize 10

Rice 9

Rolled oats 9

Peas 9

Navy beans 9

Casein 10

NaCl 1

CaCOs 3

Butter fat 20


100


11. Rats on a diet so deficient in tcater soluble B as to produce polyneuritis iccrc not anemic but showed a Icucopcnia with evidences of diminished leucopoietic activity.

The diet had the following composition:

Casein 18

Salt mixture No. 185 3.7 Agar 2.0

Butter fat 5.0

Dextrin 71.3


100.0

A. A male rat was put on this diet, at the age of 55 days, when weighing 70 grams. At the end of 137 days he weighed 6 5 grams. In a state of polyneuritis. Blood from thigh vein:

R. B. C. ... 10,320,000 P. M. N 19

Hb 100% Lymph 35

W. B. C 2,600 T 1

55 The red cells appeared normal, the polymorphonuclear cells were chiefly cells with 5 or 6 lobed nuclei. The nuclei often filled the entire cells.

B. A male rat, weighing 56 grams, was put on diet at the age of 50 days. At end of 114 days weighed 50 grams. Animal in a state of polyneuritis. Blood from thigh vein:

R. B. C 10,000,000

Hb 110%

W. B. C 1,800

The smears showed very marked leucopenia; the red cells appeared normal. The white cells were about equally divided between polymorphonuclears and lymphocytes. The polymorphonuclear cells had multilobulated nuclei.

Autoi)si€s. These rats with polyneuritis were poorly nourished rats. The spleens were of normal size. Thymus glands atrophic. Tissues of good color and the bones hard on section. Microscopic section of the bones of these animals showed that the cartilage was thin and well calcified. Few trabeculs were visible. The marrow was hfemorrhagic.**

Findlay'" described the same changes in the blood in beriberi in man as we have found in rats, namely, leucopenia and a shift to the right in the Arneth formula without important changes in the red cells and h;ruioglobin.

12. Diets which are relatively high in calcium but which are low in phosphorus and in an uncharactcrized substance tchich is present in certain fats do not produce anwmia in rats.

McCollum, Simmonds, Shipley and Park have described a very severe form of rickets which is produced by diets which have the above described characteristics. The animals whose blood was studied were fed on their diet No. 3113 which was made up as follows : f

Wheat 33.0

Maize 33.0


Salt mixture No. 185 is the same as the salt mixture above in the purified diet without iron.

The pathological condition of the bone and bone marrow of these rats with polyneuritis is described by Shipley, McCollum and Simmonds, Jour. Biol. Chem., Dec, 1921.

t The blood counts on these animals were made by Dr. A. A. Weech.


May, 19221


169


Gelatin 15.0

Wheat gluten 15.0

NaCl 1.0

CaCOa 3.0

The blood of four animals on this diet was studied. The diet did not produce anaemia iu any case and the leucocytes were quite normal.

13. Diets lore in calcium, normal or relatively high in phosphorus and low in a substance contained in certain fats caused bone changes in rats tohich were closely related to rickets. These diets also produced anwmia in both first and second generations.

A brief review of the work of Shipley, Park, McCollum and Simmonds may serve to make the following discussion clearer. These authors have found that on diets in which an organic substance, present in codliver oil and to a less extent in butter fat, which may or may not be identical with fat soluble A, is small in amount and in which there is a disproportion between the calcium and phosphorus, a condition of the bones results which is practically identical with liuman rickets. Park and Shipley have frequently noted in these animals varying degrees of enlargement of the spleen and marked pallor of the viscera particularly of the liver. The rats whose blood counts are recorded below were from their series and had been in cages made of wood and galvanized iron. The blood of rats on six diets differing in composition but with the same faults were examined.

Six rats, aged 4 5 days, whose average weight was 4 5 grams, were put on the following diet which contains a poor fat and an insufficient quantity of calcium but an abundance of phosphorus:

Wheat 30.0

Maize 19.5

Peas 8.5

Rolled oats 8.5

Rice 9.5

Navy beans 8.5

Casein 10.0

NaCl 1.0

NaHCOa 1.5

Cottonseed oil 3.0


100.0

A. One female was examined at the beginning of the experiment. Blood from her thigh vein was examined with the following result:

R. B. C 7,928,000 P. M. N 9

Hb 92% Lymph 82

W. B. C 4,700 T 9

100 Slight polychromasia, no anisocytosis or poikilocytosis.

B. A male was examined after receiving the diet for 3 months. Blood from his thigh vein showed:

R. B. C. ... 10,208,000 P. M. N 22

Hb 100% P. M. E 2

W. B. C 14,400 Lymph 74

Retic. R. B. C 8% T 2


Slight polychromasia and anisocytosis were present.


Autopsy. The spleen was of normal size. The tissues were of good color. The thorax was much deformed.

Microscopic sections. There was a great overproduction of osteoid tissue in the epiphysis and the shaft was irregularly calcified and was invaded by blood vessels. There were large numbers of trabeculiB in the shaft each surrounded by a broad zone of osteoid. The cortex of the shaft was thickened. The spaces between the trabeculfe near the marrow cavity contained large numbers of basophilic cells. The spleen showed marked congestion. The Malpighian bodies were larger than normal.

C. A female was examined after being on the diet for 4% months. She weighed 84 grams. Blood from thigh vein showed

R. B. C 9,600,000 P. M. N 10

Hb 110% Lymph 88

W. B. C 9,000 T 2

Reticulated R. B. C.

less than 10%. The red cells showed slight polychromasia, were normal.

Autopsy. The spleen was slightly enlarged. The thorax was deformed and the costo-chondral junctions were enlarged. The bones cut easily and showed broad metaphyses.

Microscopic section. The changes in the bones were similar to those just described. The Malpighian bodies of the spleen were enlarged and contained nests of erythropoietic cells.

D. A female was examined after being on the diet for 6 months. The animal was pale and weighed 80 grams. Blood from thigh vein:




Eighteen nucleated red cells were seen in counting 100 white cells. There was a marked polychromasia. The polychromatic red cells corresponded with the number of reticulated red cells {see Figs. II, III, IV). Anisocytosis and poikilocytosis were fairly well marked.

Autopsy. The spleen was large, weighing 0.8 grams. The liver and other tissues were very pale. There was marked rickets. The bones cut easily and showed broad metaphyses.

Microscopic section. The bone showed more extensive rickets than in the preceding animal. There was no calcification of cartilage, which was very irregular (see Figs. V and VI).

The Malpighian bodies of the spleen were fewer in number than in the spleen of the preceding rat. There was infiltration with round cells and large mononuclear cells. There was myeloid metaplasia of the spleen with evidences of the assumption of hematopoietic function.

Summary. A rat fed upon a defective diet for 6 months had severe rickets, splenomegaly, and actively regenerating secondary anemia.

E. A rat of another litter fed upon the above defective diet from birth at age of 2 months weighed 30 grams. Blood from the thigh vein:

R. B. C 7,360,000

Hb 110%

W. B. C 2,200

Reticulated R. B. C 10%

The red cells showed slight polychromasia. There was leucopenia with a normal differential formula.

Autopsy. The spleen was normal. There were signs of early rickets.


170


[Xo. .*}75


F. A female rat, at the age of 40 days, when weighing 55 grams, was put on the following diet which is deficient in calcium and fat:

Wheat 25.0

Maize 19-5

Rice 9-5

Rolled oats 9.5

Peas 9.5

Navy beans 9.5

Casein 10.0

NaCl 1.0

Dextrin 1.5

Whole milk powder 5.0

100.0 At the end of 7% months her weight was S3 grams. Blood from the thigh vein contained:

R. B. C 5,232,000

Hb 60%

W. B. C 10.000

Reticulated R. B. C 2 4%

The red cells showed moderate polychromasia, marked anisocytosis and some poikilocytosis. A few nucleated red cells were seen. About 7 5 per cent of the white cells were lymphocytes. There were no myelocytes in the blood film.

Autopsy. The thorax was much deformed. The spleen was slightly enlarged, weighing 0.55 gram.

Microscopic, section. The bones showed rickets in process of healing. The spleen showed evidences of hematopoiesis.

Summari/. A moderate anemia for a rat of this age with active regeneration of the red cells. Slight enlargement of the spleen. Rickets.

A. A female rat, aged 4 5 days and weighing 65 grams, was fed upon the following diet, which is also deficient in calcium and fat:

Soy bean 30

Wheat gluten 5

Casein 10

Wheat germ 5

NaCl 1

Maize 20

Dextrin 29

100 Her weight was 7 5 grams at the end of 7 8 days. Blood from thigh vein:

R. B. C 3,120,000 P. M. N.. . 22 (incl. 2 meta Hb 35% myelocytes)

W. B. C 13,000 P. M. E.. .

Retic. R. B. C. .75-80% Lymph. . . 78

100 Seventy-five nucleated red cells were seen in counting 100 W. B. C. The red cells showed anisocytosis and poikilocytosis and marked polychromasia (see Pigs. VII and VIII).

Autopsy. The spleen was very much enlarged. There were marked changes in the skeleton. The cartilage was redundant. Calcification was very defective and irregular. The metaphysis consisted of osteoid tissue in the form of trabeculae, somewhat irregularly arranged. At the periphery of the bone, processes of unchanged cartilage ran down from the cartilage into the metaphysis. There were small islets of cartilage cells in the metaphysis. The cortex of the shaft was spongy. There was a great deal of osteoid tissue around all of the trabeculce in the spongiosa. There were no signs of abnormal resorption in the metaphysis but resorption was going on actively in


the cortex and in the spongiosa near the marrow cavity (see Pigs. IX and X).

Summary. Rachitic rat; enlarged spleen; severe secondary anjemia; evidences of active regeneration of red cells.

13. Ancrmia teas caused in the necond generation of rats fed upon the followinfj dict.s that produced changes in the bonrs.

A. A male rat. aged 4 1 days, weighed 22 grams. The mother was on the following diet for 217 days before birth of her litter:

Wheat 19.3

Maize 20.0

Rice 10.0 This diet is deficient in

Rolled oats 10.0 calcium, while contain Peas 10.0 ing a fat which is not

Navy beans 10.0 strongly protective

Casein 10.0 against rickets. There

NaCl 1.0 is a relatively large

NaHCOj 1.5 amount of phosphorus.

CaCO, 0.2

Butter fat 8.0

100.0

Blood from heart:

R. B. C 448,000

Hb less than 10%

W. B. C 1,000

Reticulated R. B. C 70%

The red cells showed marked anisocytosis and polychromasia

and some poikilocytosis. A few nucleated red cells were seen.

There was leucopenia with many early forms of the bone

marrow series.

Autopsy. The tissues were pale. The liver was nearly white.

The spleen was pale, but not enlarged. There were no gross

signs of rickets.

B. A female rat, aged 24 days, weighed 18 grams. The mother had been on the following diet 60 days before the birth of her litter:

Rice 10.0

Wheat 32.5

Maize 15.0

Peas 10.0 This diet is deficient

Navy beans 10.0 in calcium and fat.

Rolled oats 10.0

Casein 10.0

NaCl 1.0

NaHCOj 1.5

100.0 Blood was taken from the heart. The blood was thin and watery, and enough to count could not be obtained. Smears were made which showed fairly well marked anisocytosis and poikilocytosis and polychromasia. Many nucleated red cells were seen. There was a leucopenia.

Autopsy. The tissues and viscera were very pale. The spleen was not enlarged.

C. A female rat. aged 16 days, weighed 10 grams. The mother had been on the above diet for 84 days before the birth of her litter. Blood from heart:

R. B. C 5,044.000

Hb 75%

W. B. C 1,200

Reticulated R. B. C 10%

The red cells showed slight polychromasia. There was a marked leucopenia, but the white cells seen appeared to be normal. No nucleated red cells were seen.


THE JOHNS HOPKiNS HOSPITAL BULLETIN. MAY, 1922


PLATE XX,


%^


"^^v


t>.


Fig. I.


Fig. IL


Fig. III.



Fig. V.



PLATE XXI



Autopsy. The spleen was small. There was no thoracic deformity. The costo-chondral junctions were slightly enlarged. The long hones were thin and cut easily.

Microscopic sections showed well calcified osteoporotic bones. D. A female rat, aged 19 days, weighed 14 grams. The mother of this animal had been on the following diet 70 days. She continued on the diet while she nursed her litter:

Wheat 30

Maize 30

Rice 10 This diet is deficient

Rolled oats 10 in calcium and fat.

Peas 10

Navy beans 10

100 Blood from thigh vein:

R. B. C 6.20S.00O P M. N 15

Hb 10% Lymph 80

\V. B. C. . . . not counted T 5

100 There was anisocytosis, poikilocytosis and a moderate degree of polychromasia. An occasional nucleated red cell was seen. The animal had a marked leucopenia.

The bones of the mother showed typical rickets. The bones of the second generation rat showed no osteoporosis and no rickets.

In five other rats of the second generation a practically normal blood picture was observed. Discussion

A diet deficient in iron alone does not produce ana>mia in the rat in the first generation. It would appear eitlier that the rat is born with a store of iron sufficient to carry him through life, or that only a very small quantity of iron is necessary to meet his requirements. On the other hand, diets deficient in an organic substance contained in cod-liver oil and to a less extent in butter fat and which also have certain improper calcium-phosphorus ratios (diets which produce rickets in the rat) produce ana-mia in some animals. Tlie ana?mia in these animals is not due simply to a deficiency of iron in tlie diet. While these diets contained only a small amount of iron, they contained as much of this element as the stock diet or the other diets used upon which no anaemia developed. It would seem that tlie disturbance in the inorganic metabolism resulting from certain diets with Avhich there is a lack of calcification of the bones also resulted in, or was accompanied by, a disturbance of the iron metabolism. It will be noted that the phosphorus content was relatively normal or increased and calcium relatively low in all the diets upon which anannia developed.

When, however, the diet contained relatively excessive amounts of calcium but was low in phosphorus, no ana'inia was produced in the rats Avhich I examined. Diets of this type cause an exaggerated form of rickets to develop in rats and the calcium and phosphate content of their blood is identical with Hint seen in many severely rachitic children.

Milk diets except those in which ca.sein was present in high percentage did not produce anitmia. Two rats which received the latter diet were the onlv animals in


the series on diets of milk or modified milk which showed prof(mnd disturbance of calcification of the bones. I have no explanation to offer for this at present.

Enlargement of the spleen appeared to be as variable a feature in rats with rickets as it is in children with rickets. Enlargement of the spleen was frequently obsei'ved in rats in association with an ansemia of the actively regenerating type just as splenomegaly is commonly observed in infants with rickets and ana'iuia. However, although rickets makes its appearance relatively early in the rat, especially when the animal is put on the faulty diet at an early age, anaemia does not make its appearance until the rat has been on the diet for a long period. For this reason it is difficult to study ana'inia produced by faulty food in rats, since they usually die from malnutrition or embarrassed pulmonary or cardiac function resulting from the marked thoracic deformity, or from pulmonary infection, before there is time for the an:pinia to become manifest. The rats in which anaemia occurred had been on the faulty diet for a long period, or the aua>mia was observed only in the second generation. Rachitic rats are usually sterile, so that second generations are not often raised.

The examination of films of the blood of the same rat, made simultaneously, the one stained with cresyl blue and Wilson's stain following the technique outlined above, the other with Wilson's stain alone, showed that if the polychromatophilic ( diffusely basophilic) red blood cells and the reticulated erythrocytes were counted, the counts were practically the same. This would seem to indicate, at least as far as the rat is concerned, that polychromasia of the red blood cells is an indication of a young cell. Because of the stimulation of the bone marrow which occurs in the course of anaemia of the type described in these rats, these immature cells are poured into the circulation in large numbers. They compose a large percentage of the red blood cells present. In the blood of the normal adult rat they are seldom encountered.

Although the au;rmic animals were not treated, it would seem logical to assume that the.se anaemias require antirachitic therapy primarily. Iron would not seem to he primarily indicated because the amount of iron in any well balanced diet is sufficient to meet an animal's needs.

Conclusions

1. The normal blood picture of the rat varies with age. Young rats have a relatively lower red blood cell count, white blood cell count, and luemoglobin percentage, and a higher lymphocytic percentage, than the adult rat. Diet and the presence of respiratory infections are factors which may influence the blood picture,

2, Well balanced diets, deficient in iron, do not produce aiKcmia in the rat in the first generation, nor do diets consisting solely of cow's milk or milk and bread. Slight ampmia may occur in rats of the .second generation on these diets.


172


[Xo. 375


3. Diets deficient in fat soluble A or water soluble B, although they cause severe nutritional disturbauces, do not produce ana'iuia in the rat. Diets so deficient in water soluble B as to produce polyneuritis diminish leucopoietic activity and cause a severe leucopenia with a shift to the right in the Arneth formula.

4. Diets low in an organic substance contained especially in cod-liver oil with a low calcium but high phosphorus content, which produce rickets-like changes in the rat, may also produce aui^mia, provided the animal is kept for a long period on the diet. Animals of the second generation on this diet may also become anaemic. This aniemia is associated with evidences of increased htematopoietic activity. There is often an enlargement of the spleen. This condition resembles the amemias seen in association with rickets in human beings.

5. A diet low in the organic substance contained in cod-liver oil and low in phosphorus with a normal calcium content, a diet that produces severe rickets with great uniformity, does not produce antemia.

LEGENDS FOR FIGURES.

Fig. I. — Blood of normal newly born rat, stained with cresyl blue and Wilson's modification of Romanowsky's stain, showing the high percentage of reticulated red blood cells present.

Pigs. II and III. — Blood of rat D, (p. 171), stained with Wilson's modification of Romanowsky's stain, showing one nucleated red cell and polychromasia of many red cells.

Fig. IV. — Blood of same rat stained with cresyl blue and Wilson's modification of Romanowsky's stain, showing reticulation of the red cells.

Figs. V and VI. — Sections of femur of same rat at different magnifications, showing rickets.

Fig. VII. — Blood of rat A (p. 170), stained with Wilson's modification of Romanowsky's stain, showing polychromasia.

Fig. VIII. — Blood of same rat stained with cresyl blue and Wilson's modification of Romanowsky's stain, showing reticulation of the red cells.

Figs. IX and X. — Sections of femur of same rat at different magnifications, showing rickets.


REFERENCES.

1. Meyer, E.: Ergebn. d. Physiol., Wiesb., 1906, V, 698.

2. Hall, W. S.: Arch, f, Anat. u. Physiol., Physiol. Abteil.. Leipz., 1896, 49.

3. Schmidt. M. B.: Verhandl. d. deutsch. path. Gesellsch. Berl., Jena, 1912, XV, 91.

4. Kunkel: Arch. f. d. ges. Physiol., Bonn, 1895, LXI, 595.

5. Cloetta, M. : Arch. f. exper. Path. u. Pharmakol., I^eipz., 1897, XXXVIII, 161; 1900, XLIV, 363.

6. Abderhalden, E.: Ztschr. f. Biol., Miinchen u. Leipz., 1900, XXXIX, 113, 195 and 487. Ztschr. f. Physiol. Chem., Strassb. u. Berl., 1901, XXXIV, 500. Lehrbuch der physiol. Chem., Berl. u. Wien, Urban u. Schwarzenberg, 1914, II Teil, 756, //.

7. Muller, F.: Arch. f. path. Anat. (etc.), Berl., 1901, CLXIV, 436.

8. Tartakowsky, S.: Arch. f. d. ges. Physiol., Bonn, 1904, C, 586; CI, 423.

9. Bunge, G.: Ztschr. f. physiol. Chem., Strassb. u. Berl., 1884, IX, 49. Ibid., 1889, XIII. 399. Ibid., 1892, XVI, 173. Ibid., 1893, XVII, 63.

10. Evans, P. A. and Happ, W. M.: Johns Hopkins Hosp. Bull., Bait., 1922 (to be published).

11. Shipley, P. G., Park, E. A., McCoUum, E. V., Simmonds, N. and Parsons, H. T. : Jour. Biol. Chem., 1921, XLV, 333. Ibid., 1921, XLV, 343.

Shipley, P. G., Park. E. A., McCollum, E. V. and Simmonds, N.: Johns Hopkins Hosp. Bull., 1921, XXXII, 160. Proc. Soc. for Exper. Biol, and Med., 1921, XVIII, 275. Ibid., 1921, XVIII, 277. Am. Jour. Hyg.. 1921, I, 492. Ibid., 1921, I, 512. Jour. Biol. Chem., 1921, XLVII, 507. Dental Cosmos (in press).

12. Klineberger, C. and Carl, W.: Die Blut-Morphologie der Laboriums-Tiere. Leipz., Johann A. Barth, 1912.

13. Powdermaker, F. : Personal communication.

14. Donaldson, H. H.: The Rat, Memoir No. 6. The Wistar Instit. of Anat. and Biol., Phila., 1915.

15. Gelling, E. M. K. and Green, H. H.: Proc. Soc. Exper. Biol, and Med., N. Y., 1921, March 16, 191.

16. Findlay. G. M.: J. Path, and Bacteriol., Edinburgh., 1920, XXIII. 490.

17. Shipley, P. G., Park. E. A.. McCollum. E. V. and Simmonds, N.: Lesions in the Bones of Rats Suffering from Uncomplicated Beriberi, Jour. Biol. Chem. (in press).



V. AN OPERATIVE PROCEDURE FOR HYDROCEPHALUS

By Walter E. Dandy


Strictures of the aqueduct of Sylvius recur after any attempt to restore the lumen. For this reason, if treat


Read by title before the Johns Hopkins Hospital Medical Society on April 3, 1922.

♦* These patients are nearly always blind in one or both eyes at the time of operation.


ment is to be successful, the fluid must be sidetracked into its normal channels. With this in mind, a procedure which apparently is anatomically correct has been devised, to supersede any direct attack on the aqueduct. This consists in removing the floor of the third ventricle. A small opening is made in the skull and dura in the frontal region, the frontal lobe is elevated until the


190


[Xo. r.T.j


bulging; third ventricle is well exposed. Usually, it is necessary to divide one of the optic nerves, for in hydrocephalus these nerves are very short; usually the chiasm lies directly on the anterior border of the sella turcica. This opening in the floor of the third ventricle affords an exit from the dilated ventricles, so that the fluid can now pass directly into the cisterna chiasmatis and interpeduncularis — the normal distributing centers for cerebrospinal fluid. AVe now have proof that an opening made in the fourth ventricle will remain permanently patent, and this encourages the hope and belief that this opening in the floor of the third ventricle will also be permanent. The ventricular wall is a very thin membrane and oft'ers a minimum of glia tissue to repair the defect.


This procedure is by no means analogous to making an opening in the roof of the third ventricle. The latter can have no beneficial result because the fluid escapes into the subdural space where the absorption is slightly, if at all, greater than in the ventricles. Moreover, the opening is through cerebral tissue which proliferates and closes it, unless a good deal of the brain has been destroyed.

AVe have employed this method 6 times. No claim is made for its success. Time alone will decide. If successful, it should also be applicable to occlusions of the foramina of Luschka and Magendie and to those cases of communicating hydrocephalus in which the cisterna chiasmatis and interpeduncularis are patent.


VL DIAGNOSIS AND LOCALIZATION OF SPINAL CORD TUMORS*


Bv W.\LTER E. Dandy


In 1"J1!), a report of intraspiiious injections of air was presented. In several cases, the air was found in skiagrams of the head and proved to be useful in defining blocks in the subarachnoid space, resulting from tumors or inflammations. The air was shown in the upper part of the spinal canal and it was suggested that blocks of the spinal canal should lie similarly demonstrable or could be excluded when not present. In five cases of possible spinal cord tumors we have made intraspinous injections of air. In four, it passed through into the cranial chamber and was demonstrable in the subarachnoid space and the ventricles, so that tumors could be excluded, and other diagnoses could be considered.

In the ca.se of one of Dr. H. Thomas's patients the upper margin of the air was shown at the fifth cervical vertebra; sharp pains were at once carried down the sixth cervical nerves on each side (the site of the tumor). Xo headache


Read by title before the Johns Hopkins Hospital Medical Society on April 3, 1922.


occurred in this case, because the air did not reach the cranial chamber. The skiagram of the head also failed to demonstrate air either in the cerebral sulci or in the ventricles. As the AVassermann for the blood and spinal fluid was positive, anti.syphilitic treatment was instituted with the result that the patient recovered completely from the paralysis. Before his discharge from the hospital, the intraspinous air injection was repeated ; headache at once appeared ; and air was shown in the skiagram of the cerebral ventricles. No pains developed along the cranial nerves at this time. It was evident that the tumor had been absorbed, and that air could pass freely through the region which previously had been blocked. This method should give the same results as the combined spinal and cisternal punctures of Ayer. It is hoped that the air shadow will be sufficiently clear not only to permit the diagnosis of a subarachnoid block but also to establish its localization. How sharply shadows in the thorax and abdomen will be defined, I do not know.


SPONTANEOUS LABOR OCCURRING THROUGH AN OBLIQUELY CONTRACTED, KYPHOTIC, FUNNEL PELVIS^


By -I. AA'iiiTRiDGE Williams.


This case seems worthy of being recorded for two reasons — first, because it aft'ords a striking illustration of the mechanism by which certain skeletal changes may aft'ect the shape of the pelvis, and secondly, because the conservatism with which the labor was conducted aft'orded conclusive evidence of the ability of the uterine cicatrix following Caesai'ean section to withstand the strain


Case report made before the Johns Hopkins Medical Society, Dec. 5, 1921.


of labor, as well as to demonstrate the fallacy of the dictum "once a Caesarean, always a Caesarean."

The patient was a twenty-one year old colored girl, who was markedly deformed by kyphotic changes in the vertebral column and by ankylosis of the left hip and knee originating from tuberculosis developing during the second year of life. The two previous pregnancies had been ended by Caesarean section in another city in liU!) and 1!)20 respectively, and she was admitted to the ward on September I'C, W21. under the belief that she was in labor at term — the last menstrual period having begun on December 20, 1920.

In view of the previous histoi-y, the woman entered the service for a third Caesarean section and, as she was anxious to avoid the possibility of its repetition, it was tentatively proposed to remove the uterus by supra-vaginal amputation after evacuating its contents. On examination, there were occasional uterine contractions, and a small child presented in R. O. P. with its head deeply engaged in the pelvic cavity. In the absence of disproportion, I declined to do a radical operation and predicted a spontaneous outcome, but advised that forceps be applied early in the second stage in order to prevent unnecessary strain upon the scars of the previous sections.

As it proved that the patient was not in labor, I examined her thoroughly the next day with the following results: a frail young woman weighing S3i^ pounds and measuring 130 cm. was lying on her back in bed. The heart and lungs were nonnal. The thorax was unusually arched and, with the large sagging breasts and pendulous abdomen, presented a peculiar picture. The fundus of the uterus reached to within three fingers of the xiphoid, and the abdominal enlargement projected in such a way that its lower part extended vertically downward. A jagged Caesarean section scar extended one-third above and two-thirds below the umbilicus. A small child lay in R. O. 1'. with the hetul deeply engaged. In the left groin just below Poupart's ligament was an old scar indicating the point of discharge of a psoas abscess. The left leg was atrophic as compared with the right, and was fixed at the hip-joint in slight flexion. The knee was flexed and comijletely ankylosed, while a long indrawn scar upon its posterior surface indicated the site of an operation which had been performed when the patient was five years of age.

Upon inspecting the standing patient from the front, (Fig.l) one was impressed by the extreme shortness of the torso, the pendulous condition of the abdomen, and the fact that the body weight was supported entirely by the right leg, while the toes of the left foot scarcely touched the floor. The abdomen was so pendulous that no trace of the abdominal cicatrix was visible, and the umbilicus lay T cm. below a line joining the iliac crests. When viewed from the rear, four striking features were noted. First, the presence of a double kyphosis, with one gibbus in the upper dorsal and the other in the lower lumbar region; second, tilting of the entire vertebral column so that the right shoulder was higher than the left ; third, collapse of the torso to such an extent that the lower-most ribs were in contact with the iliac crests, which was indicated externally by a deep transverse furrow of the skin on either side; and fourth, oblique tilting of the pelvis so that the right iliac crest was 2 cm. higher than the left.


As is shown by Figure 2, there is nothing I'emarkable about the upper gibbus, but the lower one, which is separated from the former by a moderate lordosis, is of interest, partly on account of its location, but particularly because its lower limb passes directly into the sacrum. The existence since childhood of such a lumbar kyphosis led us to suppose that the pelvis would be funnel-shaped, while the unilateral lameness would give rise to an oblique contraction. Careful examination confirmed both suppositions.

External pelvimetry gave the following nijeasurements: 21, 23, 27 and 17.5 cm., and showed that the pubic arch was asymmetrical and somewhat narrowed, with the transverse and anteroposterior diameters of the outlet measuring 7.25 and 10.75 cm., respectively. On internal pelvimetry, the diagonal conjugate was found to measure 12 cm. and the entire right side of the pelvis was flattened and pushed upward, inward and backward, while the left side presented normal curvatures. At first glance it appeared that we had to deal with a typical generally contracted funnel pelvis, associated with oblique contraction resulting from the unilateral lameness. Closer consideration showed that this was not the case, as the diagonal conjugate of 12 cm., although slightly shorter than normal, was disproportionately large for such a pelvis. On the contraiy, we were forced to conclude that it was really relatively lengthened, while both the transverse and anteroposterior diameters of the inferior strait were absolutely shortened. In other words, all of the criteria for a kyphotic funnel pelvis were fulfilled.

Figure 3, which represents an X-ray of the superior strait, clearly shows the existence of the oblique contraction and demonstrates that it had resulted from the unilateral lameness. In this instance, the patient since early childhood had supported her body weight almost exclusively upon her right leg, with the result that with each step a greater upward and inward force had been exerted through the right than through the left acetabulum, and consequently the right side of the pelvis had become flattened and slowly forced upward, inw-ard and backward, while the left side had developed normally, thus giving rise to an oblique contraction involving the entire pelvis, but more particularly the superior strait, which has assumed an obliquely ovate form. That the contraction was not more pronounced is probably attributable to the fact that the patient has always walked with a crutch, and thereby diminished to some extent the extreme mechanical possibilities of her lameness.

Reverting to the kyphosis. The presence of a "hump back," no matter what the situation of the gibbus, is always a source of anxiety to a pregnant woman and causes her to anticipate serious difficulty at the time of labor. Experience, however, shows that such fears are generally exaggerated, as the great majority of kyphotic deformities do not lead to serious dystocia. This is due


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to the fact that wheu the gibbus is .situated iu the cervical or dor.sal region, or even wheu it is dorso-lumbal iu character, the development of a marked lordosis below it results in such compensation, that the body weight is transmitted to the ba.se of the sacrum iu such a manner that the pelvis is not affected. On the other hand, as has been shown by the researches of Breisky, Chantreuil, Tarnier and others, wheu the kyphosis involves the lumbar region, there is no longer any possibility for compensation, as the lower limb of the gibbus articulates directly with the base of the sacrum, with the result that the body weight is transmitted to the upper end of the lower limb of the gibbus iu such a way that a parallelogram of forces is developed whicli resolves itself into two factors — one directed downward and the other downward and backward. The latter tends to displace the lower limb downward, and at the same time to draw it backward. As its lowermost extremity is firmly united to the base of the sacrum, this results in a rotation of the sacrum about its transverse axis so that its base becomes retropulsed, wliile its tip is displaced forwards, thereby increasing the length of the conjugata vera and diminishing that of the anteroposterior diameter of the inferior strait. At the same time the sacro-iliac joints are spread apart, with the result that the innominate bones rotate about their transverse axes, so that the ischial spines and the tubera ischii approach one another and thus diminish the transverse diameters of the plane of least pelvic dimensions and of the inferior strait with the production of a funnel pelvis, as is demonstrated by the example before us.

In most kyphotic funnel pelves which have been studied in the dry state a further change has been noted — namely, a lengthening of the sacrum from tip to base, as well as a slight diminution in its concavity arising from the fact that the traction exerted upon its base tends to draw the bodies of the sacral vertebrae out beyond the level of the alae. Whether this was present in the pelvis under discussion I am unable to state, but no evidence of it was apparent upon palpating the sacrum or in the X-ray plate.

The patient fell into labor early iu the morning of October 13, 1921, and three hours and twenty minutes later vaginal examination showed that the cervix was fully dilated and the head on the pelvic floor in R. O. A. The membranes ruptured spontaneously at this time, and low forceps were applied in order to spare the uterine cicatrix the strain incident to expulsion, and a male child weighing 2120 grams and measuring 46 cm. in length, with a biparietal diameter of 9 cm., was easily delivered without injury to the perineum.

The convalescence was uneventful except for a mild colon bacillus pyelitis, which developed three days before the onset of labor and caused a febrile reaction, which continued until the fifth day of the puerperium. The


patient was discharged in excellent condition on the 15th day and suckled her child, which on discharge exceeded its birth weight bj' 80 grams.

The labor was of interest from two points of view. First, that the small child passed through the abnormal pelvis without difficulty, and secondly, that neither the distention of the uterus incident to pregnancy nor the strain of the second stage of labor had any untoward effect upon the scars of the previous sections.

Through the courtesy of the hospital at which the previous Caesareau sections had been done, we learned that the children weighed 2880 and 2190 grams, respectively, as compared with 2120 grams in the present instance. This at once raises the question as to whether radical operative interference was necessary. While criticism is not permissible unle.s.s one is in possession of all the facts, it appears probable that the second labor, at lea.st, would in all probabilitj' have ended spontaneously had it been given a chance, and that the actual indication for the second Caesareau section was fear of the behavior of the uterine scar following the first operation. The result in our hands shows that such fears were groundless, and brings us tlo the discussion of the behavior of the Caesareau scar in general.

A pj-iori, one would be inclined to believe that the scar tissue in the cicatrix would represent a locus minoris rcsi^tentiae, which might predispose to rupture in subsequent pregnancies, and the actual occuri-ence of such accidents indicates that the danger is a real one. I shall not, however, discuss the question at any length, as an extensive article bj- Dr. Thomas O. Gamble ba.sed upon the experience of our service has just been published.

In Gamble's paper, evidence was adduced to prove that, while rupture sometimes occurs, its frequency is much less than is currently taught. This is due to the fact, which we first demonstrated, that when the uterine wound has been properly sutured, and infection has not occurred, microscopic examination shows a surprising lack of scar tissue, and demonstrates that muscular fibres cross the site of the old incision in all directions just as if it had never been made. Indeed, in a number of iiteri which W'Cre removed at a second or third section, no trace of the previous incision could be discovered, except perhaps a slight furrow upon the external and internal surfaces of the anterior uterine wall, and in certain specimens even this was lacking.

On the other hand, if the incision has not been properly sutured, and more particularly if infection has occurred, good union may not obtain, with the result that the uterine wall is thinner at the site of the old incision than elsewhere. In such circumstances, it is conceivable that, as the distension of the uterus increases, the thinning may become so accentuated as to lead to rupture with its serious consequences.


THE JOHNS HOPKINS HOSPITAL BULLETIN. MAY. 1922


lie


PLATE XXII



Fig. 1. — Showing shortened tnrsd. pendulous abdomen and body weight borne upon right leg.


Fig. 2. — Showing double gibbus and collapse of torso.



Fin. ?j. — Showing obliquel.v ovate superior strait.


May, 1922]


193


The case before us offers additional proof that the uterine scar can withstand the strain of subsequent pregnancy and labor, and again demonstrates that the dictum "once a Caesarean always a Caesarean" does not always hold good. Indeed, my experience leads me to believe that its importance has been greatly exaggerated. At the same time, it should always be remembered that,


as the Caesarean scar may represent a locus tninoris resisfentuie, this fact affords the best possible argument for restricting the employment of the operation within the narrowest limits, instead of employing it more indiscriminately as is advocated by so many obstetricians and surgeons.


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Study of Arterio Venous Fistula with an Analysis of 447 Cases. By Curle L. Callander, M. D. Price, $2.50.

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THE JOHNS HOPKINS HOSPITAL BULLETIN


The Hospital Bulletin contains details of hospital and dispensary practice, abstracts of papers read and other proceedings of the Medical Society of the Hospital, reports of lectures, and other matters of general interest in connection with the work of the Hospital. It is issued monthly. Volume XXXIII is in progress. The subscription price is $4.00 per year.

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194


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Pedicated bv his Pupils to William Henky Welch, on the twenty-Httli anniversary ot his Doctorate. This volume contains 38 separate papers.

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Twelve papers on pneumonia. By Drs. Chatard, Fabyan, Emerson, Marshall, McCrae, Steiner, Howard and Haxes.

A Study of Diarrhoea in Children. J. H. ^UsoN Knox, Jr., M. D., and Edwin H. Schorer, M. D.

.Skin Transplantation. By John Staige Davis, M. D.

Epidemic Cerebrospinal Meningitis and Serum Therapy at The Johns Hopkins Hospital. By Frank J. Sladen, M. D.

VOLUME XVI. 070 pages with 151 figures.

Studies in the Experimental Production of Tuberculosis In the Genitourinary Organs. By George Walker, M. D.

The Effect on Breeding of the Removal of the Prostate Gland or of the Vesiculffi Seralnales, or of Both ; together with Observations on the Condition of the Testes after such Operations on White Rats. By George Walker, M. D.

Scalping Accidents. By John Staige Davis, M. D.

Obstruction of the Inferior Vena Cava with a Report of Eighteen Cases. By J. Hall Pleasants, M. D.

Physiological and Pharmacological Studies on Cardiac Tonicity in Mammals. By PEIiClVAL DOITGLAS CAMERON. M. D.

VOLUME XVII. 586 pages with 21 plates and 136 figures.

Free Thrombi and Ball Thrombi in the Heart. By Joseph H. Hewitt,

M. D. Benzol as a Leucotoxin. By Lawrence Selling, M. D. Primary Carcinoma of the Liver. By Milton C. Winternitz, M. D. The Statistical Experience Data of The Johns Hopkins Hospital, Baltimore,

Md.. 1892-1911. Bv Frederick L. Huffman, LL. D., F. S. S. The Origin and Development of the Lymphatic System. By Florence K.

Sarin, M. D. The Nuclei Tuberis Laterales and the So-called Ganglion Opticum Basale.

By Edward F. Malone, M. D. Venous Thrombosis During Myocardial Insufficiency. By Frank J. Sladen,

M D., and Milton C. Winternitz, M. D. Leukaemia of the Fowl : Spontaneous and Experimental. By Harry C.

SCHMEISSER, M. D.

VOLUME XVIII. 445 pages with 124 figures. Fasciculus I.

A Study of a Toxic Substance of the Pancreas. By E. W. Goodfasture,

M."D., and George Clark, M. D. (lid Age in Relation to Cell-ovcrgrowth and Cancer. By E. W. Goodpasture, M. D., and G. B Wislocki, M. D. The Effect of Removal of the Spleen Upon Metabolism in Dogs ; Preliminary Report. By J. H. King, M. D. The Effect of Removal of the Spleen Upon Blood Transfusion. By J. H.

King, M. D. B. M. Bernheim, M. D., and A. T. Jones, M. D. Studies on Parathyroid Tetany. By D. \\"^ight Wilson, M. D., Thornton

Stearns, M D., J. H. Jannev, Jr., M. D., and JIadge DeCJ. Thurlow,

M. D. Some Observations on the Effect of Feeding Glands of Internal Secretion

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Sch.meisser. M. D. Studies on the Relation of Fowl Typhoid to Leukaemia of the Fowl. By

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Knox, M. D., H. R. Wahl, M. D., and II. C. Schmeisser. M. D. A Fatal Case of Multiple Primarv Carcinomata Bv E. D. Plass, M. D. Congenital Obliteration of the Bile-ducts. By James B. Holmes, M. D. Multiple Abscesses of the Brain in Infancy. By Ja.mes B. Hol.mes, M. D. Gastric Carcinoma in a Woman of Twenty-six Years. By R. G. Hussey,

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HlssEY, M. D. Heart Block Caused by Gumma of the Septum. By E. W. Bridgeman,

M. D., and H. C. Sch.meisser, M. D. Analysis of Autopsy Records.

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Clinical Representatives on the Staff of the Department of Pathology. Donation.

Fasciculus II. The Role of the Autopsy in the Medicine of To-day. By M. C. Winternitz, M. D. Experimental Nephropathy in the Dog. Lesions Produced by Injection

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M. D., and William C. Quinby, M. D. Mesarteritls of the Pulmonary Artery. By M. C. Winternitz, M. D., and

H. C. Sch.meisser, M. D. A Clinical and Pathological Study of Two Cases of Miiary Tuberculosis of

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M. D. The Blood-vessels of the Heart Valves. By Stanhope Batne-Jones, M. U. Equilibria in Precipitin Reactions. By Stanhope Bayne-Jones, M. V. Carcinoma of the Pleura with Hypertrophic Osteoarthropathy. Report of

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Stanhope Bavne-Jones, M. D. The Interrelation of the Surviving Heart and Pancreas of the Dog in

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Burrows, M. D. The Functional Relation of Intercellular Substances in the Body to Certain Structures in the Egg Cell and Unicellular Organisms. By

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T. Burrows, M. D., and Edw-ards A. Park, M. D. Papilloma of the Larynx Report of a Case Treated with Radium with

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VOLIIME XIX. 358 pages with 29 plates.

The Structure of the Normal Fibers of Purkin.ie in the Adult Human Heart and Their Pathological Alteration in Syphilitic Myocarditis. Bv O. Van Der Stricht and T. Wingate Todd. M. D.

The Operative Story of Goitre. The Author's Operation. By William S. Halsted, M. D.

Study of Arterio- Venous Fistula with an Anal.vsis of 447 Cases. By Curle L. Callander, M. D.

VOLUME XX. 314 pages with 82 plates.

The I'athologv of the Pneumonia in the United States Army Camps During the Winter of 1917-18. Bv Willia.m G. MacCallu.m, M. D.

Pathological .Anatomy of Pneumonia -Associated with Influenza. By William G. MacCallum, M. D.

Lymphosarcoma. Lymphatic Leukaemia. Leucosarcoma. Hodgkln's Disease. Leslie T. Webster, M, D.


CONTENTS

  • The Evolution of Human Races in the Light of the Hormone Theory. Racial Status and Form of Body. By Prof. Sir Arthvr Keith ..... 195
  • The Relation of Nutrition to Tooth Development and Tooth Preservation. I. A Preliminary Study of Gross Maxillary and Dental Defects in Two Hundred and Twenty Rats on Defective and Deficient Diets. (Illustrated.) By E. V. McCoixuM, NixA Simmonds, Ethel M. KiNNEV and Clarence J. Grieves . . . 202
  • Studies on Experimental Rickets. XX. The Effects of Strontium Administration on the Histological Structure of the Growing Bones. (Illustrated.) By P. G. Shipley, E. A. Park, E. V. McCollum, Nina Simmonds and Ethel May Kinney . . 216
  • Carcinoma of the Cervix Uteri. A Very Early Case. (Illustrated.) By Karl H. Martzloff . >< .221
  • The Capillaries of the Bone Marrow of the Adult Pigeon. (Illustrated.) By Charles A. Doan 222
  • The Effect of Slight Increase of Temperature on the Bacteriostatic Power of Gentian Violet. (Illustrated.) By John W. Churchman 227
  • Studies on Experimental Rickets. XXI. An Experimental Demonstration of the Existence of a Vitamin Which Promotes Calcium Deposition. By E. V. McCoLLUM, Nina Simmonds, J. Ernestine Becker and P. G. Shipley ..... 229


THE EVOLUTION OF HUMAN RACES IN THE LIGHT OF THE HORMONE THEORY - RACIAL STATUS AND FORM OF BODY

By I'rof. Sir Arthur Keith {Conservator of the Museum and Hnnterian Professor, Royal College of Surgeons, London, Eng.)

Herter Lecture No. 2.


Giantism, and Acromegaly. — Giants are us^ually the subjects of acromegaly as well as of overgrowth of trunk and limbs. There have been authentic cases of giantism which were free from the usual manifestations of acromegaly. There are also cases, certainly few in number, of adolescents who were the subjects of acromegaly and yet showed no increase in stature. From such instances one infers that, although giantism and acromegaly may be present in the .same subjects, they represent disturbances of two correlated yet distinct growth mechanisms. During the years of growth there must be some ever pre

Abstract of Lectures Nos. 2 and 3 of the thirteenth course of lectures under the Herter Foundation, delivered before the Johns Hopkins University, October 6, 1921.


sent process at work controlling the growth of the correlated functional systems of the body. Since the pituitaiy is enlarged in giants, who are free from acromegalic traits, we may infer that this mechanism is also represented in the pituitary — in part at least — for other centres of growth also influence stature. Giantism represents a disorder of the hormone mechanism presiding over, not the reactional, but the normal growth of parts. Localised Acromegalies. — In seeking to unravel the nature of acromegaly one is at first tempted to suppose that the condition is merely the result of the pituitary throwing increased doses of a growth-elixir or hormone, into the circulatory blood. A study of cases of localized overgrowth or partial acromegaly shows that the expla


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[No. 376


nation cannot be so simple. The most instructive cases of this kind are these seen in children in whom there is a marked unilateral hypertrophy of the apparatus of mastication. One side of the face is acromegalic, the other is normal. A minute examination of such cases shows that the overgrowth is shaqaly limited to the parts concerned in mastication — to one half of the mandible, to the corresponding maxilla and palate, and to the bones which give attachment to the cori-esponding muscles of mastication. The tympanic plate is involved in the overgrowth, showing that it is part of the apparatus of mastication. The disorder must commence in foetal life, for the teeth of the affected side are much larger than on the normal side. There is no evidence as yet that there is an anatomical change in the pituitary glands in such cases, but as we have here an undoubted manifestation of localized acromegaly, it is legitimate to infer that we must be dealing with a disturbance of the same hormone-machinery that lies behind generalized acromegaly. In such cases we see the anatomical elements of a single functional unit picked out. In generalized acromegaly all functional units are affected.

The mechanism of Growth Hormones. — These localized cases of acromegaly show that growth is not merely the result of the presence of an activating substance thrown into the general circulation by one or by a number of glands of internal secretion. A local as well as a general mechanism must be concerned. In the case of functional hormones, such as secretin and adrenalin, and also in the case of growth hormones formed within the testicle, we see that such substances, although offered to all the tissues of the body are selected and accepted only at certain definite points or regions. It is not difficult to conceive that one of these local receptive mechanisms might be heightened in avidity or susceptibility and thus give rise to a local hypertrophy. Whatever the nature of the central and peripheral growth mechanisms may prove to be, there can be no doubt that they are arranged on a physiological basis — one which bends a collection of anatomical elements to serve definite functional ends. It is this complex dual mechanism which is inherited from generation to generation and which fashions mankind into different functional types and races.

Examples of the Pitwitary Mechanism at Work in the Evolution of Types.— lip to this point I have endeavoured through a study of acromegaly to unravel the complex mechanism which regulates the growth of the human bodj', concentrating attention more especially upon the apparatus of mastication. To exemplify the manner in which the pituitary growth mechanism produces its effectSj no finer example can be chosen than man's nearest relative — the gorilla. He is the greatest and strongest meniber of the zoological group to which man also belongs— the Higher Primates. The skull of the baby gorilla is smooth and rounded like that of a child ; after


infancy there sets in a series of growth changes which transform the whole appearance of the individual; the masticatory apparatus reaches a supreme degree of development; the temporal lines rise up to form the great sagittal and lambdoidal crests of bone, the lower part of the frontal bone is extended forwards until it forms a great ledge-like bar of bone above the orbits ; the jaws and face become massive. Behind this transformation must lie the same pituitary groA\'th mechanism which is revealed in the acromegalic. In the chimpanzee the growth machinery comes to a standstill at a stage reached in the juvenile gorilla ; in man it is arrested at a stage represented in the childhood of the gorilla. In Neanderthal man we also see the pituitary mechanism vigorously at work. The ape-like supraorbital ridge, the massive jaws, and the wide expanse of the nuchal platform are evidences of this influence. But it is among peoples of the Caucasian or European type that we may best mark the peculiar effects of this mechanism among modern races of mankind. The homeland of this type extends from the western confines of India to the Atlantic shores of Europe. So far as the facial features are concerned, a long narrow prominent nose, a prominent chin with a tendency to elongation and narrowing of the face are the chief racial marks of this type. These are features which almost invariably become greatly exaggerated in the acromegalic. In the shaping of the European type, the pituitary mechanism has had much to do. In shaping the racial charactei-s of the European type the pituitary influence seems to have dominated the other centres of hormone-production ; in the Mongolian and Negro types other centres, such as the thyroid and adrenals seem to have gained in potency.

The Origin of neto racial ch<iraeters and types. — Although I have by no means exhausted the anthropological lessons to be derived from the study of pituitary disorder.s — such as are seen in giantism, Frohlich's disease (dystrophia adiposo-genitalis ) , dwarfism, infantilism, progeria — it is impoi-tant, before we proceed further, to study the manner in which new racial characters come into existence — particularly those relating to the colour of skin and hair and to the nakedness of the human body. In the Museum of the Royal College of Surgeons there has been exhibited since the conservatorship of Sir Richard Owen, the pregnant uterus of a chimpanzee, dissected so as to expose a foetus iu the seventh month of development. Prof. Louis Bolk of Amsterdam has a similar specimen. Independently he and I have realized the important bearing which such specimens have ou the theory of hormone evolution.* So human are the uterus and foetus in appearance that a superficial observer might well believe he had before him the womb and fruit of a woman. The skin of the body is nude, except for the


"The Part Played by the Endocrine Glands in the Evolution of Man." Lancet, 1921, XI, 588.


June, 1922]


197


presence of a flue lanugo; on the other hand, the scalp is covered with hair of a brown colour. The distribution of hair is human — not anthropoid. The skin is scarcely pigmented; it is ashen grey in colour; at a corresponding stage the negro foetus is not dark skinned. Prof. Bolk draws the conclusion, and I agree with him, that man has come by his naked skin and the European by his white colour by inheriting a foetal condition from his anthropoid ancestry. A transient foetal condition of the anthropoid has become fixed permanently in the human adult. We have seen that in his masticatoi-y apparatus and in his nuchal fixation, man retains stages seen in the childhood of anthropoid apes. Man's new characters have first been elaborated within the uterus of the ape; later these foetal characters have been incorporated in the make-up of the human adult.

Up to this point Prof. Bolk and I proceed together. But we must go further and ask ourselves the question. How do these foetal characters arise in the anthropoid womb? I have laid stress u]ion the fact that the pituitary mechanism — the whole mechanism of growth hormones — has been elaborated and organized on a functional or physiological basis. This was made apparent when dealing with the effects of the pituitary mechanism, in fashioning the apparatus of mastication. Nor do we make any extravagant demands on our imagination when we proceed to ^ippose that such hormone mechanisms are constituted and elaborated during the foetal life, in the same way as the mechanisms of instinct and of reflex action are given a concrete representation in the nervous s.A'stem. The nerve-machinery of an automatic action is inherited. One may hazard the presumption that this aLso may be true of the hormone machinery, which presides over the functional fashioning of the body. The long developmental intra-uterine period of higher vertebrates, particularly of the higher primates, gives the hormone system the shelter necessai'y for working out its effects in the f(i4al body and the opportunity of attempting fresh experiments. It is under the infiiience of this automatic hormone mechanism that new characters are elaborated and fresh experiments launched during the developmental stages of life. Darwin's Law of Natural Selection tests these experiments, accepting the successes, and rejecting the failures.

Hair and Skin Clutracters. — Colour is one of the principal marks of race, but in explaining why we should find all shades from the deep black of the negro to the fair skin of the Norseman, we are handicapped by our ignorance of the part played by i)ignient in the economy of the body. The facts at our disposal are these: When the medullary parts of the adrenal bodies are injured or destro.yed by disease, the skin becomes pigmented — as Thomas Addison noted in 1855. John Hunter inferred that the original colour of man's skin was black and all the facts we have gathered since his time favour this


view. The gorilla is the negro amongst anthropoids, he is deeply pigmented at birth. The chimpanzee darkens after birth, some races early, others later. In the orang the hair is red but the skin has a slaty hue. We have already seen that the foetus of man, as of anthropoid, is free from pigment.

There is thus a relationship between pigmentation and these important centres of hormone production — the adrenal glands. AVe obtain some light on our problem when we remember that the adrenals are closely connected with the important function of maintaining the body at a temperature — under all extremes of heat or cold — of approximately 98.4° Fahr. The regulation of the blood supply to the skin, the secretion of sweat, and the development of subcutaneous fat, are the important means by which this end is secured. The development of hair on the body, the amount and distribution of pigment and fat must also be accessory .circumstances in the regulation of temperature. We have seen how anatomical elements are combined into functional units by means of a hormone mechanism and I infer that future research will reveal a growth mechanism which presides over the parts concerned in the regulation of temperature and that skin-pigment will be found to be included in this system. The mechanism must be a complex one in which pituitary and thyroid are also involved, for there is no doubt that disorders of the pituitary ai'e followed by definite changes in hair and skin ; thyroid disorders also give rise to changes, but they are of another kind. From these facts we see that the skin is dominated by a hormone mechanism in which adrenal, thyroid and pituitary are combined — a mechanism of a complex nature.

Thyroid Effects. — On entering into a discussion on the effects produced by thyroid substances in shaping the racial characteristics of mankind, it is necessary to recall some of the statements made regarding the nature of the machinery lying behind the growth changes produced by hormones. I am regarding the glands of internal secretion as centres for the elaboration and distribution of hormones and believe that the organs and tissues of the body are endowed with special affinities for the hormones in circulation and that very probably these local centres have also a means of making their needs known at the centres of hormone production. In dealing with pituitai-y effects I have proceeded on the belief that we are witnessing positive results — an increased action of the pituitary producing increased activity in definite peripheral sites of growth. Thyroid substances can produce acceleration of growth ; this is strikingly shown by the manner in which extracts of the gland can increase the growth of cretinoid diildren and hasten the metamorphosis of tadpoles. 1 presume that thyroid and pituitary growth hormones utilize the same local or peripheral mechanism in producing their effects.


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Cretinism. — It is, however, not the positive or exaggerated action of the thyroid, but its minus or defective one which brings out the part played by this gland in the differentiation of races. In cretins, growth of all the tissues of the body is not only retarded but positively distorted. The effects are best seen in the cartilaginous growth discs of long bones. The cartilage cells multiply slowly and irregularly; osteoblasts cease to have the power to invade and overcome them. The cartilaginous ba.se of the skull suffers in this way ; the root of the nose becomes drawn in and widened ; the nose itself, which is built over a foundation of cartilage, is wide, short and not prominent. The face, which in acromegaly is long and narrow and wedge-shaped, is short, wide and flat in the cretin. The skin is dry, the hair scanty, and the subcutaneous tissue thickened. These are the results of a gross deficiency of thyroid substances. When thyroid extracts are administered, the characteristics previously produced disappear and are replaced by the normal. We have in cretinism positive evidence that the thyroid has to do with regulation of stature and the determination of characters of face, hair and skin — all being marks employed by anthropologists in the classification of races.

Mongolism and Achondroplasia. — Cretinism is a result of a gross deficiency of thyroid substances. There are at least two disorders of growth — mongolism and achondroplasia — which one has reason to attribute to a defective or altered action of thyroid substances. It is true that the exhibition of ordinary thyroid extracts has no effect upon such cases, nor has any one succeeded in producing these conditions experimentally in animals, yet there are clinical manifestations and structural alterations in both of these disorders, which are also seen in cretinism, and for this reason I feel justified in attributing both mongolism and achondroplasia to disturbances in the complex effect exerted by thyroid substance on the growth and differentiation of the body. Dr. Langdon Down, in 1866, recognized that a certain class of imbecile was characterized by traits which recalled those seen in the faces of Mongolian peoples. My friend, Dr. Francis Crookshank has explained the appearance of such traits as a harkback to a Mongolian ancestry. Not only is there a lack of evidence that a Mongolian people has at any time populated Europe, but there is circumstantial evidence that the Mongolian type of humanity is one of the most recently evolved of racial forms. The theory which best explains all the facts is to suppose that Mongolian features arise under a peculiar or altered action of the growth mechanisms centered in the thyroid gland.

It is not necessary to recall the bodily characteristics of achondroplasia or "bulldogism" — for the disturbance of growth is that seen in all varieties of the bulldog breed. The trunk is normal in size but all segments of the limbs, including shoulder and pelvic girdles, are retarded and distorted in growth. In particular, the base


of the skull suffers ;* the cerebellar fossa and the area of nuchal fixation are reduced in size and greatly altered in shape ; the root of the nose becomes drawn in between the ej^es, and broadened just as in cretins. The facial bones laid down over the nasal capsule are retarded in growth, so that the anterior part of the maxilla is drawn up, producing a pug-face. As in dachshunds, the limbs may be affected while the base of the skull and nose develop normally. The disorder occurs in all races of mankind and in varying degrees of severity. Darwin described a bulldog breed of cattle from the Argentine. Dr. Seligman found that the cretinoid or bulldog calves of Kerry cows had disordered thyroids. Dr. Douglas Symmers observed that in a proportion of stillborn achondroi)lastic children there was a structural disorder of the thyroid gland. In achondroplasia we are clearly dealing with a disorder of a growth mechanism which is widely distributed in the animal kingdom and the evidence points to this mechanism having its centre in the thyroid gland.

Now Mongolian peoples are characterized by limbs which are short in comparison with their trunk length. The nasal region between the eyes is wide, flat and frequently sunken in typical Mongols. The flattening of the root of the nose is even better seen in the true Negro type. In the Mongol, as in the cretin, eyebrow ridges are poorly developed or absent. It is not only amongst human races that these supposed thyroid effects can be traced. The orang is the bulldog or Mongol amongst anthropoid apes; he is pug-faced, the nasal region of his face being greatly reduced and drawn in, the supraorbital ridges are developed to only a minor extent — especially when contrasted with their development in the gorilla. With this conformation goes a silent and rather morose temperament. The orang differs in colour from other anthropoid apes just as the Mongolian colouring contrasts with that .seen in the negro or Eviropean. The late I'rof. H. Klaatsch was struck by the points of resemblance which linked the Mongolian and orang types and sought to explain the similarity by supposing that the Mongol and orang had a common ancestiy. The explanation I offer is that both have inherited a common mechanism of growth hormones and they are superficially alike because, in both, the thyroid effects of this mechanism have become dominant.

Dirarf -Races and Stature. — No anthropological problem has been more discussed than the position to be assigned to pygmy or dwarf varieties in the scale of human evolution. In finding the proper solution to this problem, it has to be remembered that all living dwarf breeds are members of the negro race and that each dwarf variety has many resemblances to the breed of negro existing in the same region. The central African pygmies are dwarf


Quart. Journ. Med., 1912, Vol. 5, p.l57.


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forms of the true negro ; the Bushman is a dwarf form of the Hottentot breed and the pygmies of the Far East are varieties of the Eastern Negroids. From their characters and distribution one infers that pygmy peoples have been produced locally from normal members of the negro race, just as dwarf races of horses, dogs and cattle have arisen locally from breeds of normal size. It is also worthy of note that the Dinkas and Shilluks of the White Nile are amongst the slenderest and tallest examples of mankind. Stature is a highly variable factor in the negro race. When we attempt to distentangle the e.xact nature of the growth mechanism which leads to the production of dwarf races, we become keenly alive to the complexity of the problem and to a need for further knowledge. All tissues of the dwarf body are fashioned on a miniature scale, but we can best realize what happens by concentrating our attention on the epiphyseal lines or growth discs of long bones which are so directly related to stature. Epiphyseal lines represent local growth mechanisms, activated and controlled by substances thrown into the circulation at centres of hormone production. Pituitary substances play on these sites of growth ; we explain giantism by an oversupply, and infantilism, such as is seen when the pituitary is compressed by a cyst, by an un(ler-suj)ply. The sexual glands can also influence the rate of growth at epiphyseal lines, but they exert their influence not directly on the cells of the epiphyseal lines but indirectly through the cortical part of the adrenals. A fuller knowledge of the part played by the growth hormones of the thyroid is likely to give us an explanation of the origin of dwarf breeds.

The Hottentot Type— The wide, flat interocuiar field of the typical Mongol is to be regarded, so I think, as an efl'ect of thyroid action ; the same inference must also hold good in the case of the Negro. Now, in the most peculiar breed of existing Negroes — the Hottentots of South Africa — the interocuiar field is particularly wide and flat; the bony skeleton of the nose is reduced ; the supra-orbital ridges are poorly developed. Many anthropologists have noted Mongolian traits in the Hottentot face, as well as the yellowish tint of their skin. These traits are also to be noted in the face and skin of the Bushman. If we admit that there is a tendency for the thyroid mechanism to become dominant in the Negro type, then we can explain the appearance of Mongolian traits in the Hottentot — undoubtedly a member of the Negi'o race. No other theory can give a satisfactory explanation of the racial features and of the distribution of the varieties of the Negro-type.

Dystrophia adiposogevltalis. — Having touched on the Mongolian traits of the Hottentot, it is convenient at this point to discuss another anthropological character by which the females of this racial type are marked — a tendency to the accumulation of fat on the buttocks and outer aspect of the thighs. This peculiar character occurred in certain peoples who lived in Europe towards the close of


the ice age, as we know from discoveries of statuettes and drawings of that distant period. These fat-buttocked women were of the European type, although their skulls do show some negroid traits. The changes which occur in modern women at the menopause help to throw some light on the physiological machinery underlying the appearance of such a feature as a race mark, for, as is well known, atrophy of the ovaries is frequently followed by the deposition of fat, particularly in the region below the waist. The same result frequently follows removal of the ovaries or testes. In the growth disorder first described by Frohlich — dystrophia adiposo-genitalis — there is a like tendency to the subcutaneous accumulation of fat, but in such instances deposition follows enlargement of the pituitarj' gland and an atrophy of the sexual system. On our present evidence, we cannot say whether the change in the pituitary or in the sex glands is the primary one — very probably it will prove to be the latter — but we may safely attribute the deposition of fat and the assumption of a female configuration of body as a direct result of the sexual atrophy. In Hottentot women the physiological process underlying the localized deposition of fat comes into play independently of any loss of sexual function and yet upon the evidence of pathology we must suppose that this process is controlled through a hormone mechanism centred in the sex glands. There is ample evidence to show that a close functional relationship exists between the centres of hormone production — the adrenals, pituitary, pineal and thyroid glands. The sex glands have apparently the power, by means of ovarian or testicular hormones, to influence and set into operation the growthcontrolling machinery situated in the glands of internal secretion. All other evidence points to the influence of the sex glands as being one which is exerted, not directly on the tissues of the body, but through the intermediation of other centres of hormone production. The deposition and absorption of fat we nuist regard as controlled by an interaction between adrenals and thyroid — the glands concerned in regulating the metabolic rate and temperature of the body. The regional deposition of fat recalls, as do localized acromegalics or overgrowths, the need for postulating a local as well as a central machinery in our explanation of the efl'orts of growth-hormones. It is worthy of remark that the profuse wrinkling of the face, so commonly seen in adult Bushmen and Hottentots, is also present in eunuchs and eunuchoid beings. In these conditions the texture and elasticity of the skin are also affected.

The Nilotic Type of Negro. — Before leaving the Negro type, from which I have selected the foregoing instances to illustrate the application of the hormone theory- to the explanation of racial features, there is a further example I may be allowed to cite. The Negro tribes along the White Nile are slender and tall, their height being largely due to length of limb. Their extremities, in opposition to


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those of the Mongolian type, are long in comparison to length of trunk. Now castration, as is well known, results in a delayed clo.sure of the epiphyseal lines. Eunuchs are taller and longer limbed than are normal men belonging to the same place and race. At puberty, when the influence of the sex glands begins to play on the other hormone centres, the body loses the slenderness of boyhood and girlhood ; in the Nilotic negro type the slenderness of youth is retained in adults — a further example of the evolution of a new racial type by the fixation of parts at an immature phase. The sex glands in this type appear to reach maturity without setting in motion the machinery which expands the framework of the body and thus undoes the slenderness of youth. It is an example similar to that cited in the Hottentot women — where menopause characters appeared without impairment of the sexual function of the ovaries.


Herter Lecture No. 3.


Sex-Glands and Adrenals. — From remote times mankind has known that removal of the sex glands completely alters the characters of body and mind of the human as well as of the animal body. What is new in this department of knowledge is the hormone theory — an explanation of how the effects of castration are brought about. Until the postulation of the theory of hormones by Starling in lOOJr, medical men had supposed that the parts and systems of the body were provided with only one means of intercommunication — a nervous or telegraphic system. By 1904 knowledge had reached a point where it could be seen that there was an infinitely older system of intercommunication — a postal or hormonic system — which as regards the growth of the body is very much more important than the nervous system.

Tumours of the Adrenal Cortex. — The important role of the pituitary gland as a regulator of growth was an accidental discovery. Clinicians and pathologists discovered that disorderly overgrowth of the body was accompanied by an enlarged and adenomatous condition of this gland. Medical science benefitted by Nature's pathological experiment. In exactly the same way we have learned that the cortex of the adrenals is an important centre of


See Keith. Journ. of Anatomy and Physiologj'. 1910, Vol. 44, p. 251; ibid. 1913, Vol. 47.

t Prof. T. R. Elliott (personal communication).

On my arrival in New York I visited Prof. Stockard's Laboratory, Cornell University, and found that he had also arrived at many of the conclusions which are given expression to in these lectures. Prof. Stockard has applied the theory of hormones to explain the various breeds of dogs as well as of human beings.

t (1) For recent literature on the pineal gland, see Krabbe: Meddel. fra Rigshosp. Borneafdeling. 1917. No. 48.


growth-control. This glandular tissue is also liable to become the site of a localized overgrowth or an adenoma in the young and is accompanied by a very definite series of growth changes in the body. Recently my friend Prof. E. E. (Jlyn * of Liverpool has rendered an important service by tabulating the data relating to cases of tumour of the adrenal cortex, adding several instances which have come under his own observation. I had an opportunity of seeing the classical case described by Bulloch and Sequeira in 1903. The occurrence of such tumours in young boys is accompanied by a premature development of all the bodily changes which should not take place until the age of puberty. A boy of five years of age will become sturdy and thick-set — an infant Hercules; his voice will break ; hair will grow on the face and pubes ; his penis becomes developed ; the testes become active. The adrenal cortex presides over the development of all of the structural parts which are fashioned into "secondary sexual characters" — the name given to them by John Hunter. Cases are recorded in which these changes have set in soon after birth — pubertal changes masking the features of babyhood. In female children or in young women, cortical tumours of the adrenal produce a different effect; they tend to bring out the sexual character of the male. With the surgical removal of such a tumour, the normal female configuration of body, voice and of mind becomes restored, the adventitious male characters disappearing.!

On the evidence of embryologj' alone one would suspect that there must be a close functional relationship between the sexual glands and the adrenal cortex ; both are differentiated at the same site and appareutly from a common embryological basis. One infers that it is through the adrenal cortex that the sex glands bring about the complex series of growth changes which transform the human body at puberty. Through the pituitaiy mechanism these same glands can play upon the general growth of the body systems.

Tumours of the pineal gland nmy be accompanied by a series of growth changes almost identical in nature and scope to those following tumours of the adrenal cortex. How such a function has come to be resident in tho pineal is an enigma at present — one which futiire research will probably clear up. J

TJie Role of the Adrenals in Raee Differentiation. — It is scarcely necessary to recall the fact that secondaiy sexual characters, which are apparently controlled by hormones elaborated in the adrenal cortex, are employed by anthropologists as marks for differentiating one race of mankind from another. The beard and hairiness of body which characterize the European or Caucasian type of man, and also, to a lesser extent, the Australoid type —


June, 1922]


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the most primitive of existing races — I look upon as an ancient and characteristic of humanity. In Mongolian and in Negro types the tendency is to a hairless face and body. In this respect these types are more highly evolved than the European. The immature stage, seen in the European youth, is tending to become fixed as the adult stage in the Mongol and Negro. The growth mechanism which determines the woolly hair and thick everted lips of the negro we do not know, but are justified in regarding both of these features as non-anthropoid and of recent origin. Large labia majora, on the other hand, are characteristic of the European type. I'rof. Bolk has pointed out that these structure;" are relatively large in foetal stages of ape and man and that the European has acquired this feature by a foetal becoming an adult char acter.

The Periods of The Life-Span. — All the evidence at our disposal points to the evolution of higher human races as having been accompanied by a lengthening of the various growth periods which make up man's normal span of life. The more civilized races of man have longer lives than the primitive races and the primitive races than the great anthropoid apes. It is quite apparent, from what occurs in children suffering from tumours of the adrenal cortex, that Nature has a hormone mechanism at her disposal for accelerating or retarding the nuituring of the body. The action of the adrenal cortex must be connected with the regulation of the periods of growth and with the span of life. We have further examples of an interference with the normal maturation of the body in the group of cases ai present described under the generic term — infantilism. There are forms such as that illustrated by a specimen in the Museum of the Royal College of Surgeons — the skeleton of Crachami — a girl, 8I/2 years of age, but in size and in many other features similar to a baby only a few months old. Dr. Hastings Gilford has distinguished this form as a fa'tal type of "infantilism." Then there is a type illustrated by such cases as that of Jeffrey Hudson who died at G3, and Bornolaski who died at the age of 98. In such cases the span of childhood covers the whole period of life. They may become sexually potent and yet retain a childish size and appearance, their epiphyseal lines remaining open late in life. Again there are those remarkable cases of infantilism to which Hastings Gilford has given the name of Progeria. Such individuals assume an aged appearance while still in their girlhood or boyhood. The exact nature of the growth disorder in these cases is not known, but it is probable that both pituitary and adrenal mechanisms are involved. Compression or atrophy of the pituitary does arrest the growth of the body — produces a condition of infantilism. In the case of progeria that I have examined, the pituitary fossa is relatively small and the neck of the gland is surrounded by a bony ring formed out of the clinoid processes. The atheromatous condition of the arteries, and


the prenmture appearance of senility suggest that the adrenals may also be involved. The evidence is sufficient to awaken an interest in the role played by hormones in regulating the length of life's span.

Conclusion

I am well aware of the imperfect nature of the evidence on which I have based a plea for regarding hormones as the agencies which control growth and evolution. Before coming to a conclusion as to whether a prima facie case has been made out, I would beg the reader to look narrowly at the position in which all who believe in the evolution of living things are now placed. Darwin gave the law of Evolution an abiding place in Biologj' ; his great difficulty was to explain how a multitude of anatomical elements became simultaneously modified to serve a single definite fuuctional purpose. A study of the growth disorders reveals the fact that the hormone .systems, centered in the pituitary and adrenal glands are organized on a functional basis. Hormone systems repre.sent automatic growth mechanism which, like all living qualities, are hereditary and variable. Hormones repi-esent the elements of an automatic .system for the control of growth. For this reason new characters do not appear at the end of a developmental stage but early in the growth of the foetus. New characters appear first in utero; later they become fixed as a new character in the adult stage. The hormone growth machineiy is just such a one as Darwin was in search of. He propounded the elaborate theory of Pangenesis as a substitute.

In these lectures 1 have been applying the theory to problems relating to the origin and evolution of human races. But the day is certainly coming when it will be made applicable to the realms of Zoology and Botany and provide a real scientific basis for these branches of knowledge.

From the experiments carried out in India by Lt. Col. Robert McCarrison it now seems possible that the substances named vitamines may influence and alter the growth mechanisms of the animal body.

Lastly, from the study of English human remains, representing samples of the inhabitants who have lived in that land at successive periods during the last 4000 years, there is a convincing body of evidence that structural changes are taking place in the jaws, palates and faces of a large proportion of the present population of England. A full knowledge of the hormone system of the human body is likely to reveal not only the cause of these structural changes but also the steps which may have to be taken to combat them.


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THE RELATION OF NUTRITION TO TOOTH DEVELOPMENT AND TOOTH PRESERVATION. I.

A PRELIMINARY STUDY OF GROSS MAXILLARY AND DENTAL DEFECTS IN TWO HUNDRED AND TWENTY RATS ON DEFECTIVE AND DEFICIENT DIETS

By E. V. McCoLLUM, Nina Simmonds,

and Ethel M. Kinney

(From the Department of Chemical Hygiene, School of Hygiene and Public Health, Johns Hopkins University, Baltimore)

and

Clarence J. Grieves

(From the Department of Pediatrics, Johns Hopkins University )



STUDIES ON EXPERIMENTAL RICKETS. XX. THE EFFECTS OF

STRONTIUM ADMINISTRATION ON THE HISTOLOGICAL

STRUCTURE OF THE GROWING BONES

By r. <i. Shh'ley and E. A. 1'akk

{From the Department of Pediatrics, The Johns Hopkins University) and E. V. McCollum, Nina Simmonds and Ethel May Kinney {From the Department of Chemical Hygiene, School of Hygiene and Public Health, The Johns Hopkins University)


CARCINOMA OF THE CERVIX UTERI

A VERY EARLY CASE

By Karl H. Martzloff

(From the Department of Gynecology of The Johns Hopkins Hospital and University)


The case reported is one of very early eartiuoma of the cervix. A panhysterectomy was performed and today the patieut feels perfectly well. Of the ultimate result, however, nothiug can as yet be said with certainty.

The patient, Mrs. M. M. (Gyn. No. 27080-271()5) is a white woman, widowed, 55 years of age, who has had two normal pregnancies and two miscarriages. Her menopause occurred five years ago.

Three months before coming to The .Johns Hopkins Hospital the patient began to have a profuse, watery, vaginal discharge which soon became blood-tinged and malodorous. On admission to the hospital, August 13, 1921, examination revealed nothing grossly abnoiiual. Nevertheless, the history of a foul-smelling, blood tinged, watery, vaginal discharge commencing five years after the menopause was so suggestive of malignancy that it was decided to perform a diagnostic dilatation and curettage.

When the cervix was dilated, a small quantity of purulent material escaped, evidently from the uterine cavity. Curettement brought away a very small quantity of tissue, and not the large amount usually obtained in definite cervical cancer. Frozen sections made at the time of this operation showed nothing that could positively be identifled as cancer, so the patient was returned to the ward. Celloidin sections made on the following day, however, showed tissue which we considered undoubtedly malignant, though by no means giving the picture of a fullblown cancer (Figs. 1 and 2).

The patient at that time refused a radical operation, and left the hospital, only to return in two weeks, when a moditied Wertheim operation was done. Her convalescence was uneventful and she is now apparently well.

On macroscopic examination, the pathological specimen with its apparently normal cervix was, to say th<j least, depr essing, for we felt that through a grave error

Read before the Medical Society of Tlie Johns Hopkins Hospital, November 7, 1921.

t This photograph was retouched in order to bring out more distinctly the smooth surface of the mucosa lining the vaginal cuff and cervix.

t In this connection it is only fair to state that when the sections represented in Figs. 1 and 6 were submitted to four well known pathologists, three considered the tissue to be cancerous and one did not consider the evidence sufficient to warrant such a diagnosis. However, after seeing the serial sections represented in Fig. 9, the opinion in favor of cancer was unanimous.


in diagnosis on our part the patient had been subjected to a serious and unnecessary operation. Figure B.f is a photograph of the specimen in longitudinal section and tells graphically why, so far as could be seen, we were in the face of a surgical success but a diagnostic failure.

Pathological Examinntioii: (Gyn. Path. No. 27099). — The specimen consists of the entire uterus with its adnexa and a liberal margin of vaginal mucosa. The uterus measures 7 x 4 x 3.5 cm. The vaginal cuff measures from 1 to 1.5 cm. in length and is normal in appearance. The lips of the cervix and the cervical canal are pale grey, smooth and glistening, and present nothing noteworthy. The uterine cavity is dilated, its walls are somewhat irregular, of a dull, dirty, grey color, and scraj)ing brings away nothing in the nature of an exudate or a necrotic mambrane. Blocks are taken from all portions of the cervix, uterus, parametrium, and adnexa.

Sections of the uterus show the myometrium infiltrated with numerous round and occasional polymorphonuclear leucocytes. No endometrium is present. The only other sections of interest are those taken from the area indicated at "A" (Fig. 3) on the anterior cervical wall. Here the cervical epithelium proximal to "A" has for the most part its usual appearance, but in certain areas it shows a definite departure from the normal. In Figure 1 both the normal and abnormal stratified cervical epithelium is seen. At x the epithelium is hyperplastic and shows a well defined metaplasia in which the epidermal cells have lost the normal gradation from stratum mucosum to stratum germinativum. Instead, one sees large oval and broad spindle-shaped cells' rather unifonn in size, with hyperchromatic nuclei which show some irregularity in staining reaction. There are some nucleoli, and mitotic figures (Fig. 5, a) are not uncommon. These cells have practically no eosinstaining cj'toplasm.

Just distal to "A" (Fig. 3.) sections .show cell-nests (Fig. 6.) in which the cells resemble those just described, although here the irregularity in size, shape, and staining reaction is more marked and the mitotic figures are more numerous (Figs. 7 and 8.).

We believe that this is an early carcinoma of the cervix uteri of a type which we have been seeing rather frequently of late and concerning the nature of which competent pathologists are not in accord. $

Because of the difference of opinion which occurred after the reading of this paper, we were glad to follow


[No. 370


Dr.MacCallum's suggestion and make serial sections of the entire cervix, in order to find, if possible, some areas of more advanced carcinomatous invasion. Serial sections made from the right half of the cervix, from which the original blocks were taken, show a condition quite similar to that which has just been described. Serial sections from the left half of the cervix show cell-nests (Figs. 9,b and 10) similar to those represented in Figure 6, but in addition there are areas of actual lymphatic invasion (Figs. 9,c and 11), small clumps of cancer cells lying free in what appear to be endothelialliiied spaces. The microscopic invasion in none of these sections goes beyond a depth of 2 mm. below the surface of the cervical canal and the entire neoplastic process is limited to the anterior portion of the cervix. This, we feel, proves conclusively that our case is one of a true malignant neoplastic process and also demonstrates the value of making serial sections, in disputable cases, when numerous routine sections do not furnish the information desired.


Discussion

Dr. Thomas S. Cullen : "Dr. Martzloff's squaiuouscell carcinoma is evidently a very early one. We have never found nuclear figures in the squamous epithelium of the cenix except in ca.ses of carcinoma of the cervix."'

After discussing briefly an early squamouscell carcinoma of the cervix, which he had reported in Surgery, Gynecology and Obstetrics, in August, 1921, Dr. Culleu added : "We fully realize that nuclear figures do occur in the squamous epithelium, otherwise there could be no reparation when defects occur. But they are so uncommon that we have not encountered them in our routine work except where malignancy exists. We have also failed to note them in the cylindrical epithelium of the cervix.

"On the other hand, nuclear figures are regularly met with in the epithelium of the body of the uterus, and in cases of hyperplasia of the endometrium nuclear figures are frequently found in the stroma cells of the mucosa."


THE CAPILLARIES OF THE BONE MARROW OF THE ADULT PIGEON


By Charles A. Doan (f?-o»i the A7iatomieal Laboratory, Johns Hopkins University)


THE EFFECT OF SLIGHT INCREASE OF TEMPERATURE ON THE BACTERIOSTATIC POWER OF GENTIAN VIOLET

By John W. Churchman

(yew York)



STUDIES ON EXPERIMENTAL RICKETS. XXL

AN EXPERIMENTAL DEMONSTRATION OF THE EXISTENCE OF A VITAMIN WHICH

PROMOTES CALCIUM DEPOSITION

By E. V. McCoLLUM, Nina Simmonds,

J. Ernestine Becker

(From, the Department of Chemical Hygiene, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore)

and

P. G. Shipley

(From the Department of Pediatrics, The Johns Hopkins University, Baltimore)


Although there is no longer any room to doubt the efficacy of cod-liver oil in curing rickets, the nature of the active principle in the oil is still unknown. It has been stated that the fat-soluble A is responsible for the beneficial effect from its administration in this disease. We have, however, published certain evidence which led us to believe that the protection against rickets which codliver oil affords is not due to fat-soluble A.

Hopkins has shown that this vitamin is readily destroyed by oxidation. Following his lead we oxidized cod-liver oil for from 12 to 20 hours at 100° C by blowing air through it. Oil which was treated in this way did not cure xerophthalmia, even though its administration was begun at the onset of the disease, when edema of the eyelids was first noticeable. On the other hand, it was just as effective in curing rickets in rats as the untreated oil. The bones of rachitic animals which were given daily a


curative dose of the oxidized oil to the amount of 2 per cent of the weight of the ration (average daily consumption of cod-liver oil about 70 mgs.) for 11 days showed quite as advanced evidences of healing as those shown by animals which had received the same amount of unoxidized oil for the same length of time.

The antirachitic effect of cod-liver oil is not due, therefore, to its content of fat-soluble A, but to some other factor which the oil contains.

The existence of this substance, which exerts its effects in so remarkable a manner on the growing bones, is now as firmly established as is the existence of any of the three hitherto recognized vitamins. From its mode of action, and the very small amounts of it which are required to exert its directive infiuence on metabolism, it must, we believe, be classed with this group of essential nutritive principles.



VOLUME I. 423 pases, 09 plates.

VOLUME II. 570 pages, nith 2S plates and fieures.

VOLUME III. 766 pages, «itli 69 plates and figures.

VOLUME IV. 504 pages, 33 charts and illustrations.

VOLUJIE V. 480 pages, «ith 3S charts and illustrations.

VOLUME VI. 414 pages, vilth 79 plntes and figures.

VOLUME VII. 537 pages with illustrations.

VOLUME VIII. 532 pages «lth illustrations.

VOLUSIE IX. 1060 pages, 66 plates and 210 other illustrations.

Contributions to tlie Science of Medicine. Dedicated Ijv his I'upils to William IIexky Welch, on the twentytifth

annivei-sar.v of his Doctorate. This volume contains 38 separate

papere. VOLUME X. 516 pages, 12 plates and 25 charts. VOLUME XI. 555 pages, with 3S charts and Illustrations. VOLUME XII. 54S pages, 12 plates and other illustrations. VOLUME XIII. 605 pages, with 6 plntes, 201 figures, and 1 colored

chart. VOLUME XIV. 632 pages, Trith 97 figures. VOLUME XV. 542 pages, nith 87 Illustrations. Twelve papers on pneumonia. By Drs. Chatard, Fabyan, Emeksox,

Marshall. McCrae, Steixer, Howard and IIaxes. A Study of Diarrhoea in Children. J. H. Mason Knox, Jr., M. D., and

EDWIX H. SCHORER, M. D.

Skin Transplantation. By John Staige Davis, M. D.

Epidemic Cerebrospinal Meningitis and Serum Therapy at The Johns Hopkins Hospital. By Frank J. Sladex, M. D.

VOLUME XVI. 070 pages with 151 figures.

Studies in the Experimental Production of Tuberculosis in the Genitourinary Organs. By George Walker, M. D.

The Effect on Breeding of the Removal of the Prostate Gland or of the Vesicula; Seminales, or of Both ; together with Observations on the Condition of the Testes after such Operations on White Eats. By George Walker, M. D.

Scalping Accidents. By John Staige Davis, M. D.

Obstruction of the Inferior Vena Cava with a Report of Eighteen Cases. By J. Hall Pleasants, M. D.

Physiological and Pharmacological Studies on Cardiac Tonicity in Mammals. By Percival Douglas Cameron, M. D.

VOLUME XVII. 586 pages with 21 plates and 136 flgures.

Free Thrombi and Ball Thrombi in the Heart. By Joseph H. Hewitt, M. D.

Benzol as a Leucotoxin. Bv I..awrence Selling, M. D.

Primary Carcinoma of the Liver. Bv Milton C, Wixterxitz. M. D.

The Statistical Experience Data of The Johns Hopkins Hospital, Baltimore, Md., 1892-1911. By Frederick L. Hoffman. LL. D., F. S. S

The Origin and Development of the Lymphatic System. By Florence K. Sarin, M. D.

The Nuclei Tuberis Laterales and the So-called Ganglion Opticum Basale. By Edward F. Malone, M. D.

Venous Thrombosis During M.vocardial InsufiBciency. By Frank J. Sladen, M. D., and Milton C. Winternitz, M. 1).

Leukspmta of the Fowl : Spontaneous and Experimental. By Harry C. Schmeisser, M. II.

VOLUME XVIII. 445 pages nith 124 figures. Fasciculus I.

A Study of a Toxic Substance of the Pancreas. By E. W. Goodpasture, M.D., and George Clark, M. D.

Old Age in Relation to Cell-overgrowth and Cancer. By E. W. Goodpasture, M. D., and G. B Wislocki, M. D.

The Effect of Removal of the Spleen Upon Metabolism in Dogs ; Preliminary Report. By J. H. King, .M. D.

The Effect of Removal of the Spleen Upon Blood Transfusion. By J. II. King, M. D.. B. M. Bernheim, M. D., and A. T. Jones. M. D

Studies on Parathyroid Tetany. By D. WItiGHT Wilson, M. D., Thornton Stearns, M. D., J. H. Jannev, Jr., M. D., and Madge DeG. Thcrlow, M. D.

Some Observations on the Effect of Feeding Glands of Internal Secretion to Chicks. By M. C. Winternitz, M. D.

Spontaneous and Experimental Leukaemia in the Fowl. By H. C. Schmeisser, M. I).

Studies on the Relation of Fowl Typhoid to Leuksemia of the Fowl. By M, C. Winternitz, M. D., and H. C. Schmeisser, M. D.

Hyaline Degeneration of the Islands of Langerhans in Pancreatic Diabetes. By M. C. Winternitz. M. D,

Generalized Miliary Tuberculosis Resulting from Extension of a Tubercular Pericarditis Into the Right Auricle. By M. C. Winternitz, M. D.

Acute Suppurative Hypophysitis as a Complication of Purulent Sphenoidal Sinusitis. By T. R. BoGGS, M. D.. and M. C. Winternitz, M. D.

A Case of Pulmonary Moniliasis in the United States. By T. R. BoGGS. M. D., and M. C. Pincoffs, M. D.

Gaucher's Disease (A Report of Two Cases in Infancy 1. By J. H. M. Knox, M. D,. H. R. Wahl, M. D., and H. C. Schmeisser; M. D.

A Fatal Case of Multiple Primary Carcinomata By E. D. Plass, M. D.

Congenital Obliteration of the Bile-ducts. By James B. Holmes,' M. D.

Multiple Abscesses of the Brain in Infancy. By James B. Holmes, M. D.

Gastric Carcinoma in a Woman of Twenty-six" Years. By R. G. HnssEY,

Subdiaphragmatic Abscess with Rupture Into the Peritoneal Cavity Fol


lowing Induced Pneumothorax for Pulmonary Haemorrhage. By R. G.

HussEY, M. D. Heart Block Caused bv Gumma of the Septum. Bv E. W. Bridgemax,

M. D., and H. C. Schmeisser, M. D. Analysis of Autopsy Records.

A. The Johns Hopkins Hospital. (Table Showing Percentage ot

Autopsies.)

B. The City Hospitals, Bay View. (Table Showing Percentage of

Autopsies.)

"The Jlonday Conferences."

Clinical Representatives on the Staff of the Department of Pathology.

Donation.

Fasciculus II.

The Role of the Autopsy in the Medicine of To-day. By M. C. Winternitz, M. D.

Experimental Nephropathy in the Dog. Lesions Produced by Injection of B. bro)wlusepticus into the Renal Artery. By M. C. Winternitz, M. D.. and William C. Quixby, M. D.


A Clinical and Pathological Study of Two Cases of Milary Tuberculosis of

the Choroid. By Robert L. Raxdolph, M. D., and H. C. Schmeisser,

M. D. The Blood-vessels of the Heart Valves. By Stanhope Bayne-Jones, M. U. Equilibria in Precipitin Reactions. By Stanhope Bavne-Jones, M. D. Carcinoma of the Pleura with Hypertrophic Osteoarthropathy. Report ot

a Case with a Description of the Histology ot the Bone Lesion. By

Stanhope Bayne-Jones, M. D. The Interrelation of the Surviving Heart and Pancreas of the Dog in

Sugar Metabolism. By Ad.mont II. Clark, M. I). Congenital Atresia of the Esophagus with Tracheo-Esophageal Fistula

Associated with Fused Kidney. A Case Report and A Summary of the

Literature on Congenital Anomalies of the Esophagus. By E. D.

Plass, M. D, Ectopia Cordis, with a Report of a Case in a Fifteen-Month-Old Infant.

By James B. Holmes. M. D. Studies in the Mechanism of Absorption from the Colon. By Samuel

G0LDSCH.MIDT, M. D., and A. B. Dayton, M. D. Report of T\^•o Fatal Cases Following Percy's Low Heat Treatment of

Carcinoma of the Uterus. By V. N. Leonard, M. D. and A. B. Dayton,

M. D. The Relationship in Typhoid Between Splenic Infarcts and Peritonitis

Unassociated with Intestinal Perforation. By A. B. Dayton, M. U. Left Duodenal Hernia. By A. B. Dayton, M. D.

Histological as Related to Physiological and Chemical Differences in Certain Muscles of the Cat. By H. Hays Bullard, M. D. A Method of Clearing Frozen Sections. By H. Hays Bullard, M. D. On the Occurrence and Significance of Fat in the Muscle Fibers of the

Atrioventricular System. By H. Hays Bullard, M. D. Studies on the Metabolism of Cells in vitro. 1. The Toxicity of a-Amino Aeids for Embyonic Chicken Cells. By Montrose T. Borrows, M. D.,

and Clarence A. Ney.mann, M. D. The Significance of the Lunula of the Nail. By Montrose T. Burrows,

M. D. The Oxygen Pressure Necessary for Tissue Activity. By Montrose T.

Burrows, M. D. The Functional Relation of Intercellular Substances in the Body to Certain Structures in the Egg Cell and Unicellular Organisms. By

Montrose T. Burrows, M. D. Studies on the Growth of Cells in vitro. The Cultivation of Bladder and

Prostrate Tumors Outside the Body. By Montrose T. Burrows,

M. D., J. Edward Burns, M. D , and" Yoshio Suzukl. M. D. The Study of a Small Outbreak of Poliomyelitis in an Apartment House,

Occurring in the Course of an Epidemic in a I^arge City. By Montrose

T. Burrows, M. D., and Edwards A. Park. M. D. Papilloma of the Larynx. Report of a Case Treated with Radium with

Resultant Chronic Diffuse Thyroiditis. By William C. Duffy, M. D. Analysis of Autopsy Records. .\utopsy Statistics.

(a) Bay View.

(b) Johns Hopkins Hospital. Report of the Photographic Department. General Improvements.

Donations.

VOLUME XIX. 358 pages -nith 29 plates.

The Structure of the Normal Fibers ot I'urkinje in the Adult Human Heart and Their Pathological Alteration in Syphilitic Myocarditis. By O. Van Der Stricht and T. Wing.kte Todd. M. D.

The Operative Story of Goitre. iTie Author's Operation. By William S. Halsted, M. D.

Study of .Arteriovenous Fistula with an Analysis of 447 Cases. By CcBLE L. Callander, M. D.

VOLUME XX. 314 pages nith 82 plates.

The Pathologv of the Pneumonia in the United States Army Camps During the Winter of 1917-18. By William G. MacCallum, M. D.

Pathological .\natomy of Pfieuiionia Associated with Intluenza. By WiLLiA.M G. MacCallum, M. D.

Lymphosarcoma. Lymphatic Leukaemia. Leucosarcoma. Hodgkln's Disease. Leslie T. Webster, M. D.


CONTENTS

  • Studies in Asymptomatic Neurosyphilis. II. The Classification, Treatment and Prognosis of Early Asymptomatic Neurosyphilis. By Joseph Earle Moore, M.D
  • The Biochemistry of Tuberculosis. By Esmond R. Long, Ph.D.
  • The Dissemination of Bacteria in the Upper Air Passages. III. The Relation of Bacteria to the Mucous Membranes. By Arthur L. Bloomfield.
  • The Changes in the Omentum of the Rabbit During Mild Irritations; with Especial Reference to the Specificity of the Mesothelium. (Illustrated.) By R. S. ClNNINGHAM
  • The Elementary School and the Individual Child. (Abstract.) By Esther Loring Richards 265
  • Notes on New Books 267


STUDIES IN ASYMPTOMATIC NEUROSYPHILIS II. THE CLASSIFICATION, TREATMENT, AND PROGNOSIS OF EARLY ASYMPTOMATIC NEUROSYPHILIS

By Joseph P^arle Moore, M.I).

Baltimore

(From the Syphilis Department of the Medical Clinic,

The Johns Hopkins Hospital)


The work of many observers during recent years has demonstrated that in a large jn-oportion of all patients who have contracted syphilis the central nervous system is invaded bj' the Treponema pallidum at the time of its general dissemination in the firet months following infection. This invasion is easily detected, in most instances, by ewly examination of the cerebrosjjinal fluid. The perqpitage of patients thus found to have

This clinical research has been aided by grants from the United States Interdepartmental Social Hygiene Board and from the American Social Hygiene Association.


early fluid abnormalities approximates the incidence of late clinical neurosyphilis — one fact which makes it probable that when central nervous system invasion does take place, it practically always occurs during the first months of the infection rather than at some later period. If this view])oint be accepted, it is essential to examine the conditions under which neurologic invasion may occur. In addition, this pai)er will discuss the clinical and laboratory methods necessary for its detection, and the response to treatment of these early forms of neurosyphilis.


[No. 377


Leaving out of consideration the occasional precocious appearance of the characteristic late forms of clinical neurosvpliilis such as paresis, tabes, etc., we have classifieil early iieurosy])Iiilis ai)pearing within a year or less from the date of infection under five clinical groups: —

1. Acute si/philitic iiirnviif/itifi, occurring in ])revi()usly untreated patients. It may be recognized as syphilitic by the accompanying signs of a recent early syphilis and by the characteristic cyto-biologA- of the cerebrospinal fluid.

2. Pncorious rdxciiltir iii'innxi/jihili.s, indicated by transitory aphasia, niono])legia, hemiplegia, etc.

o. ycurorcciirrciicr. This type occurs only under definite conditions, i.e.. in a ])atient with early syphilis who has received an amount of treatment insulticient to eradicate the disease, which is then followed by a complete lapse of treatment. The clinical manifestations are varied, but usually consist of a subacute meningitis, commonly with, but sometimes without, focal cranial nerve lesions (most off en paralyses of flie seventJi or eighth cranial nerves i. Occasionally e]>ilei)fiforni seizures occur.

4. Xriirosyi)hHif< iiKiiiiicxtfd hi/ iiiild syiiiptoiiis nr sH(jht iJn/xicdl signs, not of themselves diiif/nostir of central iicnyiiia si/stcm damage. I'atients iu this group may coini)lain of headache, neuralgic pains, insomnia, vertigo, or "nervousness," or may have no symptoms. Those with symptoms may or may not present the minor physical abnormalities which characterize the symjjfom free group, such as slight i)upillarv abnormalities (myosis. mydriasis, anisocoria, irregularity, or sluggish light reaction), and exaggeration, sluggishness, or inequalities of the reflexes. These symptoms and signs are not pathognomonic and may occur in nou syphilitic patients.

5. Asymptomatic neurosyphilis. The patienfs in this


' Zimmerniann, E. L. : Xeurorecurrences, following treatment with Arsphenamin. Arch. Derm, and Syph., V, 723, (June) 1922. This paper deals with the neurorecurrences observed in this clinic, and contains a thorough review of the literature.

Mt is recognized that nothing in this paper can he applied to the early detection of purely vascular neurosyphilis. The lesion in this type of case may be a small perivascular infiltration deeply buried in nervous tissue, and inaccessible to early clinical or laboratory methods of detection.

' Keidel, A., and Moore, J. E.: Comparative Results of Colloidal Mastic and Colloidal Gold Tests. Arch. Neur. and Psych., VI, 163, (Aug.) 1921.

' For five years, spinal puncture has been performed in the outpatient clinic, the patients being instructed to go home to bed for 48 hours. In that period, more than 3000 spinal punctures have been done, with no serious after-results. About half the patients suffer with puncture headache, but this unpleasant reaction is more than counter-balanced by the value of the information gained. Of course, this procedure is not advised it hospital beds are available.

"Weed, L. H., Wegeforth, P., Ayer, J. B., and Felton, L. D.: -A Study of Experimental Meningitis. Monographs Rockefeller Inst, for Med. Research, Xo. 12, March 25, 1920.


group have no complaint, and show uo physical abnormalities. The neurologic invasion is detected only by examination of tiie cerebrospinal fluid.

The first two of these five groups, precocious meningeal and vascular neurosyphilis, are rare, and for the purposes of this paper may be disregarded. The grotip of neurorecurrences has been comprehensively .studied by others.'

Groups -1 and 5 constitute the largest and most important class of patients. Owing to their minor character, the symptoius and signs detailed under the fourth group may be disregarded for the i)urpose of discussion, and these two groups considered together under the common name of early asymptomatic neurosyphilis. It will be shown that the recognition of the.se cases is easily accoml)lished ; that from the ranks of these patients may arise certain of the future outs])oken neurosyphilitics;- that a study of the spinal fluid abnormalities observed permits a division into three sub-groups; and tliat the adjustment of treatment to the type of case will usually bring about a clinical and serologic "cure."

This j)ai)er is based on the study of :!.")l.' i>atienfs witii l)rimary or secondary syphilis from the Syphilis Department of the Johns Hopkins Hospital. In this series are arbitrarily included those jititients whose disea.se on admission was of less than one year's duration. The available data in all cases consist of anamnesis, careful physical and neurologic examinations, accurate details of treatment, and blood and spinal fluid examinations. The last usually includes cell count, globulin test ( Tanily I , AVassermann test with 0.2, 0.4, and 1.0 cc. of fluid, with botli plain alcoholic and cholesterinized antigens, and colloidal gold and mastic curves. In a few cases cell counts or colloidal tests are lacking. Spinal puncture was performed as a routine after one or two courses of arsphenamin (from two to six months after admission),^ though in a few cases it was done much earlier or much later in the cour.se of treatiuent. I'nncture before treatment has been considered unjustifiable, ])artly because of the delay necessary in starting treatment, and partly becau.se of the theoretical danger of transferring organisms from an infected blood stream to a non-infected cerebrospinal axis, either directly through the puncture wound with hemorrhage into the subarachnoid space, or by an alteration of the meningeal periueability.'

TABLE I.

The incidence of early neurosyphilis in primary and secondary

syphilis.


I


Total cases

Acute syphilitic meningitis

Precocious vascular neurosyphilis

Neurorecurrences

Early asymptomatic neurosyphilis

Total early neurosyphilis


Number


Per cent.


352


100


2


0.56


1


0.28


19


5.3


72


20.4


94


26.4


July, 1922]


23a


Niuety-four of the 352 patients developed early iienrosyphilis, and are subdivided as shown in Table I. Of these early neurosyphilitics, 76.6 per cent were asymptomatic. The importance of this sub-group is at once apparent.

In a preliminary communication " a tentative division of early asynii)tomatic neurosyphilis into three groups was outlined, the grouping being based in part on the type of spinal Unid abnormalities (in Kid cases i, and in part on the response of the various types to treatment. Further observation of this much larger series lends support to this tentative classification, and indicates that an ai)preciation of the grouping has a real prognostic value.

The first group of this classification includes those cases in which the spinal fluid abnormalities consist of

TABLE II.

Spinal Fluid findings of Group I, Early Asymptomatic

Neurosyphilis.


Wasserniann


Colloiilal Cold Colloidal Mas<


Zx2


1 8


-t






1222110000


1000000000


Yxll


10


+ +






2211100000


2221000000


Jx20


8


±






j 1122222200


2100000000


Hx8


13 1


±






1100000000


2210000000


Bx2


9


+






1122211000


2210000000


Fx24


8


±






1111100000


2210000000


Fx6


21


4






0011100000


2210000000


E:x12


18


+ +






1222100000


2221100000


20


13


+






1221000000



Gx30


7


+ +






lllllOOOflO


lllOOOOOOO


Px21


^ 1


±






0111100000


2100000000


Nx29


20 1


+





"


1122211000


1000000000


Hx28


8 1


±






oooonooooo


2222100000


6


g 1


±






0011000000



T16


8 1


±






1221000000



Lxl6


15


+






nooooooooo


1111100000


M31


8


-f +


I





1110000000



KxlS


10 1


+


^





1100000000


1122110000


"Keidel, A., and Moore, J. E.: Studies in Asymptomatic Neurosyphilis: — I. A Tentative Classification of Early Asymptomatic Neurosyphilis. Arch. Neur. and Psych., VI, 286, (September) 1921.

' The standard of cell normality has been considered to be from to 5 cells. From 6 to 10 is considered borderline, and such a fluid has not been classed as pathologic unless there was a concomitant increase in the globulin content. More than 10 cells per cu. mm. is definitely abnormal. This standard is based on the work of W. Schonfeld, Ueber Befunde in der RUckenmarksfliissigkeit bei nervengesunden Menschen. Deutsche Ztschr. f. Nervenh., LXIV, 300, 1919.

'No stress is laid on alterations in pressure or on the isolated presence of a small amount of globulin, since both of these are frequently found in normal individuals. Both colloidal curves have been classed as negative if there was no change reading higher than 2.

'Justification for regarding these spinal fluid changes as evidence of neurologic damage is given by O. Fischer, (Die Anatomische Grundlage der Cerebrospinalen Pleozytose. Monatschr. f. Psych, u. Neur., XXVII, 512, 1910).


pleocytosis, usually slight,' a slight increase in glolmlin content, but negative Wassermann reaction and colloidal tests (Table II).* The discussion of treatment of this and other groups will be deferred till later in the jiaper, but it may be stated here that routine anti-.syjihilitic treatment suffices to clear up these alinornialities in practically all instances."

In (iroup II are included those patients whose spinal fluids show a more marked pleocytosis, with cells between 10 and 100, but usually less than 50; the globulin content also is greater than in Group I; the Wassermann reaction may be either positive or negative, but if positive, fixation ustially occurs only with large amounts of fluid ; of the two colloidal tests, either or botji may be

TABLE III.

Spinal Fluid findings in Group II, Early Asymptomatic

Neurosyphilis.


Case


Cells


Clol..


Wasserma


n


Colloidal Gold


MasliiC.lloidal


0.2


0.4


1.0


B4


56


+ -f +


4


4


4


1123311000



C13


98


-1

1


3


4


1234421100



M18


63


-f + +


4


4


4


1123332100



Fxl4


15


+ +






0011110000


3222U0O0O0


Abl4 Ru


10 12


+

+








222+32+110010112110000


1100000000 3211000000


Sy


28


+





4


1211100000


5543220000


Px22


29


±





4


1111000000


2210000000


Gxl


3


+ + +





4


1111110000


3221000000


Axl7


21


-f +






2445522100


2111000000


1x17


14


+ +-f


4


4


4


2222200000


4322200000


Ixl


2


+






1233332000


1000000000


Bx29


21


+





1


1112333000


.5432110000


C15


?


+





4


1234411000



Mx20


6







1234411000


2210000000


121


16


+ +





4




Kx9


14


+



4


4


1111100000


2210000000


Sxl2


?


±





4


1222100000


2210000000


Di


18


+ +






2223222100


5543100000


Dm


16


±






2343100000


2211000000


Fx8


2


+






1211000000


3322100000


B19


19


±


4


4


4


1222100000


5543210000


Zxl4


12


±






12*33100000


2100000000


FX12


2


-f +






1111200000


3222200000


Kx2


24


+





4


0001100000


2210000000


Nx27


5


+






0134211000


2000000000


Cx23


16


+






0123100000


4322100000


FxlO


8


+ +



1


4


3433211000


5543200000


BX15


?


-t

1


4


4


1123110000


3222100000


.1x16


2


+ +






3333210000


3221000000


Px3


7


+






2332110000


3220000000


Px5


3


+ +






1133110000


'.221000000


Dxl5


40


+ + -f





4


1123311000



Dx5 Nx20


4


-f + +

+ +








2233100000 0123322000


2220000000 2210000000


Nx30


2


-f-f +





4


1555555210


0033220000


BblG


4


±






443+3321000


4321000000


positive, the gold being of the syphilitic zone type, and the mastic curve usually to 3, or rarely paretic. Table III shows the detailed findings in 37 cases. It is ajiparent tiiat Group II is the most elastic of the three groups.


234


[Xo. 377


and that certain cases fall into this gronp, instead of into Gronps I or III, only becanse of the resnlts of the colloidal tests. It is for this reason that we do not consider a flnid examination conijplete, unless at least one and preferably both of the colloidal tests can be performed. In this group also a serologic and clinical "cure" '" may be obtained by slight modifications in the usual routine of anti-syphilitic treatment without the addition of intraspinal therapy.

(Jroup III includes those cases in which the spinal fluid ciianges are of a more advanced type. The cell count is high, ranging from 50 to 200. The globulin content is markedly increased ; the Wassermann reaction is positive with small amounts of fluid (0.2 cc. or less) ; and the colloidal gold and mastic curves are both paretic (Table IV). The abnormalities of this group are much more

TABI^E IV.

Spinal Fluid iindings in Group III, Early Asymptomatic

Neurosyphilis.



Cells


1

1 Glob.


Wassermann


Colloidal Gold







Colloidal Maslic




1


0.2


0.4


1.0




11


32


1

+ + +





5554310000



13


91


+ -f + +


i




5555540000



77


58


1 + + + +





5552110000



90


GS


1 + +





5555431000



01


12


+ + +


4




5555321000



X4


64


+ +


4




5555542000



S32


18


+ + +


i




5554210000



S47


28


-)- +


i




5555553100


Gxl8


42


+ + +





5555533200 | 5543210000


Hx5


65


+ + + +


4




2211000000 1 5543210000


Jxl


98


+ + +


4




5555543000 : 5532100000


Qx27


150


-)- + + +





5555442100


5555542100


Du


112


+ + -)


4



5441100000


5532100000


Mo


156


+ + + +


4



5555520000



Ze


131


-f + + -f





5555554100



Br


37


1 + + + +





5555521000



Ho


40


1 + + + +



4



5555421000



resistant to treatment than the preceding two; and practically no imi)rovenient restilts from the routine use of arsphenaniin and the mercurials. Intraspinal therapy as an adjunct to routine treatment offers almost the only chance of a serologic cure.

The relative incidence of these three groups is shown in Table V. Of (>7 patients with primary syphilis, 20.9 per cent had abnormal fluids (!).l jter cent fluid abnor "The word "cure" is here and elsewhere in this paper used to mean that the patient remains clinically well, and that the serology of the blood and spinal fluid becomes negative and remains so for the period of observation. It is not used in the sense of eradication of the last remaining organism. Hereafter, the quotation marks will be dispensed with.

"Particularly G. L. Dreyfus, (Miinch. Med. Woch., LXVII, 1369, 1920) who finds from 70 to 80 per cent of abnormal fluids in untreated secondary syphilis. See also U. J. Wile, and C. K. Hasley: Involvement of Nervous System during Primary Stage of Syphilis, Jour. Am. Med. Assn., 76, 8, (Jan. 1) 1921.


malities in seronegative, 26 per cent in seropositive, primary syphilis). In the 263 patients with .secondarysyphilis, 22.1 per cent of the fluids were abnormal. If argument for the early diagnosis and vigorous treatment of primary syphilis were needed, it would be fur TABLE V.

Incidence of early asymptomatic neurosyphilis in primary

and secondary syphilis.



Total


Normal


Abnormal


Fluids


falling into Croups



I


II


III


Primary


67


53


14 (20.9%)


3


9


2


Secondary


263


205


58 (22.1%)


15


28


15


Total


330


258


72 (21.8%)


18


37


17


nished by these figures ; although it is evident that in some instances neurologic invasion may occur no matter how early treatment is begun.

Under the conditions of this study (examination of the spinal fluid after a considerable amount of treatment) it appears that in (Jroup II we have the most frequent type of abnormal fluid. Of 72 cases of asymp tomatic neurosyphilis, 25 per cent fall into (Iroup 1 ; 51.3 per cent into Group II ; and 23.6 per cent into Group III. From the reports of other workers " on patients with untreated syphilis, it would seem that the minor abnormalities of Group I are much more frequent. Treatment, as we have employed it, may be considered to have accomplislied either one or both of two things: — a rapid disaiqiearance of the (iroup I changes, so that when examined the fluid appears normal; or the o]»posite eft'ect of intensifying the minor abnormalities, so that a patient who might belong to (iroup I before tre^ttment falls into Groups II or III after treatment. This assumption, for which some proof exists, constitutes an additional reason to the two already given for delaying puncture until after the administration of anti-syphilitic treatment.

The most important problems in the present day study of neurosyphilis concern the time and manner of invasion of the central nervous system. The scientific aspect of the latter is overshadowed by the practical importance of the former. If it were possible to be reasonably sure ot the time limits within which neurologic invasion might occur, the prevention of clinical neurosyphilis by means of its early detection would rest on firmer ground. The best available method of approach to this problem is by a study of the incidence of spinal fluid abnormalities at varying intervals after infection. If invasion ot the nervous system occurs within tiie first inonths of the infection, one would expect a gradually increa.sing incidence, reaching a peak during the first or second years, and thereafter remaining at approximately the same


July, 1922]



JOHNS


HOPKINS H


OSF


ITAL BULLETIN



235


The Incidence of Abnormal Spinal Fluids in time of puncture: — a comparative study.


TABLE VI. Primary and Secondary Syphilis, showing the duration of infection in months at the


Duration uf


Fordyce and Rosen



This study


Composite results


syphilis in niunlhs


Normal | Abnormal fiuiJs ; fluids


Per cenl

abnormal


Normal liuiJs


.\bDormal Percent 'I Normal llui<ls abnormal fluiils


Abnormal

fluid:,


Per cent aboormal


0—3


20 2


9.0


15


3 16.6 35


5


12.5


4—6


43 9


17.3


37


7 , 15.9 90


16


15.1


7—9


30 10


25.0


25


10 28.5 55


20


26.6


10—12


29 15


34.1


36


11 1 23.4 65


26


28.5


13—15


19 13


40.6


42


12


22.2


36


13


26.5


16—18


25


12


32.4


19—21


38


15


28.5


S^


5


12.8


24


8


25.0


22—24




48


12


20.0


level. If, on the contrary, this invasion may take place at any time, the curve of incidence should he a constantly increasing one.

In Table VI and Chart 1, we have compared the results obtained by Fordyce and Rosen '- in 248 cases with 237 comparable cases from the present study, and each of these results with a combination of the two series. These patients were all punctured within the first two years of the disease, and tlie cases are divided into three month periods, sliowing the duration of infection at the time of puncture. The conditions as to treatment are similar in the two series of cases: — in each instance puncture was deferred until after some treatment, usually one or two courses. The graphic presentation in the diart shows a rapid rise in the incidence of abnormal spinal fluids, which, according to Fordyce and Rosen, reaches its peak



18 months after infection ; according to the present series this peak is readied by the ninth month. A consolidation of the two series provides an intermediate curve, probably more accurate than eitlier alone because of the larger number of cases, in wliich the flrst peak is reached at the end of 12 months. Thereafter, except for a secondary rise at the eighteenth month, the incidence of abnormalities remains at about the same level. This is further shown in Table VII, in which our own cases are arranged according to the duration of the disease iu years at the time of spinal puncture. If the probable statistical error is taken into account, there is little or no variation


Incidence of spinal fluid abnormalities, arranged according to the duration of infection in years.


Year


Total fluids


Normal


Abnormal


Per cent abnormal


Fluids falling into Croups



I 11 III


1


149


99


35


23.3


1

11 1 17 1 7


2


95


69


19


20.0


4 1 9 1 6


3


27


20


7


25.9


1 1 5 1 1


4


28


20


8


28.5


2


2 4


5


19


16


3


15.7


1 1 — 1 2


6


9


8


1


11.1


- i 1 1


"Fordyce, J. A. and Rosen, I.: Laboratory Findings in Early

I . :riL.;f itj I ^^^ ^^'^ Syphilis:— a Review of One Thousand and Sixty-Four Qi i lT ii FI . I Cases. Jour. Am. Med. Assn.


7, 1696, (Nov. 26) 1921.


236


[No. 377


in the (lirt'ereiit years. '" These results are somewhat complicated by the amount of treatment administered, as in general those patients whose disease at the time of puncture was more than two years old had received more treatment than the remainder. This factor may account for the slightly lower percentages in the later duration groups.

Kecent e.\i)erimental work on syphilis has provided information regarding the cour.se of the infection on which an interpretation of these results may be based. Within a few days after infection Treponema pallidum may be recovered from the lymph glands '* and from the heart's blood of experimental animals.'^* In human syphilis the blood is infectious during the primary and secondary stages, though organisms are probably [)resent only in small numbers. When the period of latency has been reached, it is non-infectious except in rare instances.^" From the lJ^uph glandjs, on the contrary, virulent orgauisms may be recovered at any time during the course of the disease. This has been interpreted to mean that the lymidi nodes function as re.servoirs for the organisms.

It is probable that at the time of infection, dissemination of organisms takes place from the lymphatic system to the peripheral blood stream. The blood is not an ideal medium for their growth or continued residence, and as soon as possible, they leave it for more favorable surroundings. After a second period of incubation in their new locations, the tissues react with the secondary outbreak. At this time, it is probable that an immune reaction takes place,- " so that with the spontaneous regression of secondary lesions, the vast majority of the organisms are destroyed by the natural defenses of the body. This reaction may permit the destruction of any further organisms which may be discharged from the lymphatic system to the blood; or the actual discharge may perhaps be prevented by the local tissue reaction in the lymph glands, walling off the organisms and obstructing their egress. When this has been accomplished, the appearance of lesions of the secondaiy type ceases and latency ensues. The length of time necessary to bring about this result probably varies from three to twelve months.

If, during the first months of the disease, organisms are more or less constantly present in the blood, it is difficult to understand how invasion of the nervous system fails to occur. Evidence is gradually accumulating to show that such an invasion does take place in the majority of all cases,'" probably by vascular involvement of the meninges, parenchyma, or both. Whether or not clinical neurosyphilis will develop must partially depend on the extent to which the individual patient reacts against his infection.^" That most patients do succeed in dealing s])ontaneously with this neurologic invasion is evidenced by the comparatively snuill number of clinical neurosyphilitics.


The presence of spinal fluid abnormalities may depend on the length of time the organisms have been resident in nervous tissue. If puncture is performed at about the time that invasion occurs, the fluid may be normal, whereas if it had been delayed a few weeks or months, during which the nervous tissues had time to react, definite abnonnalities might be present. For example, it is shown bj' the study of untreated sero-negative primary,


"Moore, J. E. : The Cerebiospinal fluid in Treated Syphilis. Jour. Am. Med. Assn., 76, 769, (March 19) 1921. A study of 451 cases of late (tertiary and latent) syphilis without neurologic abnormalities showed no higher incidence of abnormal spinal fluids than in early syphilis. The conclusion was drawn that "after the disease is presumably well intrenched, about the same relative number of patients show spinal fluid abnormalities, no matter how long the disease has existed, or by what lesions it manifests itself."

" Pearce, L. and Brown. W. H. : A Study of the Relation or Treponema pallidum to Lymphoid Tissues in Experimental Syphilis. J. Exp. Med., XXXV, 39, (Jan.) 1922.

""• Brown, W. H. and Pearce, L. : A Note on the Dissemination of SpirochEeta pallida from the Primary Focus of Infection, Arch. Derm, and Syph., II. 470, (Oct.) 1920. Also Eberson, F.: Dissemination of SpirochKta pallida in Experimental Syphilis, Arch. Derm, and Syph., Ill, 111, (Feb.) 1921.

'"Engman. M. F. and Eberson, F. : A Biologic Study of batency in Syphilis. Arch. Derm, and Syph., Ill, 347, (April) 1921. This article contains a review of the literature on the infectivity ot body fluids.

"The question of immunity in syphilis is still sub judice, but enough clinical and laboratory evidence exists to make certain the presence of a defensive reaction to infection. The nature ol this reaction is as yet unknown. For a discussion of this point, see the article by Engman and Eberson, (footnote 16).

"Brown W. H. and Pearce, L.: The Resistance (or Immunity) Developed by the Reaction to Syphilitic Infection. Arch. Derm, and Syph., II, 675, (Dec.) 1920. Experimental Production of Clinical Types of Syphilis in the Rabbit. Arch. Derm, and Syph., Ill, 254, (March) 1921.

" This statement is based on the high percentage of abnormal spinal fluids found by various workers in untreated early syphilis, and on the demonstration of treponemes in the spinal fluid of early syphilitics with otherwise normal fluids. This has been accomplished by Marlnesco and Minea (Acad, de Sc, Sem. Med., 1914, p. 357), Steiner (Neurol. Centralb., 1914, XXXIIl. 132), and others. Brown and Pearce have demonstrated organisms in the spinal fluid of recently infected rabbits.

A. Steiner. (Moderne Syphilisforschung und Neuropathologie. Arch. f. Psych., 1913, L.II, 1), found pathologic alterations attributable to syphilis in the nervous systems of 16 of 31 rabbits. Six of 8 animals with generalized syphilis showed central nervous system involvement.

^' It has been suggested, on the theory of immune reactions which may vary in different body tissues, "that all tissues are not equally protected by the general reaction which occurs during the early stages of a syphilitic infection, and that certain tissues which fail to receive this protection, although less susceptible to injury or infection than other tissues, may be capahle of only a slight degree of self-protection. This is undoubtedly the case in the experimental animal, and if similar conditions obtain in man. such conditions as neurosyphilis might be explainable on this basis." Brown, W. H. and Pearce, L.: Arch. Derm, and Syph., March, 1921, III, 254.


July, 1922]


237


seropositive primary, aud secondary syphilis, that the incidence of liuid abnormalities rises with a ratio for these examples of about one to three to four, in the order nanied.^' This factor must also be considered in connection with the grouping outliued in this paper. A patient cla.ssed in Cfroup I, if punctured three months after infection, miglit fall into (Jroups II or HI if the fluid were studied six months later. The optimum time for the detection of the largest number of cases with abnormal fluids, provided no treatment were administered, would doubtless be from the twelfth to the eighteenth month after infection (see Chart I). The influence of anti-syphilitic treatment, liowever, makes this delay \UHiecessary. If treatment is regular and adequate, tlie abnormalities already present (except for Group III I will rapidly disappear, and opportunity for further neural invasion will be prevented. On the other Land, if a small amount of treatment is followed by a la])se, there is, as will be shown, an increased liability to neuro.syj)liilis. Tuncture should be employed as a routine measure, therefore, in all patients with early sypliilis at the end of the rti-«t or second course of regular treatment. If the fluid is completely negative at this time, i)uneture need not be repeated until the completion of treatment and observation. Should a lapse in treatment occur, however, the case should be managed as if it were a fresh infection, and ])uncture be repeated again following the resumption of treatment. - TABLE VIII. Arrangement of cases by race and sex.




Toldl fluids


Normal


Abnormal


Fluids falling into


Groups



I


II


III



Male


72


61


11

(15.1%)


4


5


2


Colored


1 1 Female

Total


51


43


g

(15.C%)


4


4




123


104


19 (15.4%)


g (6.5%.


9 (7.3%)


2 (l.g%.)



Male


145


109


36

(24.8%)


5


21


10


White


Female


62


45


17 (27.4%)


5


7


5



Total


207 217


154

170


53 (25.6%

47 (21.6%)


10 (4.8%


2g (13.5%)


15 (7.2%)


Males both races



9 (4.1%)


26 (11.9%)


12 (5.5%)


Females both races



113


gg


25 (22.1%)


9 (7.9%)


11 (9.7%)


5

(4.4%)


It is now necessary to consider other factors which may influence invasion of the central nervous system. In Table \'1II the available data are rearranged by race and sex. There is evident a wide ditt'erence between the colored and tlie white races in their susceptibility to early neuro.sypliilis, the incidence being lo.-t per cent as compare<l witli '2'iA) per cent. Cases of the Group I type are about equally frequent in the two races, but Group II is almost twice as common in whites and f J roup III four times as common. In other words, the negro is not only less prone to neural invasion than the white, but when his central nervous system is invaded, his defensive reactions enable him to deal with the infection so that, judging from the spinal fluid changes, his neurosyphilis is consistently milder than is that of the white. This observation is in accord with statistical data regarding the comparative Incidence of paresis and tabes, which are rare in the colored race.-^

No dift'erence is ai)parent between the sexes, of either or both races. Early neurosyphilis is as common iu women as in men, and the incidence of the various groups is approximately the same. This is a surprising result, in view of the fact that in 7(J3 unselected cases of clinical neurosyphilis, there were four times as nianj' men as women (jiaresis Ave times, tabes six times, and cerebrospinal syphilis about tliree times as frequent). The

TABLE IX.

Showing the incidence of spinal fluid abnormalities according to the age at the time of infection.


Age group


Total cases


Negative


.Uinornial


Per cent Abnormal


0—15


4


4




16—20


67


55


12


17.g


21—25


105


go


25


17.9


26—30


77


00


17


22.0


31—35


48


35


13


27.0


36—40


lOi


9


1


10.0


41—65


19


15


4


21.0


0—30


253


199


54


21.3


31—65


77


59


18


23.3


='With, C: Studies on the Different Reactions in the Cerebrospinal fluid in Cases of Syphilis. Brain, 191S, XL, 403. "Wile. U. J. and Marshall, C. H.: A Study of the Spinal Fluid in One Thousand Eight Hundred and Sixty-Nine Cases of Syphilis in all Stages. Arch. Derm, and Syph., March, 1921, III, 272.

-Moore, J. E.: The Genesis of Neurosyphilis. Arch. Derm, and Syph., July, 1921, IV, 55,

-' Zimmermann, E, L. : A Comparative Study of Syphilis in Whites and in Negroes. Arch. Derm, and Syph., July, 1921, IV, 75.


238


[No. 377


explanation is not apparent, though the iuttueuce of preynancy on the disease-* may phiy some role.

Table IX shows the incidence of spinal tlnid abnormalities of all groups arranged by age at the time of infection. There seems to be no special liability for early neurosyphilis to occur in any particular age group, thus disposing of the often expres.sed theory that if syphilis is acquired in old age the central nervous system is more liable to invasion than early in life. The variations in age groups in this series are well within the limits of statistical error.

In our prelimiuaiy paper*' it was stated that the incidence of early neui'osyphilis was the strongest argument in favor of the adefjuate treatment of all syphilis, and wo presented a series of 151 cases in which the inter-relation of spinal fluid abnormalities and the amount of treatment given prior to the examination of the fluid was studied. The further evidence provided by the present much larger series corroborates our early opinion. These data are showni in Table X and Chart II. Unless treatment is continuous over a comparatively long period of

TABLE X. Spinal fluid abnormalities in primary and secondary syphilis as influenced by treatment.



.Si ^3


1 z


a.


l£i.


Fluids falliDij into Groups


Amount of treatment


I


II


III


None or very little


57


40


17


29.8


6 (10.5%)


6

(10.5%)


5

(8.7%)


Six doses arsphenamin

mercury


115


87


28


24.3


8 (6.9%)


16 (13.9%)


4 (3.4%)


Twelve doses

arsphenamin

plus

mercury


70


52


\8


25.7


3

(4.2%)


12

(17.1%)


3

(4.2%)


Eighteen

doses arsphenamin plus mercury


38


33


5


13.1


1 j 2 (2.6%)' (5.2%)

1


2

(5.2%)


Twenty-four or more doses arsphenamin plus mercury


50


46


4


8.0



1 (2.0%)


3

(6.0%)


time, no marked effect is apparent. One or two courses of arsphenamin, with or without mercury, accomplish little. When three courses are given, and particularly if treatment is regular, a decided drop in the incidence of abnormalities occurs ; and after four or more courses the





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abiiornial percentage is very low. Reference to Chart II shows that the number of cases of the (Jroup 1 type is rajiidly decreased by treatment, the fall in jicrcentage being from 10.5 when no treatment or very little is given, to zero when f<mr or more courses are administered. A curious change is seen in the curve for the Group II type. Instead of an early and continuous drop in the incidence of this group, there is a sustained rise from 10.5 per cent, after no treatment or very little, to 13.!) per cent after one course, and to 17.1 per cent after two courses of treatment. At this point the incidence begins to decrease; after three cour.ses it is 5.2 per cent, and after four, 2 per cent. The interpretation of this phenomenon is difficult; but it is probably closely related to the question of the influence of a lapse in treatment in causing tlie appearance of precocious neurosyphilis. Two probable deductions nmy be made from this curve: — (1) a number of patients, who, if punctured before treatment, would have shown normal or (iroup I fluids, later fall into Gi'oup II because of the influence of insufficient treatment followed by a lai)se. In other words, they represent asymptomatic neurorecurrences, and fail to develop sj-mptoms only because the lesion is in a "silent area," not involving cranial nerves or other ini])ortaiit structures;-^ (2| these ca.ses plus the ones originally in


"The mild course of syphilis in pregnant women Is a well established clinical fact. Experimental evidence of the ability of pregnancy to suppress syphilitic manifestations has been furnished by Brown and Pearce: — On the Reaction of Pregnant and Lactating Females to Inoculation with Treponema pallidum — a Preliminary Note. Am. J. Syph., Oct., 1926, IV.

^Gennerich, W.: Die Syphilis des Zentralnervensystems Berlin, Julius Springer, 1921.


July, 1922]


239


Gronp II are rapidly cleared up serologically after further adequate treatment.

Cases of the Group III type are apparently uninfluenced by treatment, no matter how far ])r()longeil. Their incidence is almost as high following four or more courses of arsphenaniin plus mercury as when no treatment at all is given. It is worthy of note, that, as tlie period of treatment is prolonged, the curve representing that of all abnornu\lities begins to ai)]>r()acii that of the (irou]i 111 type; so tiiat i>resumably, if treatment were continued long enough, Groups I and II would disappear, and the Grouj) 111 curve would replace that for all abuormalities, continuing in ai)proxinujtely a straigiit line. When the treatment of these three abnornuil groups is discussed in detail, evidence supjiorting tJiese inter|iretations will be provided.

It is of interest to examine the influence of a lai)se in treatment on the inci<lence of these various abnormal groups."" The data on this point are shown in Table XI. Under the heading of "regular" treatment are classed all patients in whom spinal puncture was performed either before any treatment whatever was given, or immediately following the administration of treatment which was carried out in a regular manner. Under the heading "lapsed" are included all patients who, after taking from one to twelve doses of arsi)henamin either with or without mercury, voluntarily discontinued treatment and were punctured some weeks, months, or years later. The incidence in the latter class of patients is three times as high as in the former. Still more significant is an analysis of the types of abnonnalities found. From this angle the table shows that under the condi TABLE XI. The Effect of a Lapse early in treatment in causing the development of spinal fluid abnormalities.



ToUl


Negative


Posiliie


Per cent positive


FluiJ


falling into Groups



I


II


III


Regular


206


181


25


12.1


6 (2.9%^


17 (8.2%)


2 (0.9%)


Lapsed


124


77


47


37.9


12 (9.6% >


20

(IG.1%)


15

(12.0%)


tlons of lapsing treatment, as compared with regular treatment, the |)ercentage of Group I cases is three times as high, of Grouj) 11 twice as high, and of Grouj) III twelve times as high. It is demonstrated (Table X and Chart II) that regular treatment decreases the incidence of two of these group types. Irregular treatment, on the contrary, not only increases their incidence very markedly, even to a higher jjercentage than that observed when no treatment at all has been given, but what is much more ])ortentous of gi-avity in the situation, this faulty management often intensifies the character of the


spinal fluid changes. The explanation of this phenomenon is identical with that advanced to account for the mechanism of neui'orecurrences.-"

The existence of a neurotropic strain of organism as a possible etiologic factor was also investigated.-'* In this series of 72 cases of a.synnitomatic neuro.syphilis it was possible to examine the marital partner by the standards laid down in our first i)aper (history, ]ihysical and neurologic examination, blood Was.sennann and a study of the

TABLE XII.

The Incidence of Conjugal Neurosyphilis in the Partners of Asymptomatic Neurosyphilitics.


Sex of


Partner

syphilitic


20

C 13 M IS Px 22 Gx 1 Fx S Jx 16 D 15 11 Hx 5



M



M



M



F



F



F



F



F


III


M


III


M


Type of neurosyphilis


Yes No

Yes No

Yes Yes

No No

Yes No

Yes Yes

No No

Yes No

Yes I No

General Paresis: durYes Yes ation of infection 12 I years.


Cerebrospinal syphilis; duration of infection 3 years.


Asymptomatic neurosyphilis; duration of infection 10 months.


=° Praser, A. R. and Duncan, A. B. G. : A Possible Explanation of the Increased Incidence and Early Onset of Neurosyphilis. Brit. J. Derm, and Syph., 1921, XXXIII, July, 251; Aug. -Sept., 281.

=' This reaction is not, as has been assumed by Klauder (Early Neurosyphilis Asymptomatica, Am. J. Syph., 1919. Ill, 559), a Herxheimer reaction, as we understand the limitations of this term. The Jarisch-Herxheimer reaction follows only the first injection of a series of arsphenaniin, occurs within a few hours after the injection, and consists of an acute tissue reaction in a syphilitic lesion due to the rapid destruction of the spirochsetes which it contains. The type of response referred to above, on the contrary, occurs in the central nervous system or elsewhere some weeks after the cessation of treatment, the interval being occupied by the multiplication of organisms in the focus and the reaction of the tissues to their presence. The length of time necessary to produce this result corresponds closely with the incubation period of a lesion at the point of first inoculation.

-'Moore, J. E. and Keidel, A.: Studies in Familial Neurosyphilis: — I. Conjugal Neurosyphilis. .lour. Am. Med. Assn., July, 1921, 77, 1.


240


[No. 377


cerebrospinal fluid | in ten cases, witli positive results in only three. A summary of these data appears in Table XII. Eight of tlie marital partners of the patients in this series had sypliilis; but only thi-ee were neurosyphilitic ; The.se were partners of two (iroup II patients and one Group III patient, respectively. While it is of course recognized that this series is too small to ]iermit any definite conclusions, there is no suggestion that strain of organism is here an important factor.

That many i)atients with clinical neurosyphilis give a history of very slight or no general manifestations of secondary or tertiary .syphilis is a fact beyond dispute.-" This has led to the conclusion that the greater the early tissue reaction, the smaller the liability to late grave manifestations. In other words, those patients who react to a recent infection with a violent secondary outbreak are assumed thereby to develop a defense, ])robably of the type of tissue immunity, against later lesions. So far as neurosyi)hilis is concerned, its lower incidence in the colored as compared to the white race lends some support to this view. It has been frequently shown that secondary .syphilis in negroes is more e.xtensive and more severe than in whites. In order to discover if this factor played any role in the production of early asymptomatic neurosyphilis, the early lesions of the 72 patients in this series have been studied. It is obviously impossible to decide upon any strict basis of comi)arison for the extent or severity of early secondary syphilis because of the individual equation of the examiner. We have therefore subilivided the types of early lesions seen into three groups : — mild, average, and severe. Primary syphilis has been considered as mild if there was a single diminutive chancre, or if the proper period for the appearance of secondary

TABLE XIII.

Character of Early Cutaneous Lesions of Syphilis In Early Asymptomatic Neurosyphilis.


Group


Total cases ..


Character of eariy cuUn^ous lesions


Mild ' .Average '• Severe


I


18


5 11


2






Unselected cases of early syphilis


27 (37.5%)


23

(13.2%)


35 (48.6%)


10

(i3.8%)


123 (70.6%)


I 28 (16.0%)


lesions was much overdue, and as severe, if there was an extensive single or multiple lesion. Mild secondary syphilis is that manifested by a sparse rash of a macular or papular type, or by a few nuicous lesions only; those cases classified as severe were all specifically noted to have very profuse rashes. All other cases are classed as average. On this basis. Table XIII shows that in the 7-' early neurosyphilitic patients, mild early nianife.stations were three times as fre([uent as in 174 unselected cases of early syphilis, and that this increase takes place at the expense of the average group. It is noteworthy that 13. S per cent of these neurosyphilitic patients, however, had extensive secondary outbreaks. Although, therefore, failure to react vigorously to a recent infection seem.s to ])redis](ose to neurosyphilis, a violent secondaiy outbreak does not necessarily protect the patient against it. There is notliing in this fact to strengthen the argument of the advocates of a neurotropic strain of organism."

As brought out by this and similar studies " tliere arc certain clinical aids to the recognition of this type of early neurosyphilis. These are various subjective and objective symptoms, and the behavior of the blood Was.sermann reaction. The subjective symptoms consist of headache, which may be quite mild, but is often severe enough to cause the patient sleei)less nights; insomnia without headache; a complaint of vague neuralgic pains over the whole body, which resemble the usual prodroniata of various acute infections, especially influenza; giddiness, never severe enough to cause staggering gait or falling, and always, in those cases which we have had examined, without objective lesions in the vestibtilar branch of the eighth nerve; and nervousness. In Table XIV it is shown that about ^iO per cent of those patients with abnormal fluids com])lained of one or more of these .symptoms; while similar complaints were found in only 9.7 per cent of those witii negative fluids. It is notable


^Fournier, A.: I Paralysie Generate et Syphilis. Paris. Masson at Cie., 1905) was able to follow personally the evolution of the disease from chancre to general paresis in S3 cases. Of these, 70 had only a fleeting roseola or mucous patches or alopecia; 8 had moderate secondary lesions; and 3 had tertiary lesions. Two patients showed no specific manifestations except the chancre. On the other hand, of 243 cases of severe secondary syphilis followed for many years, not one showed symptoms of general paresis or tabes.

^"Indeed. Fournier found ( Traite de la Syphilis. Paris, J. Rueff, 1906, vol. II, part I. p. 7) the same phenomenon for tertiary lesions of any type. The figures are as follows: of 2188 cases of all types of tertiarism, 90.9 per cent were preceded by mild, very mild, or no se ondary symptoms; 7.2 per cent by a moderate, and 1.8 per cent by a severe secondary outbreak.

"Moore, J. K.: The Cerebrospinal Fluid in Treated Syphilis. Jour. Am. Med. Assn., 76, 769 (March 19) 1921. Fordyce, J. A.: Importance of Recognizing and Treating Neurosyphilis in the Early Period of the Infection. Am. J. Med. Sc, CLXI, 313. (March) 1921. Schou, H. ,T.: Nervelidesler ved tidling Syfilis og deres Behandling. Hosp.— Tid., LXIII, 57, 1920; abstr. in Med. .\bstr. and Rev., IV, 61, (April) 1921.


July, 1922]


241


TABLE XIV.

The Incidence of Neurologic Symptoms and Minor Neurologic Signs in Early Syphilis.



Subjective signs


Objective signs


C. S. F.


Absent


Per cent

Present in which

present


Ah sent


Present


Per cent in which present


Negative


233


25 9.7


235


23 8.9


Abnormal


50


22 4


30.5 22.2


54 12


18 25.0


Group I


14


6 33.3


Group 11


28


9


24.3


30


7 ' 17.9


Group III


8


9 52.9


12


5 29.3


that more than lialf of tlie Ciioiip III cases had one or more of these eomphiints, nsnally headache.

The objective signs are, in our experience, almost W'holly confined to minor pupillary and reflex changes. Careful sensory examinations, comparable to those carried out by some English observers,^- have not been made. The pupillary changes are all mild in degree, and consist of sliglit myosis or mydriasis, inequalities, irregularities, or some incompleteness or sluggishnes.s of the direct or consensual light reaction.^^ Occasionally these phenomena have been noted to develop in this class of patients while under observation. The reflex changes are usually confined to the deep reflexes, most commonly the knee and ankle jerks, and consist only of some exaggeration or sluggishness, or more rarely of inequality. Twenty-five per cent of patients with abnormal fluids showed one or more of these neurologic changes ; while 8.9 per cent of those with negative fluids showed similar manifestations.

It is obvious that both tiiese subjective and objective signs may, and frequently do, occur in normal non-syphilitic individuals, as well as in syphilitics in whom no other neurologic damage can be demonstrated. Their presence, therefore, is no more than presumptive evidence of the existence of neurosyphilis. But any or all of them, occurring in a syphilitic i)atient, shoulil direct the suspicions of the examiner towai-d this jiossibility.

"Head, H. and Fearnsides, E. G.: The Clinical Aspects of Syphilis of the Central Nervous System in the Light of the Wassermann Reaction and Treatment with Neosalvarsan. Brain, XXXVII. 1, (Sept.) 1914. This is a splendid paper which, in this country, has not received the attention it deserves.

"Nonne, M. : Uber die Bedeutung der Liquoruntersuchung fiir die Prognose von isolierten syphilogenen Pupillenstorungen. Deut. Ztschr. f. Nervenh., LI, 155, 1914.

"Stokes. J. H. and Busman, G. J.: A Clinical Study of Wassermannfast Syphilis. Am. J. Med. Sc, CLX, 658, (Nov.) 1920.


We have frequently found, as have others,^** that a persistently positive blood Wassermann reaction could be explained on the basis of neurosyphilis, and have therefore arranged this material to discover if possible the inter relation of the two factors. On the basis of about 1000 ca.ses of early syphilis, we anticipate an early reversal of the blood Wassermann reliction. As a matter of fact, a permanently negative blood Wassernuuiu occurs by the sixth weekly dose of arspheuamin in the majority of all such ca.ses. If the blood Wassermann is still positive after a second course of arsi»henamin i>lus interim mercury, we begin a careful search for the cause, paj-ing particular attention to the osseous, cardiovascular, and central nervous systems. Reference to Table X\' shows that after one course of arspheuamin without mercury,

TABLE XV. Spinal Fluid Abnormalities in Primary and Secondary Syphilis as compared with Blood Wassermann Reaction and Treatment.



1


2 E


fa _


"' 1 d 1


§ 1 1 1

Fluids falling into Croups


S i


I


II


Ill



Positive


32


21


11


34.3


5


4


2


o

si g a « i


Negative


101


77


24


23.7


7


15


2


<


Total


133


98


35


26.3





s

t->


Positive


17


11


6


35.2


1


2


3


o

1 g

5 -H


Negative


141


119


22


15.G


3


12


7


s °

<


Total


158


130


28


17.7





the blood Wassermann remained positive in 21.4 per cent of all cases with negative spinal fluids, while in the groyp of those witii abnormal fluids it was positive in 31.4 per cent. (Naturally, all patients punctured before treatment are excluded from this table.) Expressed somewhat diflerently, of those patients with a persistently positive blood Wassermann reaction, 34.3 per cent had abnormal fluids, as compared with 23.7 per cent when the blood test had become negative.

After two or more courses of arsphenaniin plus mercury, the figures are more impressive. Of 17 cases with a persistently positive blood reaction, 35.2 per cent showed


242


[No. 377


abnormal spinal fluids, while only 15.6 per cent of the negative blood cases were abnormal in this respect, lu other words, 21.4 per cent of the early neurosyphilitics had a blood AVassermann difficult to reduce, whereas this phenomenon was present in only 8.1 j)er cent of the cases with a. negative .spinal fluid. These statistics demonstrate that to the list of subjective and objective signs already detailed must be added the factor of a stubborn blood Wasserniann reaction ; and that this, when present, should at once arouse the suspicion of neurologic invasion.

The Trratmrnt of Eiirhf Asi/nipfoniotic Xciirosyphilis. — When the classification of these cases was discussed, it was stated that the grouping was determined as much by the results of treatment of the various groups as by the type of spinal fluid abnormalities. The Group I type, with only minor abnormalities in the fluid, usually yields readily to routine anti-syphilitic treatment, provided thi.s is carried out for a sufficiently long jieriod of time. Unfortunately, too few cases of the present series have been consecutively treated to furnish convincing statistical proof of this point. This statement is, therefore, based on our experience with patients who miss falling into the classification under discussion by virtue of the longer duration of their disease, on the rapidly falling curve of incidence of Group I cases after adequate treatment (see Chart III, and on the evidence of other workers. Not only do these minor abnonnalities disappear, but the patients remain clinically and serologically well.

Thirteen patients in our (Jroup II have been under treatment foi- a sufficient length of time to allow discus


sion of the results. The es.sential data are summed up in tabular form (Table X\'I i in order to economize space. Of the.se cases, one (I'x 22) has disapjieared from observation ; the renmining twelve are well after periods of observation ranging from 8 to '.id months after the first discovery of spinal fluid abnormalities. Eight patients have always been, and have remained, symptom-free. In all five instances in which various subjective symptoms were comitlained of, these rapidly disai)peare(l. Six patients were found to have the minor neurologic changes described, and in five these objective signs have remained unaltered; while in one case (Is 17), marked exaggeration of all deep reflexes has disaj)]ieared, leaving normal reflexes at ju-e.sent. If, as is jirobable, these slight abnormalities are evidence of beginning anatomical damage, it is not surprising that treatment usually failed to effect their disapi)earance.

From the standpoint of the blood Wassermann, all patients have been rendered sero-negative and have remained .so. The cyto-biology of the cerebrospinal fluid is shown in the table. In most instances there were several examinations of the fluid nnule, only the last of which is shown. A negative result was often obtained much earlier than a[)pe^irs in the chart. In ten of tue thirteen cases, a serologic cure '" has been obtained. In one instance (B 19) the cyto-biology of the fluid was markedly improved after l.j mouths' treatment, while in two cases (I'x 22 and Kx !) ) it was somewfuit worse after smaller amounts of treatment. In each of these two cases the treatment given was very scant and was especially deficient in mercury.

The treatment given to all jjaticnts in this gronj), with XVI.


TABLE The Results of Treatment of Early Asymptomatic Neurosyphilis — Group II.



Duration ol disease onad

Treatment

before first spinal

puncture.


Lapse


Interval admis spinal punC'


First C. S. F. results


Treatment after first puncture


Last C. S. F. results


Time elapsed


Clinical


Serologic



Cose No.


^

Glob.


Wa. R.


Gold


Mastic


Arsphe

Mercury


=


Glob.


Wa. R.


Gold


Mastic


first result punc- ,


result \ Remarks



^ 1


0.2 0.4[1.0


"1


0.2 j 0.4 1 1.0


1


B4 8 Mos.


5x0.6 .Neo+Hg.


6 Mos.


7 Mos. 56 1 +++


4 1 4


4 1123311000



30x0.4


16 Weeks 16~n>eks


'1 +


1 1 1 1111100000


2210O00000[19 Mos.


Well


Cure




6x0.3



1.5 Mos.| 08| +


1 3


4 1 1234421100



10x0.3


8{


1 j 1 1121100000


— 1 22 Mos.


Well


Cure Cure



M 18 U Mos.


12x0.3 & 20 wks. Hg.



7 Mos.


63 1 -f-f-1

4 4


M


32x0.3


16 Weeks


'


±


1


1 1111000000


2111000000:22 Mos.


Well








++


[0011110000


3222000000


18x0.4


18 Weeks


2


+



1 1112111000


2100000000 12 Mos.


Well


Cure 4 1 1111000000


2210000000


12x0.4


4 Weeks


29


+


2 4


4 1 1122200000 2211100000


1000000000] 4 Mos.



Worse



G«l


4 Mos.


12x0.»-)-6 wks. Hg.



4 Mos.


3


-t-l-f

4 |1111110000


3221000000


22x0.4


8 Weeks


+


1


2220000000


11 Mos.


Well


Cure



Aj17


7 Wks.


24x0.4-1-22 wks. Hg.



11 Mos.


21


+-i



2445522100


2111000000


14x0.6 (Neol


16 Weeks


4


++ 1

1


0| 1121000000

1


2222100000


14 Mos.


Well


Cure



U17


7 Mos.


None




14


-l~F-|

4


4


2222200000


4322200000


24x0.4


34 Weeks


3i ±

1 1






0222110000


100000000

12 Mos.


Well


Cure


Markedly hjfper K.K.

have disap. peared


Kji9


4 Wks.


6x0.4



1.5 Mos. I 14


-1



4


4


1111100000


2210000000


10x0.4


4 Weeks


10


-f


4 4|4


1111100000


2210000000 4 Mos. 1 Well


Worse





elsewhere





-h-l






2223222100


5543100000


12x0.4


6 Weeks


6


-f

1111000000


2100000000 14 Mos.


Well


Cure 4 4


1222100000


5543210000


22x0.3


20 Weeks


3


± 1 1 2 1 1111000000


2100000000 15 Mos. 'Well


Improved








-H-t



"


2233100000


2220000000


6x0.4


16 Weeks


3



1 1222100000


2100000000 13 Mos. 1 Well


Cure





3x0.4 7x0.4


6 Mos.



+


2

1


4


1122000000



U.S. 5x0.4 U.S.


42 Weeks


1


+



1111100000


— 30 Mos. I Well 1


Cure



July, 1922]


243


oue exception, consisted of alteiiiatiug courses of arsjilieiianiiu aud mercury (by inunction), together witli large doses of potassium iodide by mouth. Tlie average course of arspheuamin consisted of from 8 to 12 weekly injections instead of the usual six; the interim between courses was from 1 to 4 months, and was occupied by the constant administration of mercury. In only one or two instances were the two drugs used together. Intraspinal treatment was employed in only one case (D 15).

From a study of these results, it is ajiparent that patients in this group may be maintained clinically well over long periods of time, and that in the majority of instances the blood and spinal tiuid serologj- may be reduced to normal and maintained there by the u.se of arspheuamin and mercury by the usual routes of administration, without the use of intraspinal therapy. We have .so far not encountered a single jiatient in this group with whom such a result could not be obtained, provided treatment were sufficiently ])rolonge(l. It is, of course, probable that more resistant cases will be met with ; in that event, if a six months' trial of routine treatment brought about no improvement, we should at once resort to intraspinal treatment as an atljnnct.

The cases in (rroup III present an entirely different picture. The results in 8 cases of this group are presented in Table XVII. Two patients, both of whom were completely asymptomatic at the time of the first spinal fluid examination, have developed clinical neurosyphilis since that date, and in spite of energetic treatment following the discovery of fluid abnormalities. One of these patients


is now, five years after his infection, and in spite of 57 intravenous injections of arsphenamin, 11 intraspinal treatments, and a great deal of mercury, a typical dilapidated paretic. In both of these instances, however, a long period of time, punctuated by frequent lapses in treatment, had elap.sed since the date of infection. The remainder are clinically well. No patients are serologically negative, though on the average much more treatment has been given than in the Group II cases. Three are moderately improved; two slightly improved, two unchanged ; and in one the cyto-biology of the fluid is worse after 3:i intravenous arsphenamins than before. Intraspinal treatments were employed in three of these patients, resulting in a marked improvement in two.

The contrast between the results in (iroup II and in tJroup III cases is strikingly shown in Table XVIII. Of the former, 7(i.!( per cent of the patients are clinically and serologically well, as compared to none of the latter. This part of the study offers confirmatory evidence of the intei-pretation given for Chart II, in regard to the effect of the amount of treatment given on the incidence of the various gro\i]is, since in both places it is shown that Groui) II is readily influenced by treatment, and Group 1 1 1 very little if at all.

These results can be applied to the controversy over intraspinal treatment, and our attitude in regard to it, so far as this tyi)e of case is concerned, can be briefly summed np. For cases of the tyi)e of Groups I or II, intraspinal treatment is usually not necessary, and, if ajiplied at all, should not be used until the i)atient has


TABLE XVII. The Results of Treatment of Early Asymptomatic Neurosyphilis — Group III.



Duration at disease on ad

bv/ore first spmal imnclure.


Lapse


Intervai admis spinal punc

First C. S. F. results


Treatment ajter first puncture


Last C. S. F. results


Time elapsed


Clinical result


Serologic


Case No.


3


Glob.


Wa. R.


Cold


Mastic


Arsphenamin


Mercury


t


Glob.


Wa. R.

0,2 1 0.4 1 1,0


Cold


Mastic


first punc

result



mission


0,2 0.4 1,0


lure



U.


8 Mos.


2x0,3


8.5 Mos.


9 Mos.


32


-I~t-I~h



4


4


5555321000


~


16x0,35


14 Weeks


7


-1-4

1


4


4


4320000000


5443321000


20 Mos.


Well


Slightly improved


1


11 Mo3.


4x0.4 elsewhere


+


~


12


+++



4


4


5555431000


"


33x0,4


Practically


61


-t-F-l-f


4


4


5544432200


5555321000


27 Mos.


Well


Worse


X4


6 Mo9.


12x0.35-1-4 wks. Hg.


6 Mos.


11 Mos.


64


++++



4


4


5555431000


~


56x0.4


36 Weeks


1


-h-l



2


4


544433000


3321000000


31 Mos,


Well


Slightly improved


J4 1»


8 Mo9.


3x ? elsewhere 12x0.3-f 10 wks. Hg.


30 Mos.


.16 Mos.


98


+++


4|4 1 1


4


5555430000


5532100000


22x0,4


10 Weeks


70


-H-(-(

4


4


5555521000


5543210000


10 Mos.


Deep reflexes increased


Unimproved


Ze27


3 Mos.


27x0.3 and much irregular Hg.


Frequent


46 Mos,


131


++-I-I

4 4


4


5555554100



30x0,4 -Ml

spinal


Continuously for 14 Mos,


8


-t-t-l

4


4


4


5555321000



14 Mos.


Asymp at first puncture developed

G, P.

under


Unimproved


H


24 Mos.


14x0.4. Irregular Hg.


+


22 .Mos.


40


-h4-4-t

4


4


4


55555421000



23x0,3 7 intraspinal

ments


20 Weeks


2


±





4


1110000000



24 Mos.


Well


Improved


Em.


11 Mos


Ix? elsewhere 15x0.3-f 12 wks. Hg.


+


10 Mos.


156


-f-f-H


4


4


4


S5SS520000


"


67x0,3


80 Weeks



-l-l

3 ~0


4


4


5511100000



36 Mos.


Well


Slightly improved


S32


3 Mos.l2i0.4-(-8wk8. Hg.



5 Mos.


65


-t-H

4


4


4


5554210000



spinal



12


-1-4

5555321100



3 Mos.


Well


Improved


244


[No. 377


TABL.E XVIII.

Summary of Treatment of Early Asymptomatic Neurosyphilis, Groups II and III.



1


J


c o


13 J3


Serologic result



Well


Improved No change Worse


Group II


13


1


12



10 (76.9'


1 — 2


5


(23.1%)



Group III


9


1


6


2



3


1


1 (100%)!


Lad a tlioiouf-li trial of at least six months of routine anti-sypliilitif treatment. For the Group III type, it has heen shown that routine treatment aeeoniplislies little or nothing. In these cases intraspinal treatment as an adjunct to routine therapy is of value, because it offers almost the only hope of attaining a serologic cure, and because it tends to bring about improvement more rapidly than the routine method alone, thus obviating some of the dangers attending the too prolonged use of arsphenaniin '^ and mercury. In a later paper of this series, which will deal with tiie question of late asymptonuitic neurosyphilis, we hope to support our contentions in this respect with additional evidence.

The Prognosis in Early Asymptomatic ^'eiirosyphilis. — It has already been suggested that there is probably no hard and fast line to be drawn between our three groups, and that it may be possible for a patient who falls into Group I on early puncture to belong to (Jroups II or III if puncture had been carried out later. This change in grouping may also be brought about by the factor of insufficient treatment.

It has occurred to us, as well as to others, that it might be possible for the spinal fluid abnormalities of these groups to clear up spontaneonsly without the influence of treatment, or that patients siiowing these changes might remain clinically well without developing neurosyphilis.'*" If these two points could be demonstrated, the stress which we lay on early fluid changes would lose much of its empliasis. So far as the spontaneous disappearance of cyto-biologic abnormalities is concerned, we have not had the temerity to allow such patients to

'"Moore, J. E. and Keidel, A.: Dermatitis and Allied Reactions following the Arsenical Treatment of Syphilis. Arch. Int. Med.. XXVII, 716, (June) 1921.

'"Kalsiki, D. J. and Strauss, I.: The Signifteance of Biologic Reactions in Syphilis of the Central Nervous System, with Notes on Treatment, especially Intraspinal. Arch. Neur. and Psych., VII, 98, (Jan.) 1922.

"Keidel, A., and Moore, J. E.: The Treatment of Primary and Secondary Syphilis. (To be published.)


go untreated ; and we can say only that, on repuucture of those patients who had voluntarily la])sed in treatment, we have never found a negative fluid. On the contrary, in our few examples of this kind, the later puncture has always shown changes of an intenser degree than the first. We should be inclined to admit the possibility of this conception only in the Grotip I cases, and we sliould be very diffident of expression even here.

It is not surprising that adequate statistical data regarding the development of clinical neurosyi)liilis in these asymptomatic patients are lacking in the literature. Only a few years have jtassed since the importance of early spinal fluid examinations has been ai)preciatetl, and in order to provide data of any value, these patients must be continuously followed over a life time. The longest period of time for which we have been able to follow any untreated case is five years. This patient, a Group III ca.se, is apparently well at present, though she refuses to submit to re-examination. So far, no Grouj) I case, treated or untreated, has developed clinical neurosyphilis. Of our 87 (iroup II cases, one patient (Sy) whose neurologic examination was completely negative at the time of her first puncture, lapsed in treatment for five mouths. On her return, her pupils which had been normal were now widely dilated, unequal, markedly irregtilar, and reacted to light only very sluggishly. One Grou]) II patient is dead (lobar pneumonia). The others, so far as is known, renuiin well. Those with minor neurologic signs have not gotten wor.se ; those without signs have not developed them.

Of the 17 Group III patients, three have developed general paresis, one after a great deal of treatment, and two after long lapses. A fourth patient has developed outspoken neurologic signs which would now ea.sily allow a diagnosis of cerebrospinal syphilis, though no mental changes are evident. The renuiinder are apparently well.

The Prophyhui!^ of Early Aftyniptoiiiatic ycitrosyphilis. — The evidence regarding tlie origin of these various types of as.ymptomatic neurosyphilis may be applied to the formulation of a scheme of treatment for early syphilis calculated to reiluce the incidence of neurologic damage. As long as the treatment of the early stages of the disease continued to rest largely in the hands of the general practitioner rather than of the syphilologlst. we consider it unnecessary to argue that there should be a fairly rigid outline of treatment applicable to the majority of uncomplicated cases. The attempt to individualize treatment will be productive of far more harm than good.^" It is not our intention to enter into the details of our plan of treatment of early syphilis, or the factors which led to its adoittion, since this will be fully considered in a later communication.'" It is necessary, however, to point out briefly the essential features of an adequate i)lan and to indicate the arguments in its favor provided by the data of this paper.


July, 1922]


245


Anti-syphilitic ticatiiieut in early .syphilis should be continuous rather than intermittent. There should be no periods of rest between courses of treatment, because of the tendency of lapsed (or irregular) treatment to increase the incidence of spinal fluid abnormalities. In order to accomplish this, arsphenamin and mercury must be used separately. It i.s probably best to allow a little overlapping, so that the use of mercury is begun just before the last injection of a course of arsphenamin and is kept up until just after the first injection of the next course.^* There are additional cogent reasons for this method of use of the two drugs, a discussion of which is out of place in this paper.

The most indefinite part of any plan of treatment is the length of time for which it should be continued. Our own routine, at first adopted arbitrarily but now justified by the data of Table X and Chart 11, has been to treat early syphilis continuously until completely negative .serology of the blood and sjjinal fiuid has been obtained, and for one full year thereafter. While it is true that some patients will be cured with less than this amount of treatment, it is equally obvious that others will not be cured ; and there is at present no means of an early separation of the two groups. The only safe plan, therefore, is to treat all i)atients u|) to the point of cure of the nmjority.

In earlj' syphilis, treatment is begun with arsphenamin, the first course being of eight doses and succeeding courses of six doses each. Mercury by inunction is started just before the last do.se of each arsphenamin course, and continued until the first dose of the next. The interval between arsphenamin courses is at first short (four weeks), and is gradually lengthened with each course. The longest interval permitted is twelve weeks. Spinal puncture is performed as a routine after the first or .second courses of arsphenamin. If the standard of serologic cure mentioned above is adhered to, the treatment of sero-negative primary syphilis will usually last exactly one year, and will consist of at least 4 courses (26 doses) of arsphenamin and 26 weeks of mercury. In sero-positive primary and in secondary syphilis, the length of treatment in the average case is usually 1.5 months (5 courses of arsphenamin and 32 weeks of mercury). If spinal fluid abnormalities are found at the time of the first puncture, this plan is modified according to the group of abnornuility present. When treatment has been completed, there ensues a year of probation, during which there must be no syu'.ptoms, no positive and at least six negative Wassermann tests (icebox) ; and, at the end of the year, negative i)hysical and neurologic examination, negative X-ray of the cardiovascular stripe, and negative examination of the spinal fluid.


'"Stokes, J. H.: The Applications and Limitations of the Arsphenamins in Therapeutics. Arch. Derm, and Syph., II, 303, (Sept.) 1920.


If this plan of treatment could be applied to every ca.se of primary and secondary syphilis, the incidence of asymptomatic neuro.syphilis, and in all probability that of clinical neurosyphilis, might be reduced to an absolute minimum. Judging from the data supplied by this paper, the present level of 20 to 25 per cent of clinical neurosyphilis might be reduced to 5 per cent or less. The economic saving to the state, to mention only one of the advantages of this reduction, would be enormous.

Si'xiJiARY AND Conclusions

1. It has been shown that early invasion of the central nervous system in syphilis, is common, occurring in 26.-t per cent of a series of ii52 patients with primary and secondary syphilis. Of 9-1 early neurosyphilitics. 72 were asymptomatic, and were detected only by the routine application of spinal puncture.

2. Early asymptomatic neurosyphilis may be divided into three sub-groups on the basis of the spinal fiuid findings and the response of the various groups to treatment.

i Invasion of the central nervous system jtrohably occurs in the majoritj' of all patients with syphilis, and unless the course of the disease is influenced from without (by treatment), this invasion takes place in most instances within the first year after infection. The ability of the invading organisms to produce clinical neurosyphilis probably depends on the defense mechanism of the individual patient. Tlie experimental and clinical evidence bearing on these points are reviewed.

i. Early asymptomatic neurosyphilis is more common in the white race than in negroes, but is equally frequent in men and women, of either or both races.

5. I'rolonged regular treatment influences favorably the incidence of early a.symptoinatic neurosyphilis. Irregular or lapsing treatment, on the other hand, markedly increases its incidence.

6. A study of this material from the standpoint of strains of Treponema pallidum furnishes no support to the theory of the existence of a neurotropic strain of organism.

7. That the spinal fluid abnormalities of early asymptomatic neurosyphilis are evidence of actual anatomical damage to the nervous system, is indicated by the frequency of certain minor subjective and objective neurologic signs in this class of patients. An ai)preciation of these signs, and of the significance of a persistently positive blood Wassermann reaction in treated patients, furnishes a clinical diagnostic aid for the recognition of neurologic invasion.

8. Spinal puncture is an indispensable routine procedure in the management of early syphilis. Unless it is employed, many patients will be discharged as cured who are nevertheles.s candidates for clinical neuro.syi)hilis. It should be performed as a routine after the first or second course of arsphenamin, and unless a lapse in treatment


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occurs, need not be repeated (if negative i until tlie end of treatment and the probation period.

9. All three groups of asymptomatic neurosyphilis may be serologically aud clinically "cured" by appro priate methods of treatment.

10. Early a.symptomatic neurosyphilis is the forerunner of clinical neurosyphilis. One patient of our second


groui) has develojjed clinical cerebrospinal .syphilis. Of the third group, three patients have developed general paresis aud one cerebrospinal syphilis.

11. There is suggested an outline of treatment for early syi)hilis, the uniform application of which will markedly reduce the incidence of asymptomatic neurosyphilis, and probably, therefoi'e, of clinical neurosyph ills.


THE BIOCHEMISTRY OF TUBERCULOSIS

By EsiioNL) R. LoxG, Ph. D.

(Department of Pathology, UnlversHy of Chicago)


In the biocheniistrj* of an infectious disease two series of metabolism are naturally concerned, that of the invading parasite, and that of the host as modified by the former's action. The manifestations of disea.se may be looked upon as alterations in the physiology of the host initiated by the metabolism of entering bacteria, although sometimes no more than initiated, since the bacterial growth may serve to Are a train of subsequent events largely non-s])ecific in character.

There is a natural tendencj' to think of bacterial cells as simjde forms of life, and to a certain extent they are, being largely independent of each other, devoid of that correlation so conspicuous in the physiology of higher forms. But, cell for cell, tlieir life processes are probably fully as complicated as tho.se of a liver or kidney cell, or l)erhaps even one of the central nervous system. They digest their food, build ujt complex substances from simple ones, they respire, that is to say, take up oxygen and give off carbon dioxide, they excrete waste products, and they reproduce. And if they are shut off from an adequate supply of nutrition, like other cells they die and tend to autolyze.

Leaving out of present consideration those remarkable forms which snatch nitrogen out of the atmosphere, heap up monntains of sulphur, or spread vast marshes with iron, we have an almost endless variety of microorganisms whose methods of obtaining energy and manner of excreting waste are closely akin to those of cells of highly organized life. Some of these are vei-y adaptable, wonderfully equii)ped foragers, not overly particular, within reasonable limits, about the temperature at which they work or the form in which the gi-eat classes of foodstuffs are supplied to them. They grow in relatively large masses, many dying that others may live, and in their process of dissolution many kinds of them liberate powerful ferments which make more food available for the rest. In view of their relation to organic decay they have received the name of "saprophytes." Etymologically they are decay-growers. They live their day, grow, die


Read before the Ltennec Society of The Johns Hopkins Hospital, November 21, 1921.


and decay themselves, and by virtue of their involuntary sacrifice serve the high purpose of maintaining those great cycles of the elements in which a certain amount of carbon, nitrogen and sulphur is continually available in a mobile state suitable for the nutrition of other forms.

Not all bacteria by any means are so useful as these great cycle maintaiuers. Not a few, evolutionary accidents in the mighty, invisible struggle for the survival of the fittest, must eke out a more or less miserable existence, dei)endent for their nurture largely upon the unwitting, lavish generosity of the great hosts of proteo , lijx)- and cellulo-dastic forms, or upon the less willingly jirovided maintenance of more highly organized life.

Of this last set a certain lunnber iiave acquired what amounts in the end to a ])rotective adai)tation, the niysterious condition of parasitism. Tiiis mode of existence, tjie factors of which were so clearly defined in a recent address by Theobald Smith, has for its essential elements the ability on the part of invading microorganisms to multiply within the tissues of a living being, and to multiply in such place and manner that an easy exit is afforded, from which some of the inva<lers may escape to another host of the same sort. Thus parasitism as a permanent mode of life necessarily involves a cycle including more than tiie mere ability to multijily in tissues.

It is, however, only to the first of these factors of parasitism that I shall now make reference, limiting my remarks to the special case of multij)lication within the tissues of the human host. The remarkable thing is not that there are so many organi.sms that possess this cai)acity, but that there are so few, and that those bacteria which have adapted their nutritional requirements to the difficult conditions obtaining within the host, should be so helpless in the region of bounteous plenty outside, while those which attack dead organic material so energetically seldom establish a home beyond the first barrier of living cells. The typhoid bacillus ])ursuing its increasingly lonely way down densely populated sewer pipes is a pitiful object beside the infinitely


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more vigorous proteus family; so poorly equipped for the jouruey is it that after Ave or six days few members of its group survive to tell of a different phase of their existence, the tale of deadly destruction wrought so short a time before in an environment impregnable to the surrounding billions of saprophytes.

Among these bacteria which, so far as I can see, serve no useful purpose in the great scheme of life, is a group of organisms characterized by the protoplasmic possession of a large amount of non-sapouitiable lipoid, which so modifies their reaction to some of our chemical reagents that they can be readily picked out from the rest by a simple application of appropriate stains. This is the acid-fast groui>. Within it are several varieties, including parasites adapted to life in different hosts, namely, the tubercle bacilli, and saprophytes unable to maintain such existence, although flourishing more or less widely outside.

Time does not permit us to puzzle long over the evolutionary relationship of these forms, but that there is a relationship, remote though it may be, is at least suggested by certain facts. The most outstanding of these is their intei'changeability as antigens in the so-called biological reactions, which indicates, according to the more general coiicei)ts of immunology, that they possess in common certain proteins, specific to the degree that they are present in no great concentration, or not at all, in the bodies of other bacteria. The "tuberculin," if 1 may use the term, formed by the growth of a grass bacillus in bouillon, will elicit a typical reaction in a tuberculoiis animal, as the similar product from a hay bacillus will not. and there seem to be more investigators who believe in a common "acid-fast fixation of complement" than who do not. This refers to qualitative conditions and does not mean that the organisms of the group may not be separated by biologic tests made on a careful quantitative basis.

Certainly these organisms are closely enough allied in their chemical makeup so that it would be difficult or almost Impossible to distinguish them as dead chemical material. Give a man an autoclaved culture of grass bacilli and one of tubercle bacilli and he would be a keen analyst if he could distinguish between them. He might filter them off and stain them, noting that they were resistant to acid decolorization, and he might extract them with chloroform and find a content of waxy substances in the neighborhood of oO per cent, and be able to say, "These are not hay bacilli or typhoid germs ; they belong to the acid-fast group." He might go further and make emulsions of the dead bacteria and test them out against tuberculous guinea-pigs bj' skin inoculation, and confirm his previous conviction on the group to which the organism belonged, and he might perhaps notice that one of his preparations brought out a distinct reaction in


quantities definitely less than those required with the other, but he would be fortunate if he could be sure.

It is only with the living material that he could stand up without fear and say, "These are tubercle bacilli, and these are something else in the acid-fast group." And he woulil have one or perhajis two lines of fire on which he could hit his nuirk. The first of these would be the establishment of the condition of parasitism in an appropriate host. The second would be the proof of a characteristically different metabolism in non-living culture media. Of these the first would be a bull's-eye. made on a shot through the dark. The second would be made through the light, in so far as known chemical reagents are clear to our understanding, as a guinea-pig is not, but it would probably not be a bull's eye — at present at least. I am convinced that a time will come when a relation of virulence to metabolic activity will be established, but little progi-ess has been made to date. So far as I know, the surest chemical criterion at present for a tubercle bacillus is that discovered by Nocard thirty-five years ago, its marked glycerophilism.. Our hypothetical solver of bacteriological unknowns could. I believe, find that for one of his strains glycerol was a practical necessity, while for the other eth^-l alcohol or the neutral salt of lactic or jtyruvic acid would serve equally well. Or he might nuike up a medium of neutral reaction containing phosphoric acid and certain inorganic salts, with a single amino acid like leucine or alanine as the sole organic material, and find that the grass bacillus grew readily on this, whereas the tubercle bacillus did not. In either case he would l)e dealing with a distinction in carbon metabolism.

If he attempted to distinguish them on the basis of nitrogen metabolism, he would be working on a more difficult problem, for he would find that, if hard enough pressed, either one could satisfy its nitrogen requirement with ammonia alone or with the nitrogen of simple amino acids or acid amides, as alanine or propionamide. He might be able to make some distinction on the readiness with which they abstracted that ammonia from different nitrogenous combinations, however, although he has little enough infornuition to go on at present. For instance, in their action on amines, certain differences might be noted, inasmuch as some of the grass bacilli have been found to deaminize ethyl amine. The tubercle bacillus as well as a good many of the saprophytic acid fasts do not. Again, in their utilization of imino nitrogen, as seen in the compound creatinine, a moie nmrked difference is shown, as this nitrogen seems unavailable for the tubercle bacillus, while a larger number of the saprophytes, including several strains of grass and smegma bacilli, in my experience, are able to grow on this compound as sole source of nitrogen, presumably hydrolyzing it and thus securing amino nitrogen. Also he might find that certain combinations of nitrogen and carbon,


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while not uecessai-y, were favorable, liidii-atioiis have been found that the dianiino acids and tryi)to)>liane favor growth, just as they do in animal life, perhaps because they are a necessao' part of bacterial protein molecules, and their .synthesis is a matter of some difficulty. That both organisms possess the capacity for syntliesizing the aromatic rings, however, if forced to do so, may be readily proved by growing them with an ammonium salt as the sole source of nitrogen and testing the product some generations removed, for tryptophane, tyrosine and purine bases. The tests would be positive.

All in all, much is yet to be done to fnrnisli an adeiiuate basis for correlating metabolism and virulence. Some of the lines are so open, and so obviously important, that I feel like apologizing for coming before you tonight, as a worker in the field, to report that some of the most promising have been scarcely touched. For instance, to mention just one, much of natural resistance of the tubercle bacillus to digestion infra vitain has been attributed to its high wax content. But the avirulent acidfasts have a high wax content too — at least the few of them so far examined. What is the difference?*

So far we have considered chiefly intake and synthesis in our brief reflection on the metabolism of the tubercle bacillus. The other side, catabolism and excretion, is at least as important from our point of view. As the tubercle bacillus grows in a bouillon culture, or in the disintegrated tissue of a caseous tubercle, diffusible molecules of the medium must be passing through its semipermeable tissue all the time, there to meet certain enzymes which knock off various parts of the molecule,some of which are retained by the cell, caught on the fly so to speak, while others diffuse back into the medium. The chief blocks so chipped off are ammonia, various acids from the amino acids, and oxidation products of the carbohydrates. The variation in the amounts cast out leads to those more or less characteristic changes in reaction depicted in the Theobald Smith curve.

Now these simple substances are not the only materials getting out of the bacilli into the medium. For the medium of growth, freed from bacilli, now possesses certain properties not conferred by the products of metabolism just mentiouefl. It contains something which is poisonous to a tuberculous animal. Inasmuch as the symptoms elicited by this broth are the same as those called forth by any sort of watery extract of tubercle bacilli, carefully washed though they be from adhering broth, it may be assumed with some confidence that it is some product of the bacteria in the broth, and not the reverse, which causes the reaction. Now what of their own substance have the bacilli contributed to the sur


It has been learned, since this address was given, that while the total quantity of lipin in tubercle bacilli and saprophytic acidfast bacilli is about the same, the proportion of that lipin as wax is much higher in the tubercle bacillus.


rounding medium in tlie weeks or months of tlieir growth? A not unnatural supposition is that some have died and autolyzed, furnishing a certain amount of the products of protein autolysis. Somewhat against this hypothesis is the well known fact that tubercle bacilli do not autolyze to any great extent. Still a very little might be l)roduced of a very potent substance.

That tuberculin is not a product formed extracellularly by action on the constituents of the medium, is practically certain, inasmuch as strong tuberculins can be made from simple media, like ammonium phosphate plus glycerol. In fact such media furnish the best material for a study of the nature of tuberculin, since there are no complex interfering substances. Now if tuberculin is protein in nature, like other materials concerned in immunity, it seems as if such media should be ideal for proving that fact. And yet just here there is the most amazing discrepancy of results. A half a dozen men, including Lockemann, report growing tubercle bacilli on such media, filtering them off cleanly, obtaining a true tuberculin with the material, and precipitating by ammonium sulphate or alcohol a substance in it which gave all the protein reactions, and was itself active as tuberculin. And a half a dozen other men, including Lowenstein and Pick, get no such thing. They get an active tuberculin, to be sure, but it gives no protein reactions. As far as my own experience goes, I have twice made tuberculins, once by growth on an ammonium chloride-glycerol medium, and once on an alanine-glycerol medium, which were active on tuberculous guinea-pigs, but gave no protein reactions, not even the simple biuret test.

In favor of the view liiat it is protein in nature, are the experiments of a ntnnber of investigators who have weakened or destroyed tuberculin by the action of such proteolytic fenneuts as pepsin, trypsin and erepsin. Perhaps, when the problem is solved, it will be found that tuberculin is a polypeptid, an autolytic product of the tubercle bacillus, protein in its nature in that it is derived from protein, but not sufficiently complicated in its structure to give the usual protein tests. This finding would force us to modify some of the prevailing concepts of immunity. Certainly an attractive problem here awaits solution.

One more word before 1 i>ass to the subject of the metabolism of the infected host. That concerns the conditions under which bacteria, even parasites, may multiply within a living body. The researches of Kettger and his associates have i)retty definitely brought out the fact that bacteria are unable to grow on pure proteins or coagulated proteins, or in many instances even on pure proteoses or pei)tone. They need a certain amount of still simpler luodncts of protein digestion from which to construct their enzymic unichinery for the cleavage of larger molecules. This is a significant point in their


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relation to live cells. Living piotoplasni contains no great amount of the simple molecules which are favorable to bacterial growth ; consequently, living tissues are a poor feeding ground for germs. Once these tissues die, however, or, in other words, once the rate of catabolism greatly exceeds anabolism, or the latter ceases altogether, autolytic products accumulate, and the conditions for bacterial multiplication are at once established. This point will be referred to again when we come to consider the relation of the growth of the tubercle bacillus to necrosis. Doubtless many other important factors are concerned, such as the oxygen and carbon dioxide tensions, as has been so well brought out in the worlc of Wherry and Ervin and in the very interesting recent investigation by Corper. Death of the tissues probably plays a role here too.

So much for the present concerning bacterial metabolism.

A discussion of the other side of the picture, namely, the altered physiology of the host, properly begins with a consideration of the most characteristic lesion of tuberculosis, caseation. In this type of necrosis, tissue becomes pale grey or yellowish in color, with a certain resemblance to cheese, which is indeed closer from the point of view of chemical composition than that of appearance to the eye, the necrotic mass consisting, like cheese, essentially of coagulated protein and finely divided fat. The protein is chiefly simple protein, not nucleo — or otherwise conjugated. In fact the lessened amount of nuclear material, such as phosphoric acid and purine bases, is one of the striking chemical differences between caseous and normal tissues. Autolysis, which is on an extremely low plane, has at least gone far enough to bring these nuclear components into diffusible form. The total amount of protein is about the same as in the corresponding area of normal tissue, as is tliat of lijiins, although certain qualitative differences are to be noted in each group. The proportions of cholesterol and lecithin are altered, and the iodine number, or index of nousaturation of lipins, is changed, but the variations from normal are not so great as to cast doubt on the assumption that the caseous matter consists almost entirely of disintegrated pre existing tissue, modified to a slight extent by slight autolysis and the introduction of a small amount of foreign protein and lipin from the bodies of tubercle bacilli.

The cause of caseation necrosis in tuberculosis is still unsettled. Some investigators have claimed specific coagulating properties on the part of substances in the body of the tubercle bacillus. Auclair years ago reported producing caseation with a certain fraction of the fatty material. Morse makes a similar report today. Rnppel obtained a protamine, which he called "tuberculosamine," which like other protamines caused a precipitation of proteins from their solutions. Others liave considered


caseation as allied to the Arthus phenomenon, the i-esult of the lodgment of a foreign protein on highly sensitized soil. In this case we are perhaps to think of cleavage of the foreign protein with the production of a toxic, coagulating fraction. It seems to me, however, that too much emphasis cannot be laid upon the unquestioned condition of local anemia as a causative factor in necrosis. The mechanism of its production offers room for argument, although .several plausible explanations are readily thought of. The entering bacillus may produce substances causing an initially minute amount of capillary throml)osis, enough to produce necrotic material capable of giving sustenance to the bacilli present. However, the anemia may be more simply explained. Every entering mass of tubercle bacilli is promptly met by a perfectly non-specific foreign body reaction, the proliferation of a fixed phagocyte, the so-called epithelioid cell. The mere multiplication of these cells in a given area must be sufficient, through the pressure exerted, to shut off some of the capillaiy circulation, as Mallory explains necrosis in typhoid fever. Thus a local anemia may be readily established, with subsequent death of the cells of the anemic area. Then, and not until then, the conditions for bacterial multiplication are established, the bacilli obtaining the nutriment which they were unable to obtain from living cells from the autolytic products of the dead ones. After this start bacilli are transported, one way or another, to neighboring areas and an endless chain of foreign body reaction, anemic necrosis, and bacillary proliferation established.

A more difficult problem is to explain the long continued chemical status quo of the caseous tissue. In some waj- this must be dependent upon the presence of the tubercle bacillus. The like is not often seen in other forms of necrosis. An anemic infarct shrinks as its substance is slowly absorbed and replaced by entering fibroblasts. A cancerous nodule becomes umbilicated as its central anemic mass degenerates and diffuses away. But in the tubercle, the fibroblasts, althougli they may ring the lesion in leathery form, have little tendency to enter on radiating lines, while solution of the central, morphologically disintegrated tissue by autoenzymes is conspicuous by its absence. The lack of chemotaxis for motile cells is perhaps explained by the failure of the cellular enzymes to liberate diffusible products. Certainly extractives are pre.sent in but minute amount. Even in extreme cases of fluidity, as in cold abscesses, the crystalline products of the autodigestion of proteins are almost absent. Nor can proteases be demonstrated by the MtiUerJochmann plate method. Why this fundamental failure of autolysis? Have the autolytic enzymes been destroyed, or are they merely held in a state of inactivity? Jobling and Peterson have furnished some interesting information on this point. They noted first, that proteolytic action in general, and autolysis in particular, were inhib


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ited by the soaps of unsaturated fatty acids, while not affected by the saturated ones; secondly, that caseous areas, which characteristically did not autolyze rapidly, contained such soaps in appreciable amount ; and thirdly, that the saturation of these soaps with iodine removed their autiproteolytic action, following which autolysis pursued a normal course. Finally, they were able to demonstrate the presence in the tubercle bacillus it.self of unsaturated fatty acids, the soaps of which are more active in proportion to their iodine value in inhibiting trypsin and leucoprotease than soaps of linseed and olive oil, and that these soaps too can be deprived of their inhibitory action by iodine. Their conclusion naturally was that the unsaturated fatty acids of the tubercle bacillus are a very important factor in the failure of absorption of caseous tissue. I have always felt that this piece of work is very much in need of amplification. For instance, it would be desirable to know if the leprosy bacillus, which excites a foreign body reaction without caseation, is devoid of these anti proteolytic properties.

We may next consider this caseous mass for its bearing upon the physiology and metabolism of the host. It has its specific and non-specific elements, as far as tuberculosis is concerned. In the first place, it contains tubercle bacilli, which, chemically considered, are foreign proteins, proteins which by their continued presence within the body have so sensitized the tissues of that body that they react, more or less violently with hyperemia and exudation, to the same material, whether it arrive at a given point by dissemination from the original focus, or by fresh introduction from without. Yet it is unlikely that this reaction to "tuberculin" accounts for all, or even the greater part of the toxemia of tuberculosis. For, secondly, the tissue of the caseated area, once homologous, before its coagulation, has now become a foreign protein too. Whether the body is allergic toward it, as it is to tuberculoprotein, is a question. Be that as it may, the substance is appreciably toxic per se. The toxicity of the products of incomplete protein digestion, proteoses and peptones, is too well known to require comment. But is much of such diffusible substance present in the caseous tubercle? We have noted before that self-digestion of the caseous mass occurs on but a small scale.

In this connection may we not refer to a very instructive set of experiments by Dr. Krause? You are doubtless quite familiar with these; yet they seem to me so important for our conception of the chemistry of the disease that I cannot refrain from repeating their story in some detail. Krause started out with the idea that foci at the height of reaction probably contained more toxic material than non-reacting foci. Accordingly, to prove this he gave large doses of tuberculin to tuberculous guinea-pigs, killed them off during the acute stage


of the subsequent illness, and found, as he knew he would, that the tuberculous foci were in a condition of inflammatory reaction. He then emulsified these inflamed foci in physiological saline, centrifuging the product to remove mas.ses of a size sufficient to act as emboli, and injected the clear juice intravenously into normal animals. He found, as he anticipated, that the material was highly poisonous. Then he went a step further. He injected emulsions similarly prepared from the non-reacting foci of animals which had never received tuberculin. Somewhat to his surprise, I believe, he found that such emulsions too were markedly toxic, as much so as the first ones. He next took tissue from healthy guinea-i)igs, emulsifying and injecting normal organs, and found that this material too was toxic for normal animals, and furthermore, just as much so as that from a tuberculous focus. If any of these emulsions were filtered, however, through a Berkefeld caudle, the product was no longer toxic. These experiments indicated to Dr. ICiause that an animal is poisoned by any kind of cellular material which gets into its blood stream in large amount in unit time. An abundance of confirmation of these experiments on the effects of tissue juice in the blood stream of normal animals has accumulated in the literature of fields other than tuberculosis. The recent experiments of Karsner and Hanzlik and of Mills are instructive. It is especially interesting that lung tissue furnishes exceptionally toxic substances. All these investigations prove, I believe, that a storehouse of disintegrating protein, even though inhibited in its autolysis to such an extent that no great amounts of toxic proteose and peptone are ab.sorbcd, is yet a source of poison to the organism containing it. On the periphery of each caseous mass in the body of a consumptive a certain amount of this material must be all the time being swejit into the circ\ilation. Any condition which will increase the circulation at that point naturally will favor absorption. Such a condition occurs in the focal active hyperemia which follows the administration of tuberculin, or the dissemination of tubercle bacilli to fresh but sensitized soil, the latter being a process constantly occurring in progressive tuberculosis. Thus, the toxemia of progressive tuberculosis may be thought of as to a considerable extent non-specific in character, dependent upon the absorption of disintegrating protein, but it must be continually borne in mind that it is aided, in the manner noted, by one whicli is quite specific in nature, the focal inflammatory reaction to tuberculoprotein.

Let us now take uj) tlie alterations in the piiysiology of the host, dependent upon absorption from his focus, regardless of the site of the latter, so long as it is in a sufficiently unprotected state, leaving out of consideration those changes in metabolism which depend upon the location of the lesion, such as the diminished gas exchange following obliteration of lung volume, or dis


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turbed absorption resulting from iutestiual ulceration, which also have sequehe of abundant chemical interest.

One of the most striking clinical manifestations of this absorption is fever. And in a perfectly non-specific manner to a certain extent changes are produced in the patient's metabolism, not because he has tuberculosis, but because he has fever. This is hardly the place for a discussion as to the cause of the fever in tuberculosis, but we may simply recall the fact that, since the time of Traube, fever has been attributed to a diminution in the ability to discharge the heat produced, the regulatory control being lost through the action of some poison upon the vasomotor centers, and we may state further that according to modern opinion, developed largely from the work of Vaughan, that poison is frequently protein in nature. Indeed there is much ground for believing that the fever in tuberculosis is, like the fatality in the case of Dr. Krause's guinea-pigs, due to the presence, in the blood stream, of foreign protein or protein cleavage products, and that these in turn have two sources, tuberculoprotein and the protein of the disintegrating caseous mass, of which two the latter is by far iu excess.

Whatever be the mechanism involved in the production of fever, once fever is established, metabolism may be modified more or less profouudlj' as a result of it. "Warmed tissue metabolizes more material than cooled tissue," writes Lusk, citing the classic experiments of Pfltiger, who curarized rabbits, thus paralyzing their voluntary muscles and capacity for increased work, at the same time applying external heat, and noted that as the rectal temperature rose from 39° to 41° the oxygen absorption increased 10 per cent for each degree. Plenty of confirmation has appeared since the time of Pfliiger, and a large literature has accumulated on the nature of the material metabolized. That basal metabolism is increased in tuberculosis has recently been shown by Grafe, who found in twenty patients a raising of heat production per kilogram varying between 10 and 80 per cent, the higher figures being for febrile cases. Whether such metabolism can be considered "basal" is a technical question discussed by McCann and Barr, who also note an increased metabolism in fever, which at 40° may be 30 per cent above normal.

The effect of fever upon protein metabolism and nitrogen elimination is more complicated. One of the most interesting investigations on the subject was that of F. Voit, who found that artificially raising the temperature of a fasting dog to 40 41° for twelve hours increased the nitrogen elimination 37 per cent above normal. That the action was not a selective one upon protein, however, was indicated by the fact that a preliminary feeding of 30-40 grams of sugar prevented this increase in protein metabolism, which was thus .seen to be simply a part of the general increase, occurring only when the store of more readily burnable glycogen was depleted. That a


loss of nitrogen occurs in infectious fevers has long been known, and that this is greatest during the height of fever is also generally true.

How much of it, however, is due to the fever and how much to more direct action of the absorbed material which causes the fever, remains to be determined. Many writers speak of a toxic destruction of protein. May, for instance, says, in the conclusion of his comprehensive review of the subject of metabolism iu tuberculosis : "The wasting of tuberculous subjects is due primarily to poisons absorbed from tuberculous nodules. Large doses cause a direct injurj- to cell protoplasm. The amount of protein destruction attributable to this cause is not very large, but becomes of importance when continued for a long period of time and when there is no compensatory regeneration. It appears that the power to regenerate on the part of these cells injured by toxins is greatly reduced and in severe cases entirely lost." He goes on to write, however, "Higher degrees of toxic action are usually accompanied by fever, and the metabolism of the febrile consumptive follows the same laws as metabolism in the fever of other causes. There is an increased protein destruction, but not an increased fat metabolism, except as may incidentally result from dyspnea, chill or muscular activity."

Graham and Poulton, in Friedrich Muller's cliuic, have showed that in man a body temperature of 40.2°, brought about through the influence of a steam bath, does not of itself cause an increase in the metabolism of protein. In their carefully controlled experiments the abnormally high body temperature was maintained for several hours and yet there was never any increase in the breakdown of body protein as the result of hyperthermia. Coleman and Dubois, in their investigation on the metabolism in typhoid fever, found it impossible to escape the conclusion that the destruction of protein was caused by the toxins of the disease, noting that in some cases protein destruction continued for several days after body temperature had reached a low level. In their recent work on tuberculosis McCann and Barr ad«pt a similar point of view concerning the cause of protein destruction, and find that in this disease the toxic destruction is conspicuously less than in typhoid fever. An interesting finding in tuberculous patients was normality in the specific dynamic action of proteins, indicating that ingested protein was undergoing normal catabolism, and not serving merely as partial replacement of greatly wasted tissue proteins, as seemed to be the case in Coleman and Dubois' typhoid patients, who, probably because of the high level at which protein metabolism was being maintained, failed to show the normal specific dynamic action of ingested protein. That some toxic destruction occurred, however, was shown by their results on the minimum nitrogen excretion of the tuberculous organism, 5-6 grams per day, somewhat more than that which


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may he obtained iu healthy men, although distinctly less than the lowest obtainable iu typhoid fever. I may add that Ahlquist has found that while normal children, four to nine years of age, showed a retention of at least 20 per cent of the nitrogen absorbed, tuberculous children of the same age retained only 8-14 per cent. This is confirmatory experimental evidence for an impression we have long held concerning the growing tuberculous child.

Summarizing the results of the investigations cited ^^-h may conclude that in progi-essive tuberculosis the rate of metabolism is increased, as a result of the operation of two factors, hyperthermia and the toxic effect of a foreign protein upon protoplasm. As far as protein destruction is concerned, the second of these seems to be the more important, for hyperthermia per se does not increase nitrogen elimination in the nonnal subject. Yet it may well be that in the increased general metabolism induced directly by fever, the tissues become impoverished in tho.se protein-sparing constituents which serve to prevent protein destruction at similar temperatures in health, and that protein may for this reiison be drawn upon to maintain the high level of total energj' transformation.

In less severe, afebrile tuberculosis such tissue destruction as does take place is probably the result of the action of some poison upon the cells. McCann and Barr's results justify the conclusion that in such cases basal metabolism is but little altered, and protein metabolism but slightly above the normal. Even Grafe, who noted an increase in basal metabolism which at times reached 80 per cent, comments on the marked conservation of the


afebrile tuberculous organism for its protein nitrogen.

Going back to reconstruct our chain of chemical factors involved in the progression of tuberculosis we have the tubercle bacillus as the first link, a wax-armored microorganism, maintaining itself in necrotic tissue, picking and choosing its nutriment from the heterogeneous mass set before it, utilizing the glycerol of hydrolyzed fats, and probably building its wax therefrom, taking ammonia from certain of the amino acids produced in the digestion of dead protein, utilizing others directly to speed up the process of synthesis of its own protein, autolyzing to a slight extent, sufficiently to sensitize the surrounding host to its difl'usible protein products, being carried by the lymph, by jihagocytes or otherwise, to new soil, there to be met by a non-specific foreign body response, which in the end operates to produce anemia and death of the isolated cells. Then we have the failure of that dead tissue to autolyze, perhaps because of the presence of ferment-inhibiting substances within the bacillus, the phenomenon of caseation. Finally, there is more or less absorption of foreign protein from that focus, that of the bacillus itself and that of the disintegrating tissue, both toxic to the body protoplasm, both capable of causing fever and stimulating the metabolism of the host, so that in severe cases the typical picture of consumption ensues. I feel that this is a very sketchy treatment of a tremendous subject, and that some of the conclusions drawn may prove incorrect, but they may sene to draw attention to the intricate chemical mechanisms involved in the disease.


THE DISSEMINATION OF BACTERIA IN THE UPPER AIR PASSAGES

III. THE RELATION OF BACTERIA TO THE MUCOUS MEMBRANES

By Arthi'r L. Bloojifield

(From the Biological Division of the Medical Clinic, Johns Hopkins University and Hospital)


In the preceding papers of this series ' it was shown that bacteria and foreign particles introduced into the mouth are carried back by definite currents and rapidly eliminated. In certain cases, to be sure, foreign organisms may persist for long periods of time, but in such instances they are usually found in a focus of diseased tissue such as an infected tonsil. The free nuicous surfaces of the mouth and tongue, on the other hand, preserve a fairly simple and constant flora and foreign organisms deposited thereon are usually rapidly eliminated. The question, therefore, arises as to what prevents the removal and elimination of the "normal" flora. Why does the tongue not rid itself of its habitual flora just as it does of foreign organisms? It has usually been assumed that the harmless "mouth bacteria"' were growing freely in the buccal secretions just as they might in


a test-tube of broth. But the above considerations would seem to indicate that some flrmer union must exist between the microorganisms and the mucous membranes of the host. The present communication deals with the nature of this union and its mechanism.

Methods The general method of approach was based (ui the following considerations. If the nu'mbers of the liabitual "uormal"' flora of a healthy mucous membrane are merely growing freely on that surface in the mucous layer, it should be possible by some process of intensive cleansing to remove a large part or all of such organisms. If bacteria still persist in spite of such procedures, one must assume a very close affinity, both functional and anatomic, between the organisms and the mucous mem


July, 1922]


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brane, quite analogous to that which obtains in the case of a foreign pathogen and a focus of diseased tissue.

In the first group of experiments attempts were made to remove the bacterial flora of the tongue by washing with water. The individuals studied were all healthy men with clinically normal buccal cavities. The tongue was the point of attack because it lent itself most readily to the experimental procedure.

Exp. I. Dec. 8, 1921. The mouth was thoroughly rinsed with water and the anterior half of the tongue was then alternately flushed and scrubbed with sterile gauze pledgets for five minutes. Cultures were made before and after the procedure from approximately the same spot on the tongue and they were plated by similar methods on various media. Results of Cultures: (aerobic methods only).


Culture before washing

( 1 ) *> Gram-negative cocci

(2) Many M. Tetragenus

(3) «> non-hemolytic strepto cocci

(4) Many hemolytic influenza

bacilli


Cullure after ^cashing

(1) °° Gram-negative cocci

(2) Many M. Tetragenus

( 3 ) '^ non-hemolytic streptococci

(4) Many hemolytic influenza bacilli


Comment: The cultures showed no qualitative or quantitative change in the flora after the above procedure.

Exp. II. Jan. 3, 192 2. The same procedure was employed, but the washing was kept up for fifteen minutes.


Culture before washing

(1) °o M, Tetragenus

(2) A few Gram-negative cocci

(3) Many grey streptococci

(4) Many green streptococci

(5) A few p i n-p o i n t alpha

hemolytic colonies (not identified)


Culture after washing

(1) <» M. Tetragenus

(2) A few Gram-negative cocci

(3) Many grey streptococci

(4) Many green streptococci

( 5 ) A few p i n-p o i n t alpha hemolytic colonies (not identified)


Comment: After washing the tongue with water for fifteen minutes there was no qualitative change in the flora. In this experiment there seemed to be a slight quantitative decrease in the number of colonies but it was within the limit of error of swab-culture technique.

Exp. III. Jan. 5, 1922. In this experiment it was decided to push the effect of washing with water to the limit. Three procedures were carried out successively. First the mouth was thoroughly rinsed, then the anterior part of the tongue was scraped with the teeth and finally it was scrubbed with sterile gauze. These three manoeuvers were repeated continuously for thirty minutes. At the end of this time the tongue was irrigated with five liters of water. Cultures were made from the same spot on the anterior tongue before the experiment, after the thirty minute period of washing, after thirty-five minutes, and after sixty minutes.


Culture before washing

(1) =0 M. Tetragenus

(2) °° Gram-negative cocci

(3) Many green streptococci

(4) Many grey streptococci

(5) A few hemolytic influenza

bacilli

(6) A few Staph, aureus


After 30 minutes

(1 ) =° M. Tetragenus

(2) °° Gram-negative cocci

(3) Many green streptococci

(4) Many grey streptococci

(5) A few hemolytic influenza

bacilli


After 35 minutes

( 1 ) =0 M. Tetragenus

(2) °° Gram-negative cocci

(3) Many green streptococci

(4) Many grey streptococci

(5) A few hemolytic influenza

bacilli

(6) A few Staph, aureus


After 60 minutes

(1) 00 M Tetragenus

(2) =» Gram-negative cocci

( 3 ) Many green streptococci

(4) Many grey streptococci

(5) A few hemolytic influenza

bacilli

(6) A few Staph, aureus


Comment: After washing for thirty minutes there was no qualitative or quantitative change in the cultures save that Staph, aureus which was present in very small numbers in the control was absent. Inasmuch as it was recovered in the two following controls, this absence in one culture must be an experimental error which is to be expected with the crude swab method where only a few organisms are present.

Exp. IV. Jan. 10, 1922. The mouth was thoroughly rinsed with water and the tongue was then scrubbed with water and a stiff toothbrush with frequent irrigations. At the end of fifteen minutes the scrubbing had progressed to a point where the tongue began to bleed, and the experiment was discontinued. Cultures were made before and after the scrubbing from the same site.


Culture before scrubbing

(1) Many M. Tetragenus

(2) A few hemolytic influenza

bacilli

(3) Many Gram-negative cocci ( 4 ) Many green streptococci

(5) Many grey streptococci

( 6 ) A few Gram-positive

diphtheroids Comment: There was no quantitative change in the flora


Culture after scrubbing

(1) Many M. Tetragenus

(2) A few hemolytic influenza

bacilli

(3) Many Gram-negative cocci

(4) Many green streptococci ( 5 ) Many grey streptococci

( 6 ) A few G r a m-p o s i t i v e

diphtheroids demonstrable qualitative or after the above procedure.


Discussion ok Experime.n'ts I-IV.

It will be seen from the control cultures that the organisms present on the tongue at the start of the experiments were members of the "normal flora" — that is to say, organisms which are present in practically all healthy mouths and which are found widely spread throughout the mucous membranes of tongue, cheeks, tonsils and pliaryux — with the exception of the hemolytic influenza bacillus, and the staph, aureus in Experiment III. While some of the bacteria were undoubtedly removed in the washing process, the number was relatively so small that there was no demonstrable change in the appearance of cultures made after the most vigorous sort of a scrubbing process. It becomes clear, then, that the members of the "normal habitual" mouth flora are not simply growing free in the mouth secretions but that they are intimately bound to the mucous membrane just as are foreign organisms in a focus of infection. Their complete removal is apparently no more readily ett'ected than that of diphtheria bacilli, for example, whicJi are breeding in the depths of an infected tonsil. The nature of this adhesion to the mucous membrane will be discussed below, but further attempts at removal by other means will first be described.

The preceding experiments had indicated that the actual breeding place of the mouth flora must be beneath


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the layer of mucus and very uear to the actual cells of the mucous membranes. Means of drawing the organisms out and making them more accessible to the washing effect of irrigations were considered. It seemed possible that strongly hypertonic solutions might produce the desired result. Sodium chloride was therefore tried.

Exp. V. December 13, 1921. The mouth was washed and the tongue was then scrubbed with saturated salt solution for ten minutes. At the end of this time it was irrigated with two liters of water.

Culture before procedure Culture after procedure

(1 ) =» M. Tetragenus ( 1 ) =» M. Tetragenus

(2) Many Gram-negative cocci (2) Many Gram-negative cocci ( 3 ) Many n o n-h e m o 1 y t i c ( 3 ) Many n o n-h e m o 1 y t i c

streptococci streptococci

(4) Many hemolytic influenza (4) Many hemolytic influenza bacilli bacilli

Comment: No demonstrable qualitative or quantitative change in flora.

Exp. VI. A piece of rubber dam with a window about 3 cm. square was placed on the tongue. This allowed manipulations without contamination of the experimental area by the saliva. A paste of sodium chloride was applied to the tongue. The subject almost immediately experienced a strong burning sensation. After three minutes the salt was washed off and the tongue thoroughly irrigated with water. The salt pack was then reapplied and the above procedures repeated for fifteen minutes. Cultures were made before and at the end of the experiment.

Culture before salt application Culture after salt application

(1) ^ M. Tetragenus (1) °° M. Tetragenus

(2) Many Gram-negative cocci (2) Many Gram-negative cocci

(3) A few hemolytic influenza (3) A few hemolytic influenza

bacilli bacilli

(4) A few green streptococci (4) A few green streptococci

(5) A few grey streptococci (5) A few grey streptococci

(6) 1 colony Staph, albus

Comment: No quantitative or qualitative change made out. Exp. VII. The tongue was scrubbed and irrigated with a saturated solution of sodium bicarbonate for five minutes.

Culture before bicarbonate Culture after bicarbonate

(1) °° M. Tetragenus (1 ) >» M. Tetragenus

(2) Many Gram-negative cocci (2) Many Gram-negative cocci (3)0° non-hemolytic strepto- (3)°° non-hemolytic strepto

cocci cocci ( 4 ) A few G r a m-p o s i t i v e ( 4 ) A few G r a m-p o s i t i v e diphtheroids diphtheroids

Comment: No qualitative or quantitative change made out. E.\p. VIII. Similar to Experiment VI, except that a pack of bicarbonate of soda was applied for fifteen minutes.

Culture before procedure Culture after procedure

(1) » M. Tetragenus (1) °° Tetragenus

(2) Many Gram-negative cocci (2) Many Gram-negative cocci

(3) A few hemolytic influenza (3) A few hemolytic influenza

bacilli bacilli

(4) A few green streptococci (4) A few green streptococci

(5) A few grey streptococci (5) A few grey streptococci Comment: No qualitative or quantitative change made out. Exp. IX. It was thought that possibly the adhesive qualities

of mucus might have prevented to some extent the removal of organisms by the foregoing procedures. Sodium carbonate


which is a solvent for mucus was therefore tried. The tongue was scrubbed and irrigated for fifteen minutes with two per cent sodium carbonate — the greatest concentration that could bfe endured.

Culture before procedure Culture after procedure

( 1 ) =0 M. Tetragenus ( 1 ) =° M. Tetragenus

( 2 ) Many Gram-negative cocci ( 2 ) Many Gram-negative cocci

(3) A few green streptococci (3) A few green streptococci

(4) A few grey streptococci (4) A few grey streptococci Comment: No demonstrable qualitative or quantitative

change.

Summary

In summary then it appears that it was found impossible to alter the character of the flora of the tongue as revealed by aerobic surface cultures by any of the above procedures. Many organisms were undoubtedly removed, but the experiments show clearly that the normal mucous membranes of the tongue can not be washed free of bacteria. It seems fair to conclude, therefore, that the organisms which are found in the normal mouth so uniformly as to constitute in a .sense a normal flora are not growing free in the secretions but are in actual and vital contact with the mucous membrane.

A study of stained sections of the tongue shows a mucous membrane not unlike the skin in structure. The covering layer of stratified squamous epithelium is thrown up into microscopic folds and the actual living surface is more or less covered by desquamated shrunken cells which again suggest a cutaneous keratosis. Furthermore, the mucous membrane is everywhere perforated by the orifices of the minute mucous glands. We believe that the actual niduses of growth of the bacterial flora is in these crevices in the epitlielium and in the gland orifices. Here there is a localization of the bacteria. As they multiply, certain organisms are thrown oft' and are removed just as are foreign particles or bacteria experimentally introduced. Hence we have a constant stream passing out from a source which is relatively fixed and therefore allows the persistence of a bacterial species on the normal mucous membranes of the mouth for indefinite periods of time.

If the above conclusions are correct, it seemed probable that a foreign organism placed on the tongue could be readily washed away, inasmuch as there was no actual growth in the sujicrficial layers of the mucous membranes. The next experiments were aimed at settling this point.

Exp. X. The entire twenty-four growth from an agar slant of Sarcina lutea was smeared on the anterior part of the tongue. A culture was immediately made. The tongue was then vigorously scrubbed with water for five minutes and another culture was made.

Culture immediately after Culture after washing

application of Sarcina for five minutes

(1 ) Innumerable colonies of (1)2000 colonies Sarcina Sarcina — plate covered lutea.

with a confluent yellow (2) The usual tongue flora in growth. normal proportions.


July, 1922]


255


Comment: In contrast to the preceding experiments it was possible in ilve minutes to wash away the vast majority of the artificially implanted organisms.

Exr. XI. A similar experiment was performed, a twentyfour hour agar slant of Staphylococcus albus being employed.

CuUure immediately after Control culture applying albns

Usual tongue flora Innumerable colonies of albus

(no Albus) — confluent growth

After washing for o minutes After washing for JO minutes

About 200 colonies albus + About 100 colonies albus +

usual mouth flora usual mouth flora

Comment: Washing for five minutes removed almost all of

the Staphylococci introduced, although even after ten minutes

a few colonies still were present.

Discussion of Experiments X and XI.

It is seen that even ten minutes vigorous washing did not serve to remove all the colonies of a foreign organism experimentally ai)])lied. The reduction being so vast ami striking, however, in contrast to the response of the indigenous organisms under similar treatment, it may fairly be concluded that an essential difference exists in the relation to the mucous membrane of an organism adapted to growth in the mouth and a foreign organism experimentally introduced. The latter behaves like an inanimate foreign particle.

The next point to be tested was the following. If the actual growing and multiplying fraction of a mouth inhabitant sucli as Streptococcus viridans is located in the mucous membrane and if there is a balance of power at this point among the various members of the flora which regulates their relative proportions, the reintroduction of an autogenous strain in large quantity should be followed by a rapid disposal of the organisms intro duced just as is the case with foreign bacteria. This question was approached as follows.

A culture was made from the tongue of Individual B. The predominating strains of green streptococcus and of M. tetragenus were isolated.

Exp. XII. A culture was made from the tongue and immediately thereafter the twenty-four hour growth from a bloodagar plate of the autogenous strain of S. viridans was placed on the tongue. Cultures were made immediately after application and two hours later.

Culture immediately after Control culture before inoculation loith

inoculation S. viridans

Usual tongue flora Plate almost pure S. viridans

CuUure two hours later

Same organisms as in control and

in about same proportions.

Comment: The autogenous strain of Streptococcus, when re-introduced, did not colonize, but was eliminated just as were foreign organisms.

Exp. XIII. Procedure similar to that of Exp. XII, an autogenous strain of M. tetragenus being employed.


Culture immediately after Control inoculation icith M. tetragenus

Usual tongue flora Plate almost pure Tetragenus

Control two hours later

Same organisms as in control and in

about same proportions

Comment: Result similar to that in Experiment XII.

Discussion of Experiments XII and XIII.

In previous studies in which foreign organisms (B. coli, Staph, albus, B. influenza', B. Friedliinder) were experimentally applied to the tongue, it was found that such organisms were rapidly eliminated, usually within a few hours.- From the present experiments it appears that a culture of an autogenous strain, when reintroduced, is eliminated just as is a foreign organism. It may be that even a single subculture on artificial media alters the bacteria in such a way that its adaptation is lost, but it seems more likely that as soon as the organism is withdrawn from its actual nidus of growth it is no longer able to reattach itself but is doomed to elimination. Be this as it may, we wish to use this experiment merely to emphasize further the point that the persistence of any organism on the buccal mucous membranes implies a close anatomico-biological reaction at the site of growth.

The facts brought out so far deal mainly with the so-called "normal" flora of the mouth — namely, groups of organisms so constantly and persistently present in healthy people that one must assume a high degree of general adaptation between the race of organisms as a whole and the population at large.

There exist, however, other groups of bacteria which are present much less frequently, and yet are often encountered in the normal mouth. Pneumococci, for example, are found in from thirty to fifty per cent of the general population, influenza bacilli in from twenty to thirty per cent, and hemolytic influenza bacilli in from thirty to fifty per cent. Now, although these organisms as a whole are clearly less highly adapted to growth on human mucous membranes than those considered above, the question remains open as to whether in the individual harboring them a ditterent type of adaptation exists. More specifically stated, can the influenza bacillus or the pneumococcus, when growing in the mouth of a healthy person, be more easily removed than the members of the "normal" flora?

This point was a rather difficult one for systematic experimental approach, but certain suggestive observations were obtained. It was found that individual B was carrying hemolytic influenza bacilli on the tongue. Various attempts were made to remove this organism as follows :

December 8 — Tongue scrubbed with water five minutes. Decem-ber 12 — Tongue scrubbed with saturated salt solution five minutes.


256


[No. 377


December 13 — Tongue scrubbed with saturated salt solution fifteen minutes.

December 16 — Tongue scrubbed with saturated sodium bicarbonate for fifteen minutes.

Cultures made before and after the above procedures showed hemolytic influenza bacilli in similar numbers. On January 3, however, these organisms disappeared spontaneous!}-, nor were they recovered thereafter.

This observation is in accord with the assumption which seems inevitable to us that any organism which persists on a normal mucous membrane for more than a few hours is in vital biological relationship to the mucous surface. Were this not so, it would be removed as a foreign particle by the normal eliminative mechanism. The bearing of this principle on the significance of the "carrier in the spread of respiratory disease will be discussed at another time.

Summary The above experiments clarify some of the puzzles of mouth bacteriology. They explain, in the first place, why the organisms which have been regarded as members of the "normal"' flora are able to persist and are not eliminated as are foreign bacteria experimentally introduced. They persist because their nidus of growtli is actually in the mucous membrane — the organisms which are disdiarged from these niduses are already doomed to be removed. The situation is crudely analogous to that of the blood cells which are swept away from the parent cell-nests in the bone marrow. Indirectly, we also get the explanation for the fact, which at first seemed remarkable, that foreign organisms experimentally introduced into the mouth are so completely and rapidly eliminated, for it becomes apparent that to persist such organisms need more than a favorable salivary medium. — an actual adaptive reaction with the mucous membrane, which is doubtless a biological process of the highest complexity and subtlety, must take place. The question of the importance of the saliva in the growth of organisms in the mouth is also clarified. The saliva, as a rule, plays no significant part either favorable or inhibitory. It now becomes intelligible why bacteria, such as colon bacilli, which thrive in saliva in vitro, are not found in the


normal mouth, whereas organisms such as pneumococci or influenza bacilli, which die in saliva in vitro, are frequently present. The colon bacillus, despite its growth in saliva, is unable to react with the mucous membrane so that colonization there can take place; pneumococci, on the other hand, are able to do so, and their actual growth processes probably occur in the mucous mem branes largely or entirely independently of overlying secretions. It is readily apparent why an organism located in a focus of infection does not spread from such a focus over the entire mouth and throat, and it is clear why an excess of an autogenous strain when reintroduced is promptly eliminated. No sooner has the organism been discharged from its nidus of growth than it is already altered to such a degree that return to colonization is unlikely. It becomes clear, then, that these biological adaptations are most delicate and sensitive, and further analysis may throw light on the essential mechanism of infection and of the production of the carrier state.

Furthermore, it becomes apparent that to speak of a "normal" flora is onlj' appropriate in a broad clinical sense, indicating groups of organisms widely and constantly disseminated and usually harmless. From the biological point of view any organism which has established actual growth on a normal mucous membrane is, for the time being at least, just as normal an inhabitant as any other bacterium similarly adapted.

Conclusions

1. No bacteria of any soi-t entering the moudi can persist free in the secretions for more than a few hours.

2. More permanent presence implies a biological adjustment to growth on the mucous membrane whereby the organisms become localized at the site of growth.

3. Such adaptive power is relatively constant for large groups of bacteria in relation to the general population, but varies widely in the case of individual organism and individual host.

REFERENCES

1. Bloonifield, A. L.: Bull. Johns Hopkins Hospital, 1922, (April); Amer. Rev. Tuberculosis, 1922, V, 903.

2. Bloonifield, A. L.: Amer. Rev. Tuberculosis, 1920, IV, 247.


THE JOHNS HOPKINS HOSPITAL BULLETIN


The Hospital Bulletin contains details of hospital and dispensary practice, abstracts of papers read and other proceedings of the Medical Society of the Hospital, reports of lectures, and other matters of general interest in connection with the work of the Hospital. It is issued monthly. Volume XXXIIl is in progress. The subscription price is $4.00 per year.

(Foreign postage, 50 cents.) Price of cloth-bound volumes, §5.00 each.


July, 1922]


257


THE CHANGES IN THE OMENTUM OF THE RABBIT DURING MILD

IRRITATIONS; WITH ESPECIAL RFERENCE TO THE

SPECIFICITY OF THE MESOTHELIUM

By R. S. Cunningham

(From the Anatomical Laboratory of The Johns Hopkins University)


The ease with which cells can be observed in the omentum, without the necessity of subjecting them to much niani])ulation, has caused this structure to be widely utilized in studying special characteristics of individual types of cells and their interrelationshii)s, under both normal and experimental conditions. In a general way the histological investigations which have been conducted on the omentum can be divided into two groups. The investigations in one of these groups have been concernetl with the genetic classification of the cells which constitute tlie taches laiteuses, particularly with regard to their lupmatological relationships. The other group includes numerous studies which have been made on the characteristics and relationships obtaining between the three great groups of cells which constitute the general framework of the organ — the clasmatocytes, the fibroblasts, and the serosal lining cells. The sero.sal lining cells have received particular attention in this regard and much work has be^n done on their reaction to irritants and on the role played by them during the formation of adhesions.

The stutly of the lining cells covering the omentum has three salient features : the relation of these cells to the production of the free cells which are found iu the exudate of the peritoneal cavity during iuflanunatious, the relation of the serosal cells to the clasmatocytes, and finally, the relation of these lining cells to the fibroblasts which constitute the greater part of the framework of the organ. The first two questions are closely related and have been discussed elsewhere;** and the conclusion was reached that the majority of the free cells of the exudate were derived from the clasmatocytes, which, in the omentiim, occur chiefly iu the neighborhood of the blood-vessels and in the taches laiteuses, though they are also to be found scattered through the entire omental network. The serosal lining cells were found to have no part iu the formation of the true jihagocytic mononuclears of the exudate, and no genetic relationship with the clasmatocytes.

In regard to the relationship of the lining cell to the fibroblast there has been much diversity of opinion; many of those who have studied the omentum during inflammatory reactions have concluded that these cells are capable of definite interchange in morphological and functional characteristics. Others have described similar


appearances in the omentum but have left unsettled the question of the interchangeability of fibroblasts and serosal cells. And finally, a few have maintained the specificity of the serosal cells and the fibroblasts in the general serous membranes, but without any direct consideration of the omentum itself.

Ranvier -"• -' was the first author to discuss the relation of the lining cells of the omentum to the other cells constituting tlie framework of that organ. His views have been widely accepted, and his conclusions supported by the observations of many workei-s ; Cornil," Marchaud, '""' RJoloff-^-'* Borst,' and others. Ranvier describes the omental lining cells as changing during a mild inflammation from characteristically outlined i)avement cells to star-formed cells with long processes wliich lie considers as wholly indistinguishable from the fibro blasts, and he concludes from such observations that these two types of cells are identical, at least so far as their reactions to irritation are concerned, and hence must be capable of easy interchange.

Among the more modern workers on this subject are Weidenreich "" and Schott;-" these observers, after long and minute study of the reactions of the peritoneal membranes, conclude that the lining cells of the omentum cannot be distinguished from the fibroblasts and that the two types of cells are entirely interchangeable. Schott, in his study on the cells of the serous cavities, derives the free cells of the peritoneal exudate from the serosal lining cells and the fibroblasts indiscriminately.

Kiyono,'^ on the other hand, after study of omental spreads and sections from aninuils vitally stained with carmine and trypan blue, states that he could never find any indication of a transition from lining cells to fibroblasts. He says that normally the lining cells form a continuous, characteristic cell-series over the entire omental surfaces, and that they can easily be differentiated from the fibroblasts. But he .states that during inflammation a portion of the lining cells assume a form which greatly resembles that of the fibroblasts, and he therefore admits the possibility of a close relationship between the two. He is unable to state with certainty if the lining cells do, or do not, participate in the formation of new connective tissue.

Marchand's '^ description of the omentum in fresh preparations is very exact and deserves considerable


258


[No. 377


attention nnd credit. He studies the omentum after the introduction of a suspension of lycopodiuni spores, and describes the mesothelial cells, seven hours after the injection, as rounding up and their cytoplasm becoming finely vacuolated; these cells stand out clearly from the surface of the omental network. Twenty-four hours after the injection he found many delicate protoplasmic structures of various forms: Hat, irregular masses of jirotoplasm with numerous processes which appeared and disajjpeared and long spindle-formed cells with finely vacuolated protoplasm. He states that the origin and relationship of these large, variously formed elements could not be determined in fresh preparations. From stained sections and preparations from later stages he concludes that all of the lining cells proliferate to form fibrillary structures, and later a part of the.se form fibrils and new connective tissue, the other part forming more lining cells. The former are the star-formed irregular cells, to which he assigns amoeboid capabilities and which he believes, participate with the clasmatocytes in the formation of giant cells, while the latter become again the usual flat mesothelial elements.

Biittner introduced cultures of stai)h_ylococcus aureus or weak silver solutions into the peritoneal cavity of guinea-pigs to produce inflammations. He studied the omentum carefully at varying intervals following this procedure and says, because of the complicated structure of the omentum, it is difficult, after irritation, to differentiate between the types of cells or decide whicli groups are undergoing mitosis. Although he leaves open tlie question of a possible transformation of lining cells into fibroblasts, he thinks his experiments show that the omentum is covered by a single layer of cells which react during inflammations as true epithelium. This was jiarticularly well demonstrated in his sections cut perpendicularly to the surface where the dift'erences between the cells on the surface and those constituting the subserosal tissue are very striking. In sections taken three days after the commencement of the inflammation he found areas showing evidences of regeneration of the lining cells. After seven days he found adliesions wliere the lining cells had entirely disappeared. Where they liad regenerated there were no adhesions.

Doniinici "• '- thinks the cellular part of tlie omentum is composed of several .syncytium.s ; the syncytium of liuing cells, of fibroblasts, of adventitial cells, of fat cells, etc. These syncytiums are not entirely separated from one anotlier, but are connected by means of anastomoses between the cells. Scattered through the whole structure are free cells among which he classes the macrophages. These macrophages ari.se by differentiation from the various syncytiums which belong collectively to tiie connective-tissue series. According to him, the endothelial syncytium of lining cells is to be considered as modified


connective tissue, which during inflammation gives rise to a large number of macrophages.

Von Bungner * thinks that both serosal lining cells and fibroblasts participate in the formation of granulation tissue, but he found that they I'etained their individual characteristics during the i)rocess, and that tlie cells derived from them could be easily difterentiated into young connective-tissue elements and young lining cells. He could not detect any transition of lining cells into fibroblasts, but he thinks that the lining cells play a more imjiortant role in new tissue formation than is usually attributed to them.

Yon Brunn,-- ' after minute study of inflammatory processes in the peritoneal cavity, concludes that the lining cells are of a specific epithelial type which never gives rise to fibroblasts and that young lining cells are never formed from fibroblasts during the processes of inflamnuition and the formation of new tissue. In referring to tlie work of the French authors he suggests that perhaps their views were so unanimously in support of a genetic relationship between lining cells and fibroblasts, because they based their conclusions principally upon observations made on spread preparations of the omentum, in which material. Yon Brunn states, it is extremely difficult, if not impossible, to obtain a clear picture, the underlying fibroblasts becoming easily con fused with the sujierimposed lining cells.

Tschaschiu, -*'•-" after a prolonged study of the serous cavities with vital dyes, concludes that the .serosal cells, because they renuiin totallj' unstained, are specific ; but in the omentum this specificity is extremely difficult to establish. In connection with this question it is interesting to note that he quotes Maximow as believing that the fibroblasts in the omentum and mesentery retain a more embryonic character than in the genei-al connective tissues, and that it is more difficult to dift'erentiate tiie fibroblasts from the resting wandering cells in the omentum than elsewhere.

Finally the work of Foot" on wound healing in the omentum must be referred to in this connection as it has a certain bearing on the results of these observations. In his experiments he found a verj' remarkable reversal of type in the case of the connective-tissue cells and of the endothelium to an undifferentiated mesenchyme, from which there arose new vessels, connective tissue, and phagocytic endotheliocytes." These conclusions are of interest in regard to the relation of serosal lining cells to fibroblasts, because, if these two groups of cells are very closely related, it would .seem nu>st likely tliat the reactions described by Foot would involve the serosal lining cells as well as the connective-tissue cells.

In general, then, it seems that the majority of those investigators who have studied tlie omentum witii particular regard to the question of the changes which take place during irritations have concluded that the flbro


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blastic and luesotlielial eleiueutis are morphologically and functionally interchangeable. But this view has not been accepted by others, and the ijrincipal evidence in favor of tlie specificity of these cells has been obtained from the study of sections of the serosal URMnbranes covering the various organs. The contradictory conclusions arrived at may have been the result of tlie various methods employed, or due to some fundamental ditt'erences between the cell potentialities as they appear in the omentum and in the general subserosal tissue, as suggested by Maximow.

It has been long known that tlie serosal lining cells underwent ciianges in size, shape, and general appearance when subjected to irritations. In attempting to determine the exact nature of these reactions it has .seemed essential to reinvestigate both the omentum and tlie other serosal membranes with evei"j' available methoil. The reactions of the mesothelial cells are obviously important in two connections; the fundamental relation which they bear to the fibroblast and in regard to the role which they play in the formation of adhesions. The present communication reports tlie results of the examination of the omentum in an effort to determine whether tlie changes which occur in the morphological and cytological characteristics of the .serosal lining cells, during mild irritations, indicate any genetic relationship, or functional similarity, between these cells and the tibrobla.sts.

The structure of the omentum of the rabbit is very <-haracteristic. There is a framework of blood-vessels wliidi anastomose freely without the intervention of a very elaborate capillary bed, that is, much of the circulation is arteriovenous in character. The larger vessels are surrounded by layers of adipose tissue and, in this way, the omentum presents the appearance of a flat membrane marked off by vessels and fat into irregular sections, or areas, of quite diverse sizes. Rich -' has shown, however, that there is a capillary bed which is not apparent with the usual methods of observation, but which becomes very prominent after the administration of histamine, which by dilating the capillaries makes them far more conspicuous. But not even after the administration of histamine can a very elaborate plexus of capillaries be observed in the thin membranous areas ordinarily outlined by the larger and medium-sized ves.sels. That these areas are non-vascular is ftir from true, but the vascularity is sufticieiitly decreased at these jilaces to make studv of the membiaiie as a whole entirely satisfactory.

The thin membranous part of the omentum consists of numerous cells surrounded by a mass of interlacing connective tissue fibrils and covere:l by a layer of flattened mesothelial cells, whose outlines, in the normal state, can always be demonstrated by the mosaic pattern which thev evidence after treatment with silver nitrate and sub


sequent exposure to sunlight. The special structures which are so evident in the omentum of the rabbit, called the taches laiteuses, are scattered more or less indiscriminately throughout the entire omentum. The taches laiteuses are collections of various types of free cells grouped about areas of increased vascularitj'. These cells have been widely studied in connection with the formation of free cells during inflammatory reactions in the peritoneal cavity, and in relation to general ha?niatological problems.

The omenta upon which the observations reported here were made were from a series of rabbits which had bad their peritoneal lining cells irritated by the injection of heterogeneous laked blood. Some of these rabbits were vitally stained by the intravenous administration of trypan blue, and a few received the vital dye intraperitoiieally in order to give the irritated serosal lining cells every possible ojjportunity to absorb and store the dye.

When the animal was readj' for study it was an;x?sthetized and the abdomen opened ; the omentnm was observed with regard to the general aiiiieaiance, occur rence of adhesions etc. Several small pieces were transferred to coverslips, which were then immediately sealed to the slide with vaseline; these preparations were sometimes stained with neutral red by Pajipenheim's method. In many cases small amounts of fluid were found in the peritoneal cavities of the rabbits and in such cases a small drop of this fluid was used to mount the fragment of omentum; in others Locke-Lewis solution or homologous .serum was used.

The remainder of the menibranous ]iart was carefully spread without touching the surface, a coverslip was dropped on it and the area cut out ; the coverslip with its layer of omentum,,was then immersed in a weak solution of silver nitrate for a few minutes, transferred to distilled water and exposed to direct sunlight until it showed a light brown; it was then flxed in 95% alcohol. Other preparations were rinsed carefully before being placed in silver nitrate, and still others were fixed immediately in alcohol, sublimate, or osmic vapor. From each animal at least three ]ire])arations were always made, one to be studied fresh, one silvered, and one fixed directly for control.

In the normal omentum, when the fresh ]jreiiaratioii, the ordinary stained spread, and the sjuead treated with silver nitrate were compared, the remarkable fact was noted that in the two former the mesothelial layer of cells could not be made out at all. There were nuclei which were suggestive, but these were in no case sufficiently distinctive to be diagnostic. As soon, however, as silver nitrate was applied, tlie superficial layer became entirely ajipareiit and distinctly separated from the other cells which were subjacent to it. It is entirely obvious that if in the normal, fresh omentum the cells could not be distinguished with ease, those in the irritated omen


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tuiii would be still more difficult to classify, so that the application of the silver method becomes eveu more important after irritation. In all studies on the fresh omentum it was impossible to determine the outline of the serosal lining cells, and hence the various types of cells present were very difficult to distinguish. In fresh unstained omenta from animals which had received two or three doses of laked blood the microscopic appearance was quite bewildering. In such a preparation only the nuclei and large numbers of granules surrounding them could be seen clearly. The cellular outline could be made out only in rare instances, and analysis of the type of cell depended entirely upon the appearance and reactions of their granular content. But by means of various vital and supravital dyes and by numerous control methods applied to the fixed material, the characteristics seen in the fresh could be analyzed to a considerable extent.

Three types of cells could always be observed. The first two were quite similar in nuiuy respects and were sharply differentiated from the third by certain characteristics of the vacuoles and granules which they contained. The two that resembled each other so closely had in common large, pale nuclei with one or two very highly refractive nucleoli, and contained varying numbers of highly refractive granules, all of which were of tlie same size and character. The arrangement and number of granules, however, within each cell permitted a division of these cells into two groups; one group having many granules which were usually distributed in one solid mass of quite varied shape, sometimes a crescent about one side of the nucleus, sometimes a belt surrounding the nucleus, and in a few cases they were arranged in several clumps about the nucleus. In all of the cells of this type the highly refractive granules were associated closely with the nucleus and were never seen branching out through the cell in any process-like arrangement. la the other type, this finding was just the reverse; the granules were scattered in quite irregular fashion from the nucleus, sometimes in a single, long, slender thread extending from each end of the nucleus, and again, in several irregular process&s of uneven size and length, suggesting the spread legs of some arachnida. In this last type the granule.s were fewer in number and often formed very slender and detached threads extending throughout the finer processes of these cells. These two types of cells were, respectively, the serosal liniug cells and the fibroblasts.

The third type of cell was the clasmatocyte, a cell whose character was indicated quite definitely by the great variation in the size of the vacuoles and granules which it contained. There were only a very few of the fine, highly refractive granules, but there were vacuoles of various sizes, and masses of hyaline material which were evidently phagocytic enclosures. The arrangement of these cellular enclosures was quite irregular, but


seldom extended as in the fibroblast into fine lines or threads; they were rather blunt and rounded as though in true pseudopodia. Most of these cells were rounded, however, and appeared as balls or spheres of massed granules and vacuoles surrounding the nucleus.

When a preparation such as that described above was mounted on a coverslip previously i)rei)ared by being coated with neutral red, the three types of cells presented a very different appearance. The clasmatocytes took up large amounts of the dye into their vacuoles and became masses of red globules of different sizes with a few interspersed, fine clear granules; while the other two types had only a few, fine, red droplets scattered among the clear granules. In the fibroblasts there were more red bodies than in the serosal cells and they were especially prominent in small enlargements of the fine processes.

The application of silver nitrate to the study of the characteristics of the omental cells during inflammatory changes proved to be extremely difficult to carry out successfully, because it was found that a certain amount of fibrinous and cellular debris always accumulated on the surface of an irritated area and cavised irregular staining with the silver. The use of silver nitrate to outline the margins of endothelial cells was first described by Von Eecklinghau.sen -- and has since proved a very important part of the technique in the study of bloodvessels and the linings of the serous cavities. What the nature of the reaction is, is yet in doubt. It was first thought to represent the impregnation of some intercellular cement substance which had the i)ower of combining with the silver. It has since been suggested that the reaction was more likely due to a peripheral concentration of chlorides in the cells an<l that the silver oxychloride which is formed is reduced in sunlight so that metallic silver is precipitated in the borders of the cells. This entire question is thoroughly reviewed by Macallum.^'

The cellular debris wliich is formed during the cour.se of an inflammation increases as the intensity of the irritation is augmented, and in this way the ajujlication of the silver reaction to the irritated omentum becomes increasingly difficult. It must be noted here that the successful application of this method could not have been considered as plausible until the theory of ])eripliei-al salt concentration had heeji suggested. I have found that if the technique be carried o)it veiy carefully the method may be u.><ed to great advantage in the study of mililly irritated omenta. When the silver nitrate was applied in very we^ik solutions, i/^ to %%, and the spread i>reparations were agitated gently while in the silver solution, and washed carefully before being exposed to the light, in some of the specimens the entire surface of each serosal cell had a brown appearance with a darker outline, and the entire layer of rounded or irregular cells became apparent. The use of this reaction to demonstrate


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the stages in the changing niesothelial cells, during the progress of an irritation, has proved quite successful and has established certain definite characteristics as most specific.

The first change observed in the layer of serosal cells was an irregular widening of the line which marked their l)oundaries. This took i)lace very iri-egularly so that there was no longer a perfect apposition of the cells each to each. In an omentum which had been subjected to a mild irritation there were many places where one could see groups of surface cells with perfect api)osition, side by side with other groups in which considerable gaps occurred between the cell margins. This is shown in Figure 1 ; on the left of the figure there are .several mesothelial cells which have outlines, dianged only very slightly from the normal ; while on the right the cells are beginning to sejiarate and there are numerous spaces between them. This type of reaction is entirely natural in a structure like the omentum, because consistent changes could never be expected over the entire surface at one time. Every stage of separation of the cells from small gaps in the thin, continuous, brown line of silver precipitate, seen in the normal omentum, to areas the width of nuclei were observed, but in these early stages of irritation there were almost always points of contact except where an occasional cell had been desquamated. In many places the cells were bound together by numerous, tine jirocesses as seen between the two cells shown in Figure ;*.*

At the same time that the withdrawal of the borders of tiie cytoplasm of the serosal cells was taking jjlace there was a beginning increase in the thickness and density of tlie cytoiilasm. This rounding up of the serosal cells has been often observed in the serous membranes in general, but in the omentum the changing niesothelial cell has usually been confused with the branching fibroblast. The cells shown in Figure 2 have separated in part from each other and have increased somewhat in thickness


This brings up the question of whether the surface cells form a true syncytium, as claimed by Kolossow, or whether these cells are merely in apposition, as is the case with many tissues. Kolossow " describes the lower part of the serosal cells as anastomosing with the neighboring cells by means of fine processes, while the upper part of the cell merely lies in apposition to the surrounding cells. If this opinion prove correct, it will be easy to understand the drawing apart of cells with the formation of multiple, small openings between the anastomosing processes. It is evident that the observations cited above, at first sight, strongly support Kolossow's idea that the surface cells are bound together by numerous processes. But long and careful study of large numbers of preparations has not convinced me that these are anastomoses In the sense of syncytia, but rather served to indicate a union of a less intimate character. There is, however, very little doubt but that there is some type of junction which binds these cells together more closely in some places than in others. The nature of this union has so far not been analyzed, and is a most difficult question to settle.


but have not changed very materially from their normal, flat morphology. Here and there in early stages a single cell was found to have been desquamated and a larger area than could have been formed by mere separation was discovered, hut other places were to be seen in every prejjaration where the cells were regularly spaced as if they had each retained their normal positions and merely had become more and more rounded up. In these early stages of irritation the processes which the serosal lining cells manifested were very striking and were quite characteristic, varying from cell to cell only in the matter of size. They were .seldom very long, though they occasionally reached 8 to 10 microns in length. They were sharply pointed, somewhat similar in shape to the old-style church steeple. They generally extended at right angles to a line drawn at their base tangential to the periphery of the cell, and in cases of more rectangular cells were perpendicular to the sides. These projections were usually fine, very sharply-pointed, and about 3 to 5 microns long, although, as already stated, they were sometimes even longer. Very occasionally the ends were blunt or forked instead of pointed, but this was not very common, and neither did these processes have bulbous enlargements on them, nor were they divided to form two, definite, characteristic branches. These processes are well illustrated in Figures 2, 3 and 4. Figure 3 is a microphotograph of a prejiaration in which a large rectangular cell was becoming separated from a smaller one, and the processes which still hold them together are well shown. In Figure 2 there are to be seen the fine, long processes characteristic of cells which have but recently separated from each other. It is important to note here that almost every individual jireparation of an omentum from an animal with relatively mild peritoneal irritation showed several stages of the progressive changes which represented the course of an irritation. This is easily understood because the omentum may be exposed in part, and protected in part at the same time. In every specimen, however, there was a general prej)onderance of a certain type, and in this way it was easy to establish the characteristics which represented the several different stages in the process. I mention this because it is so veiy easy to misinterpret histological appearances and it is esseutial to be assured that the particular area which is being examined represents a definite stage in the series of events which are being studied.

In the stage following tlie one described above the niesothelial cells had continued their increase in thickness and the rounding up of their cytoplasm, and therefore the edges of the cells were more widely separated. As this rounding up progressed, the number of processes on the cells diminished in number, but those that remained were in general somewhat larger. One cell in Figure i has a process of this type. In this stage of the irritation the omentum was often covered in large areas


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by cells scattered very regularly, but separated from each other by quite considerable areas of denuded tissue. Figure i is from such an area and here it is quite certain that no cells have been desquamated. In Figure 3 it is probable that cells have been desquamated, as the distances between the cells appear greater than usual for so early a stage. In preparations from animals which had been exposed to still longer irritations the mesothelial cells had rounded up still further, they had been desquamated in a greater degree, and were in large measure obscured by the numerous clasmatocytes which had reached the surface and were adhering to it, as well as by some cellular debris. The processes of the serosal cells had not materially changed in type, but were somewhat more irregular than in earlier stages, probably due to the attempt of some cells to flatten out and recover the denuded areas while others were continuing to round up as the result of the continued irritation. Mitotic figures were extremely difficult to find in jireparations of spread omenta, and the evidence of cellular division was based upon sections taken from the same omenta and appropriately staine<l. Active mitotic figures were relatively few even in these sections but many cells were to be seen in various stages of division.

When the silver preparations are successful, they are very brilliant and demonstrate that the morphological changes which take place in the mesotiielial cells are wholly characteristic and indicate the specificity of this cell-type. When the technique outlined above was combined with previous vital staining the definition of the serosal lining cell as a specific type became even more exact, since the method of storing the vital dye was equally as specific as the morphological changes.

In all of the omenta from animals vitally stained with trypan blue, the mesothelial cells showed very interesting reactions. In the normal these cells have been found to store the dye in a rather characteristic manner, the dye being collected in a small clump in a definite part of the cell ; on progressively increased staining the dyegranules extended around the nucleus in a perinuclear rosette. The normal staining of the serosal lining cells has been described in detail elsewhere." Studying the reaction of the cells which had been irritated, the deposits of blue were found to deviate somewhat from those in the normal. As the cells began to separate and round up there was a tendency of the blue droi)lets, which had been rather specifically located in one end of the cell, to be dispersed somewhat more widely in the same general part of the cell. In Figure 2 the blue granules can be seen as groups of definite black spots in one end of each cell, i.e. opposite one pole of the oval nucleus. But the granules are somewhat irregular in distribution, as may be noted especially well in the two cells in the lower right corner of the photograph. The silver ])recipitate in a large measure obscures the blue granules in the bodies


of the cells. With increased irritation there was a more general tendency to even distribution in those parts of the cell where there was much blue and a gradual though uneven widening of the belt of blue constituting the perinuclear rosette. In general the effect of irritation was found to be a more even distribution over a larger area of cytoplasm, sometimes but not always, accompanied by a diminution in the concentration in that part of the cell which originally contained a small ring or mass of granules. In Figure 5, which is from an omental spread that had been treated with osmic acid ami had not been silvered, there is, in the upper center, just above the round, heavily stained clasniatocyte, a mesotiielial cell which is somewhat out of focus, but which nevertheless illustrates the distribution of the vital dye granules as irritation progresses. The cell is irregularly triangular in shape, the upper part on the left being entirely out of focus, while in the upper right there is a very distinct ring of blue granules. This ring represents the first dye deposited in the cell while the patches of granules in the remainder of the cell have been formed during the progress of the irritation.

It is extremely important to note that in no case were there any granules of blue to be .seen in the processes of the serosal cells. In fact, in those cells having the largest processes there was not the least tendency of the blue granules to enter them, and in this particular there was a very sharp contrast to the fibroblasts. In Figure i there are two cells which have very characteristic processes, and these show very clearly that they contain no dye inclusions. These clear processes should be contrasted with the long, branching, dye-filled processes of the fibroblasts in Figures 5 and 6.

The reactions of the fibroblasts and the clasmatocytes in these omenta from animals having mild irritations were wholly characteristic. Scattered throughout the omentum characteristic fibroblasts were to be found everywhere. The typical fibroblast has been studied and described so often that it seems useless to enter into the especial characteristics of this cell at any length. Schott,- Btittner," Kiyono '^ and Tschaschin -"• -^ have figured the fibroblasts in the omentum as large cells with smaller nuclei than the serosal cells, fading outlines, and fibrillary cytoplasm. Evans and Scott '^ have studied them in subcutaneous preparations with numerous vital dyes and figure their vital dye content quite extensively; it is probable that these would be similar in the omental fibi-oblasts, even though Maximow has suggested that they retain their embryonic characteristics in this locality during adult life. In ])reparations from a normal omentum which has been vitally stained with trypan blue and then silvered, the fibroblasts were seen in between the two layers of serosal cells as faintly outlined cells with fine blue granules scattered through the cyto


July, 1922]


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plasm. These cells were often branched and even though the outlines were difficult to indentify exactly, they could be made out to some extent because the trypan blue was distributed tliroughout the processes. When subjected to the effects of irritation, the fibroblasts instead of becoming more compact developed much more extensive processes which in many cases were quite remarl^able. They reminded one very much of the branclied cells of the tadi)ole's tail as figured by Clark and Clark ' and in a quite similar manner contained tlie trypan blue in those enlargements which are found on their processes. In my preparations these cells were especially characterized by the fact that the vital dye extended to the tips of tlieir branches (Fig. 5), although the amount of dye in the fibroblast was very much less than that found in the dasmatocyte. As has been stated, the fibroblasts lie scattered throughout a meshwork of fibrils between the layers of serosal cells. During earl}- stJiges of any irritation the.se two groups of cells undergo specific changes without much departure from tlieir definite layer-like arrangement. The mesothelial cells of the entire surface layer have, in many omenta, separated and rounded up, so that each cell was divided from its neighbor by a considerable area ; and at a different level there were large numbers of variously shaped, richly branching fibroblasts. This layer arrangement is well illustrated in Figures -t and 6. Both are photograph.s from the same omental spread. In Fgure 4 there are shown seven mesothelial cells wliich have separated from each other and rounded up to a considerable degree; if the photograph be studied carefully the shadows of other cells can be seen in the depths. In Figure 6 the camera was focused on the fibroblastic layer and the mesothelial cells may only be seen as faint outlines; one of these can be fairly well made out in the lower left corner, while two others cast somewhat less well defined shadows in the upper right corner. When the irritation was more advanced there was more tendency for arms of the branched fibroblasts to extend into tlie spaces between the separated mesothelial cells. In Figure 5 the single mesothelial cell is .seen to be only slightly out of focus, while there is considerable variation in the relative clearness of the dift'erent processes of the two numerously branched fibroblasts. The distribution of the dye in these long, tine, streaming processes was most remarkable, some of them extending entirely across the oil-immersion field, and often they appeared to be attached to or following fibrils of some kind. Tlie contrast between the fibroblasts and the clasmatocytes in Figures 5 and G is very striking, ]iarticularly in Figure 5; and it must be noted that the characteristics are about equally as specific for the two types of cells in both figures.

In the omentum of a vitally stained rabbit there are, in addition to the fibroblasts, many large cells, most of them long and narrow and loaded with blue granules.


Among these cells are some that are round or oval in shape; these latter are especially numerous in the taches laiteuses. These cells are typical clasmatocytes and have been carefully studied by many observers. Marchand ^^ notes their e.special affinity for the perivascular regions and calls them "adventitial" cells. Long lines of them are always arranged along the course of blood-vessels and are at times in most intimate contact with the endothelium of the capillaries. This distribution of the clasmatocytes is used by Shipley " as an argument for concluding that they represent a type of digestive organ, functioning intermediately between the blood-vessels and the tissue-cells proper. In preparations of the normal omentum the clasmatocytes were usually either long and slender cells with centrally located nuclei, or else round or oval cells with somewhat excentric nuclei. The former type is the one invariably associated with the bloodvessels, and so striking is this morphology that they have been termed "trailer" cells by Buxton and Torrey." In the taches laiteuses the more compact type was often met with, but even here, there were large numbers of the long and slender cells loaded in a striking manner with the vital dye.

When the omentum was irritated, the first change that could be noted in tlie clasmatocytes was a tendency to round up and change from the long, slender cells to round or oval masses, with or without, short, blunt processes. The surface of any irritated omentum was dotted with these round cells loaded with trypan blue and large numbers could be seen between the serosal layers. Figure 5 shows the typical rounded dasmatocyte, while Figure 6 shows one with blunt processes filled with trypan blue inclosures.

In the study of the omentum during mild irritations by means of the methods outlined above, it has become entirely clear that the serosal lining cells and the fibroblasts react dift'erently both as regards their morphological appearance and the distribution of their vital dye-content. The serosal lining cells increase in thickness and are much more compact than the normal, flat, plate-like structures. They ditter considerably in size and shape, being round, oval or square and some of them have a few, fine-pointed processes which vary in size but not in character. The vital dye content is more dift'use than in the normal cells, but there is never any dye to be seen in any of the fine processes. On the other hand, the fibroblast during irritation becomes an elaborately branched structure, forming a marked contrast to the compact serosal cell. The processes of the fibroblasts vary enormously in size and shape; some are long and slender, others branched into two or more subdivisions, and many have bulb-like enlargements, especially where they branch. The vital dye is distributed throughout these processes, the granules often being collected in the bulblike enlargements.


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It is thus obviou.s that during the course of a mild irritation the changes which talie place in the mesothelial and fibroblastic elements indicate that they become even more widely separated from each other in their groupcharacteristics. The cell-bo<lies and processes of these two cell-species are mori)hologically quite distinct in regard to size, shape and general characteristics. It seems entirely justifiable to conclude that such marked differences must denote some variation in the structure of the cytoplasm, and hence indicate some especial adaptation for certain definite physiological activities. In addition to the.se morphological criteria, it has been demonstrated that the vital dye content of the fibroblast and the mesothelial cell presents a sharp contrast. Here again the principal interest is attached to the wide distribution of dye granules in the processes of the one cell and the total absence of the dye from those of the other. That differences in the way in which vital dyes are stored represent corresponding differences in physiological adaptation seems most probable, and perniit.s further security to be placed in the conclusion that the mesothelial and fibroblastic elements are wliolly specific.

It is hoped that the observations reported above, which differentiate the reactions of the mesothelial cell and the fibroblast quite sharply during mild irritations, will be useful in determining the roles perfonned by each of these two types of cells in the healing of wounds, the regeneration of denuded areas, the fonnation of adhesions, and finally aid in establishing the genetic derivation of the so-called "endotheliomata" of the lining membranes of the various serous cavities.

EXPLANATION OP PLATE

All the figures are micro-photographs of omental spreads from rabbits which had had continuous, mild irritations induced by the repeated introduction of laked blood into their peritoneal cavities. The negatives have not been retouched.

Fig. 1. — Exposure to mild irritation for 36 hours. Preparation silvered and stained with carmine. Shows relatively normal mosaic pattern on the left, and beginning separation of the cells on the right, x 500.

Fig. 2. — Exposure to irritation for 60 hours. Preparation from rabbit which had been vitally stained with trypan blue. Silvered and stained with carmine. Shows partial separation and beginning rounding up of the mesothelial cells. The blue droplets are seen as irregular patches in the cells, x 700.

Fig. 3. — Exposure to mild irritation for 4 days. Silvered and stained with carmine. Sliows the final stage in the separation of two mesothelial cells and the general characteristics of these cells when definitely modified by the irritation. X 1100.

Fig. 4. — Exposure to mild irritation for 5 days. Vitally stained with trypan blue. Shows the even distribution of the serosal cells on the surface of the omentum; there has probably been no desquamation, x 850.

Fig. 5. — Exposure to mild irritation for 6 days. Vitally stained with trypan bine. Fixed in osmic acid and stained with carmine. Shows two clasmatocytes heavily loaded with blue granules, two branching fibroblasts, and one mesothelial cell. X 700.


Fig. 6. — Same preparation as Fig, 4, Shows one clasmatocyte with a large content of trypan blue and pseudopodial-like processes, one characteristically branched fibroblast, and the shadows of three mesothelial cells which are out of focus. X 750.

LITERATURE

1. Borst, M,: Zur Pathologie der Serosen Deckzellen. Virchows Arch., 1900, CLXII, 94-99.

2. Brunn, M. (von): Ueber die Entziindung der serosen Haute mit besonderer Beriieksichtigung der Rolle der SerosaDeckzellen. Beitr. z. Path. Anat., 1901, XXX, 417.

3. Idem: Zur Histologie der Epithelien der serosen Haute. Centralbl. f. allg. Path. u. path. Anat., 1900, XI, 604.

4. Bijngner, O. (von): Ueber die Einheilung von Fremdkbrpern unter Einwirkung chemischer und mikroparasitarer Schadlichkeiten. Beitr. z, path. Anat. u. allg. Path., 1896, XIX, 33-126.

5. Biittner, F.: Untersuchungen iiber das Verhalten der Peritonealepithelien bei Entziindung. Zieglers Beitr., 1899, XXV, 453.

6. Buxton, H. & Torrey, J. C: Absorption from the peritoneal cavity, IV. and V. The function of the omentum. Journ. Med. Research, 1906, XV, 55-87.

7. Clark, E. R. and Clark, E. L. : Phagocytosis of carbon and carmine granules in the transparent tails of tadpoles. Anat. Record, 1918-19, XV, 151-163.

8. Cornil, M. V.: Des modifications que subissent les cellules endotheliales, dans les inflammation, et en particulier dans les adherences des membranes sereuses et dans la pneumonie. Arch. med. exper. d'anat. path., 1897, s. I, IX, 9.

9. Cunningham, R. S. : On the origin of the free cells of serous exudates. Amer. Jour. Phys., 1922, LIX, 1-36.

10. Cunningham, R. S.: The reaction of the cells lining the peritoneal cavity, including the germinal epithelium, to vital dyes. Amer. Jour. Anat., 1922, XXX, No. 4.

11. Dominici, M.: Macrophages et cellules conjonctives. Compt. rend. Soc. de biol., 1901, LIII, 890-S92.

12. Dominici, M.: Polynucleaires et macrophages. Arch, de med. exper., 1902, XIV.

13. Evans, H. M. and Scott, K. J.: On the differential reaction to vital dyes of the two great groups of connective tissue cells. Contrib. to Embryol. No. 4 7, vol. X, Carnegie Inst, of Wash., Publ. No. 273, 1920.

14. Foot, N, C: Studies on endothelial reactions. V. The endothelium in the healing of aseptic wounds in the omentum of rabbits. Jour. Exper. Med., 1921, XXXIV, 625-642.

15. Kiyono, K,: Die Vitale Karminspelcherung. 1914, Jena, G. Fischer.

16, Kolossow, A.: Ueber die Struktur des Pleuroperitoneal und Gefassepithels, Arch, f. Mikr. Anat., 1893, XLII, 318.

17. Macallum, A. B. : Die Methoden und Ergebnisse der Microchemie in der biologischen Forschung. Ergeb. d. Phys.. 1908, VII, 552.

15. Marchand, F. : Ueber die Veranderung der Peritoneal Endothelien bei der Einheilung kleiner Fremdkorper. Stzungsb. d. Gesellsch. z. Marburg, 1897, No. 3, 29-41.

19. Marchand, F.: Ueber Klasmatozyten, Mastzellen ,und Phagozyten des Netzes. Verh. d. Deutseh. Path. Gesellsch., 1901, IV, Tag.

20. Ranvier, L, : Des clasmatocytes. Arch. d'Anat. Micr., 1899, III, 123-139.

21. Ranvier, M. L. : Anatomie genSrale de I'endothelium du peritoine et des modifications qu'il subit dans rinflammation experimentale; comment il faut comprendre la gu^rison


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July, 1922]


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des plaies par reunion immediate. Compt. rend. hebd. de Sci., 1891, CXII, S42-S45.

22. Recklinghausen, F. T. (von): Die Lymphgefasse und ihre Beziehung zum Bindegewebe. Berlin, Hirschwald, 1862.

23. Rich, A. R.: Condition of the capillaries in histamine shock. Jour. Exper. Med., 1921, XXXIII, 287-297.

24. Roloff, F.: Ueber die Rolle des pleuroperitonealendothels bei der Enstehung bindegewebiger Adhasionen. Habilitationschr. Rudolstadt, Hofbuchdriickerei von F. Mitzloff, 1894.

2 5. Roloff, P.: Ueber die Rolle des pleuroperitonealepithels bei der Entstehung bindegewebiger Adhasionen. Arb. a. d. path. Inst. z. Tubingen, 1896, II, 171.

26. Schott, E.: Morphologische u. experimentelle Untersuchungen iiber Bedeutung und Herkunft der Zellen der Serosen Hohlen und der sogenannten Makrophagen. Arch. f. mlkr. Anat., 1909, LXXIV, 143-216.


27. Shipley, P. G.: The physiological significance of the reaction of tissue cells to vital benzidene dyes. Amer. Jour. Phys., 1919-20, XLIX, 284-301.

28. Tschaschin, S.: Ueber die "ruhenden Wanderzellen" und ihre Beziehungen zu den anderen Zellformen des Bindegewebes und zu den Lymphocyten. Folia Haemat., 1913. XVII, 317-397.

29. Tschaschin, S.: Ueber die Herkunft und Entstehungsweise der lyraphocytolden (leukocytoJden) Zellen, der "Polyblasten" bei der Entziindung. Folia Haemat., 1913. XVI, T. I, 247-294.

30. Weidenreich, P.: Ueber die zelligen Elemente der Lymphe und der serosen Hohlen. Anat. Anzeiger, 1907, XXX, Verb. d. Anat. Ges. z. Wurzburg, 51~,56.


THE ELEMENTARY SCHOOL AND THE INDIVIDUAL CHILD

(ABSTRACT)

By E.STHER LORING RiCHARUS

(From The Henry Phipps Psychiatric Clinic of the Johns Hopkins Hospital)


In Mental Hygiene for April, 1920, the writer described a fifteen months" study of "Some Adaptive Difficulties Found in School Children." The work was a bit of private research in School No. 70 of the Baltimore schools, and quite divorced from any organized activity in the public school .system.

Of the forty-six children who came under observation, thirty-five were rei)orted a.s having dittic\ilty in keeping up with their grades in one or more subjects. In each of these ca.ses where there was a suggestion of retardation, the Biuet-Simon test was applied. As a result, sixteen of the above-mentioned thirty-five were found to have a mental retardation of from three to six years. The academic trouble,s of the remaining nineteen were associated with, if not the disguised expression of, such faulty psychobiological reactions as shyness, laziness, inattention, and vicious tendencies, sensitiveness to criticism, daydreaming, hypochondriacal fears, with resulting irregular attendance. The eleven remaining from the total forty-six were referred for the more overt adaptive difficulties of temper tantrums, sullenness, cn'ing spells, twitching, indift'erence, excitability, poor coordination with the hands, quarrelsomeness, etc.

In reviewing the data accumulated, one fact stood out; in practically every case the peculiar characteristics for which the child was referred could be easily traced to their first appearance in the early years of school-kindergarten and first and second grades. In the luajority of


Read before the Mental Hygiene Section of the National Conference of Social Work, Milwaukee, June 23rd, 1921. Published in Mental Hygiene, Vol. 5, No. 4, pp. 707-723, October, 1921.


cases, also, the unhealthy luibits of adaptation began in the home, and were carried into and through tiie school life, handicapping the eft'orts of teacher and child to get together on the business of early training in the classroom; and the commonest result of this handicapping was the repetition of grades. Accordingly, it seemed best to devote some time to the first grade, with the special object of examining the problem of backwardness at its source.

By way of experiment, eighteen children were selected from the first grade for special study. Their ages ranged from six to ten, and their years of repeating the first grade from one to three. These children were studied from the standpoint of the storj- of the home, tlie story of the school, and the stoi^ of the child himself, as recorded under the headings of complaint, school historj% habit data, personality traits, and a rough estimate of physical status as obtained from the health records, weight curve, and brief examination. Aside from the Binet-Simon tests, no special technique was used, either in the examination of the children or in the sifting of facts. The Binet-Simon findings were as follows: In two cases the actual age and tested age were exactly the same; in seven cases there was a difference of only one year between the actual age and the tested age ; in seven cases there was a difference of two years between the actual age and the tested age; one child objected so violently to examination that it was thought best not to push matters; and one child showed a difference of three years between tlie actual age and the tested age. It was felt that the difference of from one to two years between tlie actual age and tested age of fourteen of these


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cliildivn represented no real backwardness, but was the exiire.ssion of various factors in the background, development, early training, and personality traits of the individual children. That these were the facts responsible for the lodging in the first grade seemed more probable than deficient mental eqnijjment.

Turning from the facts of standardization to those of physical condition and living arrangements, there was nothing striking in the story of these little people. There was one mouth breather, one child with definite eye strain, and two tuberculosis suspects. Several children appeared pale and undernourished, but only two weight curves were below the normal. The habit data were characteristic of this industrial section — comparatively late bedtime, with two, often three sleeping in a bed ; a diet of coffee, buns, and soup ; movies two nights a week. Unideal as the.se conditions are, it must be remembered that they have not proved a serious hindrance to the school progress of hundreds of other boys and girls.

These eighteen candidates for reconstnictive therapy were put under a special teacher in September, 1920. In June, 11)21, ten members of this class were qualified for the third grade and seven for the .second grade. Only one child of the class failed to respond to special study.

To what have these results been due? Before giving my own impression of the matter, let me say a word about the school background of the above experiment. For several years School 76 has been studying the problem of repetition in its midst. For three years a special health worker, privately financed, devoted her whole time to following up minor ailments — common colds, postcontagious disease conditions, and anj' other factor influencing regular attendance. The result was an attendance record of from 96 to 98 per cent, but the repeating of grades remained in statu quo. This same worker directed a campaign towards the relation between progress in school and the clearing up of tonsils and adenoids. The results, over a period of three years, were that 50 per cent of the children operated on showed no difference in their school work, 25 per cent showed some improvement, and 25 per cent appeai-ed to make a poorer showing than before operation.

The plan tried in the first three grades of dividing the class so that each half of a room rotates between playgi'onnd and class work resulted in a certain amount of gain in school progress, due to the fact that each teacher worked with smaller groups. It did not, however, eliminate repeaters such as have been described above, the majority of whom had been accorded the benefit of the scheme just outlined.

In view of these facts, it would seem that the success obtained with the experimental class was due not so much to the extra time spent on them as to the fact that this time was devoted to a study of the individual needs of these children. Here were eighteen school failures


according to the criterion of grading, and eighteen individuals whose pedagogical record and BinetSimon findings would be credentials enough to admit them without question to that mysterious order called the backward child." They could not be poured into the ordinary mold of school curriculum because of certain traits of personality that had to be discovered, understood, and wisely handled. The same psychobiological characteristics of shyness, indolence, fear, sensitiveness, daydreaming, etc., acted as conflicting factors in the BinetSimon tests, obscuring the native capacity to such an extent that a difference of from one to three years appeared between the child's actual age and tested age.

A year of school training directed by an understanding of these characteristics has resulted in restandardization data with the BinetSimon tests in June, 1921, that run parallel with the academic progress of these children as recorded in their grading reported above. The seven children who showed a difference of two years and over between their chronological age and mental age according to intelligence tests made in March, 1920, now show a mental level that coincides with their actual phy sical age.

And for those of us who examine such children, there is subject for reflection. Are we using the BinetSimon scale as a yardstick to measure off' lengths of intelligence, or are we using the tests as a help towards the sizing up of individual child problems'? Are we confusing the term "mental age" with a diagnosis of the home situation in question'? Do we see the personality with its setting of life story behind the intelligence level'? These are possibilities of view that at times .seem strangely remote from the productions of psychiatrists and practicing psychologists. As such, we should beware of agitating ourselves to decimal points of determination concerning qualifications for the groups of subnormal and dull, lest the object of our research becomes buried beneath the mound of his own case.


THE JOHNS HOPKINS HOSPITAL BULLETIN


It is issued monthly. Volume XXXIU is in progress. The subscription price is $4.00 per year. (Foreign postage, 50 cents.) Price of cloth-bound volumes, $5.00 each.

A complete index to Vols. I-XVI of the Bulletin has been Issued. Price, 50 cents, bound in cloth. Orders should be addressed to

The Johns Hopkins Press, B.u.timohk, Md.


July, 1922]


267


NOTES ON NEW BOOKS


studies in the Paleopathology of Egypt. By Sir Marc Armand RuFFER. Cloth, $7.50. (University of Chicago Press, 1921.)

This is a collection of Sir Armand Ruffer's papers published after his death evidently with the loving aid of Lady Ruiter who worked over many incomplete manuscripts and brought together notes and literary references to complete them. The papers treat of an extraordinary material so romantic as to compel the interest of everyone to whom the names of Thutmose, Amenhotep and Raraeses call up the ancient glory of Egypt.

The author had great opportunities for the study of the mummies of all the periods of Egyptian history and utilized them for the purpose of determining the character of the diseases from which the people suffered. This was relatively simple in the case of bony lesions, among which various forms of osteoarthritis deformans stand out prominently. Spondylitis with bony ankylosis of many vertebrse seems to have been common and periarticular exostosis hardly less so. Tuberculosis occurred and there is a case of Pott's disease with a psoas abscess, but there were no evidences of syphilis. The teeth furnish also much accurate information and it is readily seen that the ancient people took little care of them and that there were no efficient dentists. The teeth were often greatly worn and in an advanced state of caries with destructive abscesses which martyrised with toothache even such an exalted person as Amenhotep III.

It was found that the soft tissues of these mummies, although dried into unrecognisable form, could be softened and made pliable and even embedded in paraffin so that microscopical sections could be cut and stained. The tissues are by no means destroyed and cells can be recognised with their nuclei; muscle fibres with striations, elastic fibrils, areas of calcification and even bacteria and the eggs of Bilharzia hsematobia can be clearly seen. The methods of embalming which are fully described were such as to destroy many of the tissues, often leaving others tightly wrapped round little statuettes which were placed in the body. The arteries were not used for injection of fluid, but were slashed away everywhere, and even most of the muscles were removed and replaced by all sorts of material. But although no connected examination of all the organs could be made, isolated observations of extreme arteriosclerosis, cirrhosis of the liver and renal disease were frequently possible.

Dwarfs, including some which were evidently chondrodystrophic, and various malformed persons are unmistakably recorded in the ancient sculptures. The Queen of Punt looks as though she were the victim of congenital dislocation of the hips rather than rickets or elephantiasis.

There are various other studies such as that of a lower miocene crocodile with arthritis, an osteosarcoma of the pelvis, a variola-like eruption of the skin together with many nondescript ulcers and wounds, the traces of which remain and sometimes, as in the case of Saknounra, throw some light on history. These diseases aie largely repeated in the case of the Copts, the Sudanese and the Nubians.

Most interesting are the studies of the Royal mummies which are, however, rather restricted owing to the divinity which hedges around these personages even now as they lie in the Cairo Museum. Everyone who has dwelt with pleasure upon the portrait busts of Ikhnaton, with their eager yet dreamy delicacy, must feel a shock of disappointment upon


learning that he was a ridiculously obese man even in a household where all the members were very stout. The rest seems not very important. Rameses II was bald and had blackheads on his forehead. Thuthmose III and Amenhotep II had a macular eruption on their legs, and two or three queens were bald.

On the whole though, they were pretty well — all these royal people — and in spite of their consanguineous marriages they were preeminently strong, fertile, intelligent and vicious.

It is an extremely interesting account in spite of the rather wearisome repetitions, which are perhaps unavoidable in a collection of papers published at considerable intervals, and one is impressed by the infinite pains required, in handling these crumbling remains, to snatch from oblivion a few meagre hints of the discomforts and sufferings of these vivid people of thousands of years ago.

W. G. M.

The Care of the Eyes. By Robert Henry Elliott, M.D., B.S., Sc.D., F. R. C. S. (Eng.). (London, Henry Frowde and Hodder <{ Stoughton, 1921.)

This book is an elementary treatise, intended especially for nurses, on the subject of ophthalmic nursing and diseases of the eye. Of the three sections into which it is divided, the first deals with the special anatomy and the general and special therapeutic procedures used in ophthalmology. The second part contains a very brief description of the commoner diseases of the eye. The third, in the form of an appendix, is probably the most valuable section of the book It illustrates the special instruments used in eye surgery, and gives excellent lists of those employed in the various operations.

Inasmuch as various ophthalmic clinics develop individual technique, nursing and therapeutic procedures within fairly wide general limits, the general value of any treatise, which outlines the technique and nursing procedures used in one individual clinic, will not be great. There are, however, many excellent chapters, and many essential points clearly made, which are of undoubted value. The second part of the book, dealing with diseases of the eye, is too brief. The appendix, describing instruments and lay-outs for various operations, is unusually complete. With such minor additions as any surgeon would make to suit his own individual preferences, this section will be of distinct value to the operating room nurse.

The book is well gotten up and quite readable.

A. C. W.

Obstetrics and Gynaecology. Edited by John S. Fairuairn. Cloth, $20.00. (London: Oxford University Press, January, 1921.)

The purpose of this book is evidently the very laudable one of bringing together in one volume the subjects of obstetrics and gynecology. This is in accord with the growing opinion that the two subjects are so closely related that the successful practice of one requires a thorough knowledge of the other. The arrangement of the subject matter is excellent and is proof that no sharp line of separation can be drawn between the two branches. The editor has wisely included many topics, such as public health and social and medico-legal problems, which are ignored in most texts.


268


[No. 377


Although this volume is the work of fifty-five authors, there is remarkably little overlapping of description and discussion. However, of necessity each topic is brief, and therein lies the chief weakness of the book. This brevity limits the mention and discussion of varying opinions to a minimum and, in many instances, to a more or less dogmatic expression of the author's individual views. This is particularly true of the sections on operative procedures.

It Is unfortunate that the illustrations are so inadequate. More of them would be helpful, and many of those used are so reduced in size as markedly to limit their value.

J. W. H.

The Treatment of Common Female Ailments. By Frederick John McCann, M.B., M.R.C.P., F.R.C.S. {Edtcard Arnold d- Co., London. 1922.) 145 pp. fS.OO.

This book of one hundred and forty-five pages has been written to guide the general practitioner in the general care of gynecological patients. The author deals with the various subjects in a concise and practical manner, giving many useful hints and prescriptions and covering a phase of gynecology which is too often overlooked by the modern specialist.

In spite of its merits, this little book is open to several criticisms. Like most works of this sort, it leaves one unsatisfied. The physician who is acquainted with the fundamental principles of gynecology usually does not need such an elementary text, and one who does not possess this acquaintance, needs far more. In the second place, we question the advisability of including in such a work decriptions of technical procedures such as the performance of operations, and the administration of radium, or the extended discussion of abstruse subjects like endocrinology, all of which are attempted in this manual. Furthermore, we feel that the author has been unfortunate in recommending certain procedures which seem to be antiquated, while failing to mention some of the noteworthy recent contributions. In general, although this book contains much that is practical and useful, it may prove disappointing to the practitioner who needs an up-to-date guide in the treatment of genito-urinary diseases in women.

L. R. W.

Syphilis and Its Treatment. Wilfrid S. Fox, M.A., M.D. Cloth, $9.00. [NeiD York, Paul B. Hoeber, 1921.)

In this work intended for the use of medical students and practitioners of medicine, the author has limited himself chiefly to a discussion of the skin lesions of the disease. The illustrations consist of numerous water color and black and white drawings. There are few references to recent or remote literature, and the material represents the personal experiences of the author, treated in an informal way. Partly for this reason, but chiefly because the book presents only one side of the disease — and that perhaps the least important, from the standpoint of the patient — it falls far short of being a desirable text for students. Many writers on syphilis inadvertently (or purposely, as in this instance) fail to deal with syphilis as a disease affecting all portions of the bodily mechanism and stress only its manifestations as applied to certain organs or systems. This method of handling the subject, while satisfactory for those already expert in syphilology, is distinctly a faulty approach for the novice.

The text contains many rather dogmatic statements which either overlook the established facts or are not substantiated by them. For example. It is stated that except in monkeys, animal "inoculation experiments have been negative or doubtful. In rabbits there is a disease nearly related to it, if not identical, and Noguchi claims to have inoculated the testicles


of rabbits with syphilis." The existence of true rabbit syphilis has of course been thoroughly established by many observers, chief among whom are Neisser, Uhlenhuth and Mulzer, Nichols and Reasoner, and Brown and Pearce. The statement that mercury given by inunction is absorbed in considerable quantities through the lungs by inhalation is hardly in accord with known facts. The discussion of the paternal transmission of syphilis, will meet with many objections from the syphilologists of this country. The failure to mention routine spinal puncture as an indispensable accompaniment of the management of early syphilis constitutes a grave omission.

J. E. M.

Heart Disease and Pregnancy. By Sir James Mackenzie. (London, Henry Froicde, and Hodder if Stoughton, 192 1.)

This small volume adds another to the list of works bearing the impress of the long clinical experience and research of the distinguished author, and has all the interest and charm one has learned to expect from the writings of Sir James Mackenzie.

While essentially a restatement of the newer knowledge of cardiac disease, as so often emphasized by the author, it constitutes an appeal for the application of this knowledge to all the problems relating to the question of marriage and the dangers involved in pregnancy and confinement for women the subjects of cardiac disease.

Written with the purpose of presenting his own views, based upon a long personal experience, to the obstetrician as the individual vitally concerned, one can not help feeling that the author has failed to appreciate, in a measure at least, the real extent to which the modern knowledge of cardiac disease has been made familiar to the obstetricians as well as to the profession generally. That just such a statement of the underlying principles involved will, however, prove of great value to many cannot be denied.

It is refreshing to read the author's description of the sequence of events in mitral stenosis of rheumatic origin and to get in his clearly put words the statement of the method for the correct evaluation of the physical signs in this condition as well as in aortic insufficiency. The chapter devoted to the neurotic heart covers this phase of the subject in a most satisfactory way.

Although lacking any exhaustive discussion of a number of points involving the pathological physiology concerned, and dealing chiefly with the clinical phases of the subject, there can be no question of the soundness of the views advanced.

A very complete index adds much to the value of the volume.

E. P. C.

Diseases of the Eye. By Georue E. De Schweinitz, 9th Edition. (Philadelphia and London, W. B. Saunders, 1921.)

That De Schweinitz' "Diseases of the Eye" has reached a ninth edition is a sufficient proof of its merit. In a book of this kind it is very difficult to meet the wants of everyone. Nevertheless, all in all, even the most critical should be satisfied. The revision of certain parts of the book and the foot-notes add materially to its value. The new edition is up-to-date, and includes a discussion of the more recent subjects in Ophthalmology — Localization and Organization of the Cortical Centers of Vision, The Dermic Grafts. The Epithelial Inlays. Jennings' Self Recording Tests for Color Blindness. Ophthalmoscopy with Red Free Light, Electric Desiccations, etc. Credit is given to other authors for their work or suggestions. In fine. Dr. De Schweinitz has given us another book which will render good service to the practising Ophthalmologist and the special student.

L. J. G.


JlLY, 19l.'2J


269


Kpideiniology and Public Health. By Victor C. Vaughan, Henry F. Vaughan and George T. Palmer. Cloth, $9.00. (St. Lojiis, C. Y. Moshy Company, l!>:i2.) Progress in the study of infectious disease has been marked by a series of disappointments in the application of specific therapeutic measures following the early brilliant successes in diphtheria and meningitis. Today the immunologist concerns himself largely with matters only remotely related to the practical management of disease; the actual fight has been left to the epidemiologist and the public health worker. We lay more and more stress on the prevention of disease as a whole, as well as in the individual, and the recent blight, especially, cast by the influenza pandemic has impressed on those concerned with the health of the nation the essential importance of understanding and grappling with the conditions which underlie and promote the occurrence of such scourges.

It is with interest, therefore, that one approaches the new work on Epidemiology and Public Health by Victor C. Vaughan of which the first volume on Respiratory Infections is before us. After reading this section which contains an excellent summary, both of the historical development and of the contemporary status of epidemiological ideas, one feels that an era is closing. Despite the vast amount that has been accomplished, the hope of the future seems fully as great as the performance of the past. It becomes clear that with no more than our i)resent knowledge the next influenza epidemic will blast us like the last; poliomyelitis and encephalitis are as baffling as ever from the standpoint of prevention, and in tuberculosis the ground is barely cleared for rational and concerted attack. The interesting fact that healthy carriers may harbor pneumococci or meningococci or diphtheria bacilli has as yet led to no fruitful practical method of checking epidemics due to these organisms. Epidemiology, in short, has thus far brought out mainly generalities about the spread and incidence of disease. In the succeeding era it may be hoped that the application, still incomplete and abortive, of these generalities will mature into a practical and certain control of the scourges yet unchecked.

In sumimary, Vaughan's book states the problem and brings it up to date, the dry facts being varied by sections in which the author's well-known ideas on the "protein poison" are

elaborated. a t n

A. L. B.

Human Parasitology. By Damaso Rivas. B.S.Boil., M.S., M.D., Ph.D. (1st edition, W. B. Saunders Company, Philadelphia and London, W.llt. 715 pages, 422 figures. 18 plates.)

The reviewer purchased this book with a sense of comfort in having discovered the long needed comprehensive English treatise on parasitology. The appearance of the book is attractive and its size seemed designed to permit a certain degree of completeness. It was a pleasure to note in the preface that the author had "kept himself informed of changes in nomenclature" and was aware of the fact that "the ever changing nomenclature of parasitology is a continued source of emibarrassment." This seemed to promise a discriminating criticism of the names given to parasites, and the adherence to valid terms.

This prefatory satisfaction, however, was not long sustained. In the text for Fig. 12, "linen threads" are noted in the diagram of the cell, and at this point the reader picks up a strand of error which runs through the fabric of the book. This, like the word "amiboid" on p. 606. may have been a typographical error. Throughout the book, however, there are many similar examples of inaccurate uses of words, uncritical compilations and contradictions. An insect is at one tim'e described as the intermediate host, and at another the definitive host, of the same parasite. Although the author protests


his allegiance to the International Committee upon Zoological Nomenclature, he disregards the rules of that Committee, and indeed, revises the Latin language. He omits diphthongs not only when he uses the current simplified spelling of words derived from' Latin, but also in proper generic and specific names. Thus Amo:l)a becomes Amcha, and Twnia becomes Tenia — terms which have no validity under the rules of nomenclature. The embarrassing confusion of names given to one parasite is well illustrated by the different terms for the nematode "whip-worm," which the author scatters through the text. These are not grouped as synonyms under the valid designation, but occur as follows: Trichocephalus, p. 352, Trichiuris, p. 353, Trichiuris trichiurus, p. 358, Trichiiiris trichiuras, p. 392, Trichiuris trichiuris, p. 449, Trichocephalus trichiura, p. 393, Trichocephalus trichiuris, p. 394, Trichim-is trichiura, p. 713.

Although the book bears the date 1920, there is little evidence to show that the author has kept up with recent work in this field. This is exhibited particularly in the section on the Enfamrrba-. Dobell's work is not mentioned, while the fanciful conceptions of Schaudinn and Craig are repeated. Entamaba histolytica and Entamaba tetragena are described as distinct species, and their differences are set forth in a table. The use of emetine in the treatment of amoebic dysentery is not mentioned.

The section on the Flagellata is confused and inadequate. The spirochaetes, however, are included in this section without hesitation. Their inclusion leads to this statement on p. 220: "Noguchi and Pareja claim to have found a flagellate in the blood of yellow fever patients." Undoubtedly, Leptospira ictrroides is referred to here. The statement is, to say the least, misleading, since it has not been determined whether the spirochcetes are bacteria or protozoa.

The chapters on Helminthology are the best in the book. Here also contradictions occur in the text. In the table opposite p. 242 the genital pore of Schistosoma is said to be anterior to the ventral sucker, whereas on p. 259 it is said to be posterior to it.

The descriptions and figures of the insects are excellent in some respects, but inadequate for the differentiation of species. Several errors and misleading statements quickly strike the attention of the reader. On p. 552, Glossina palpalis is said to transmit Trypanosoma hrueei, and on p. 514 it is mentioned without conviction that the "louse has been said to transmit the virus of typhus fever."

A great variety of fungi, and bacteria, together with the technic of mycology, bacteriology, microscopy, haematology, and serology are described in the final 100 pages of the book. The futility of this sort of compressed compilation is shown by the description of the microscope, which is contained in a paragraph of 22 lines. The yeast-like organism Monilia is classed with the Hyphomycetes along with Actinomyces (which is incorrectly called Discomyces) . Many of the laboratory procedures described are antiquated, and the directions given are too sketchy to be of value.

There are many excellent pictures in the book, and also many which are drawn to such a small scale that the details of the structures supposed to be represented cannot be seen. These are, therefore, of no assistance in the elucidation of a text which is often obscure.

It would be unprofitable to continue to catalogue the errors found in this book. Much correct information is contained in it, and the book may serve as a means of orientation in parasitology. But its authority is destroyed by its errors and omissions. It disappoints expectations and does not supply the need of a comprehensive and accurate text-book in English on the subject of Parasitology. S.B-J.


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[No. 377


THE JOHNS HOPKINS HOSPITAL REPORTS


VOLUME I. 423 pases, 09 plates.

VOLUME II. S70 pnees, with SS plates and figures.

VOLUME III. 766 partes, Tilth 68 plates and figures.

VOLUME IV. 504 paces, 33 eharts and Illustrations.

VOLUME V. 480 pages, with 33 charts and Illustrations.

VOLUME VI. 414 paces, with 79 plates and flEures.

VOLUME VII. 537 pages with Illustrations.

VOLUME VIII. 552 pages with Illustrations.

VOLUME IX. 1060 pages, 66 plates and 210 other Illustrations.

Contributions to the Science of Medicine. Dedicated bv his Pupils to William Henry Welch, on the twenty-Hfth

annivei'sary of his Doctorate. This volume contains 38 separate

papers. VOLUME X. 516 pages, IS plates and 25 eharts. VOLUME XI. 555 pages, with 38 eharts and Illustrations. VOLUME XII. 548 pages, 13 plates and other Illustrations. VOLUME XIII. 605 pages, with 6 plates, 201 figures, and 1 colored

chart. VOLUME XIV. 633 pages, with 97 figures. VOLUME XV. 642 pages, with 87 illustrations. Twelve papers on pneumonia. By Drs. Chatard, Fadyan, E.merson;

Marshall, McCrae, Steiner, Howard and Hanes. A Study of Diarrhoea In Children. J. H. Mason Knox, Jr., M. D., and

Edwin H. .Schorer, M. D. Skin Transplantation. By John Staige Davis, M. D. Epidemic Cerebrospinal Meningitis and Serum Therapy at The Johns

Hopkins Hospital. By Frank J. Sladen, M. D. VOLUME XVI. 070 pages with 151 figures.

Studies in the Experimental Production of Tuberculosis in the Genitourinary Organs. By CiEORGE Walicer, M. D. The Effect on Breeding of the Removal of the Prostate Gland or of the

Veslculse Semlnales, or of Both ; together with Observations on the

Condition of the Testes after such Operations on White Hats. By

George Walker. M. D. Scalping Accidents. By John Staige Davis, M. D. Obstruction of the Inferior Vena Cava with a Report of Eighteen Cases.

By J. Hall Pleasants, M. D. Physiological and Pharmacological Studies on Cardiac Tonicity in Mammals. By Percival Douglas Cameron, M. D. VOLUME XVII. 586 pages with 21 plates and 136 flgures. Free Thrombi and Ball Thrombi In the Heart. By Joseph H. Hewitt,

M. D. Benzol as a Leucotoxin. By Lawrence Selling, M. D. Primary Carcinoma of the Liver. By Milton C. Winternitz. M. D. The St.itlstical Experience Data of The Johns Hopkins Hospital, Baltimore,

Md., 1S!I2-1911. By Frederick L. Hoffman. LL. D., F. S. S. The Origin and Development of the Lymphatic System. By Florence R.

Sabis, M. D. The Nuclei Tuberls Laterales and the So-called Ganglion Optlcum Basale.

By Edward F. Malone. M. D. Venous Thrombosis During Myocardial Insufficiency. By Frank J. Sladen,

M. D., and Milton C. Winternitz, M D. Leuksemla of the Fowl : Spontaneous and Experimental. By Harry C.

SCH.MEISSER. M. D.

VOLUME XVIII. 445 pages with 124 figures. Fasciculus I.

A Study of a Toxic Substance of the I'ancreas. By E. W. Goodpasture, M. D., and George Clark, M, D.

Old Age in Relation to Cell-overgrowth and Cancer. By E. W. Goodpasture. M. D„ and G. B. Wislocki, M. D.

The Effect of Removal of the Spleen Upon Metabolism In Dogs ; Preliminary Report. By J. H. King. M. D.

The Effect of Removal of tlie Spleen Upon Blood Transfusion. Bv J. H. King, M. D.. B. M. Bernheim, M. D., and A. T. Jones, M. D

Studies on Parathyroid Tetany. By D. ^X'ltiGHT Wilson, M. D., Thornton STEARN.S, M. D., J. H. Janney, Jr., M. D., and Madge DeG. Thurlow, M. D.

Some Observations on the Effect of Feeding Glands of Internal Secretion to Chicks. By M. C. Winternitz. M. D.

Spontaneous and Experimental Leukaemia In the Fowl. By H. C.

SCH.MEISSER. M. D.

Studies on the Relation of Fowl Typhoid to Leukaemia of the Fowl. Bv

M. C. Winternitz, M. D., and H. C. Schmeisser, M. D. Hyaline Degeneration of the Islands of Langerhans In Pancreatic Diabetes,

By M. C. Winternitz. M. D. Generalized Miliary Tuberculosis Resulting from Extension of a Tubercular

Pericarditis Into the Right Auricle. By M. C. Winternitz. M. D. Acute Suppurative Hypophysltis as a Complication of Purulent Sphenoidal

Sinusitis. By T. R. Boggs, M. D., and M. C. Winternitz, M. D. A Case of Pulmonary Moniliasis In the United States. By T R. Boggs.

M. D , and M. C. Pincoffs, M. D. Gaucher's Disease (A Report of Two Cases In Infancy). By J. H. M.

Knox, .M. D., H. R. Wahl, M. D., and H. C. Schmeisser, M. D. A Fatal Case of Multiple Primary Carcinomata. By E. D. Plass, M. D. Congenital Obliteration of the Bile-ducts. By Ja.mes B. Holmes,' M, D. Multiple Abscesses of the Brain In Infancy. 'By James B. Hol.mes, M. D. Gastric Carcinoma in a Woman of Twenty-six Years. By R. G. Hussei,

Subdiaphragmatic Abscess with Rupture Into the Peritoneal Cavity Fol


lowing Induced Pneumothorax for Pulmonary Hremorrhage. By R. G.

HussEY, M. D. Heart Block Caused by Gumma of the Septum. By E. W. Bridgeman,

M. D., and H. C. Sch.meisser, M. D. Analysis of Autopsy Records.

A. The Johns Hopkins Hospital. (Table Showing Percentage ot

Autopsies.)

B. The City Hospitals, Bay View. (Table Showing Percentage of

Autopsies.) "The Monday Conferences."

Clinical Representatives on the Staff of the Department of Pathology. Donation.

Fasciculus II.


ICxperimental Nephropathy In the Dog. Lesions Produced by Injection

of B. bronchisepticus into the Renal Artery. By M. C. Winternitz,

M. D., and William C. Quinby, M. D. Mesarteritis of the Pulmonary Artery. By M. C. Winternitz, M. V., and

H. C. Schmeisser, M. D. A Clinical and Pathological Study of Two Cases of Mllary Tuberculosis of

the Choroid. Bv Robert L. Randolph, M. D., and H. C. Schmeisser,

M. D. The Blood-vessels of the Heart Valves. By Stanhope Bayne-Jones, M. U. Equilibria in Precipitin Reactions. By Stanhope Bavne-Jones, M. D. Carcinoma of the Pleura with Hypertrophic Osteoarthropathy. Report of

a Case with a Description of the Histology of the Bone Lesion. By

Stanhope Bayne-Jones, M. D. The Interrelation of the Surviving Heart and Pancreas of the Dog in

Sugar Metabolism. By Ad.moxt H. Clark, M. D. Congenital Atresia of the Esophagus with Tracheo-Esophageal Fistula

Associated with Fused Kidney. A Case Report and A Summary of the

Literature on Congenital Anomalies of the Esophagus. By E. D.

Plass, M. D. Ectopia Cordis, with a Report of a Case in a Fifteen-Month-Old Infant.

By Ja.mes B. Holmes, M. D.


Report of Two Fatal Cases Following Percy's Ix)w Heat Treatment of Carcinoma of the Uterus. By V. N. Leonard, M. D. and A. B. Dayton, M. D.

The Relationship In Typhoid Between Splenic Infarcts and Peritonitis Unassociated with Intestinal Perforation. By A. B. Dayton, M. D.

I^ft Duodenal Hernia. By A. B. Dayton, M. D.

Histological as Related to Physiological and Chemical Differences in Certain Muscles of the Cat. By H. Hays Bullard, M. D.

A Method of Clearing Frozen Sections. By H. Hays Bullard, M. D.

On the Occurrence and Significance of Fat in the Muscle Fibers of the Atrioventricular System. By H. Hays Bullard, M. D.

Studies on the Metabolism of Cells in ri'fio. 1. The Toxicity of o-AmlnoAclds for Embryonic Chicken Cells. By Montrose T. Burrows, M. D., and Clarence A. Neymann, M. D.

The Significance of the Lunula of the Nail. By Montrose T. Burrows, M. D.

The Oxygen Pressure Necessary for Tissue Activity. By Montrose T. Burrows, M. D.

The Functional Relation of Intercellular Substances In the Bodv to Certain Structures in the Egg Cell and Unicellular Organisms. By Montrose T. Burrows, M. D.

Studies on the Growth of Cells in vitro. The Cultivation of Bladder and Prostrate Tumors Outside the Body. By Montrose T. Burrows, M. D., J. Edward Burns, M. D . and' Yoshio Suzukl. M. D.

The Study of a Small Outbreak of Poliomyelitis in an Apartment House. Occurring in the Course of an Epidemic in a Large Citv. By Montrose T. Burrows, M. D., and Edwards A. Park. M. D.

Papilloma of the Larynx Report of a Case Treated with Radium with Resultant Chronic Diffuse Thyroiditis. By William C. Duffy, M. l>.

Analysis of Autopsy Records.

Autopsy Statistics.

(a) Bay View.

(b) Johns Hopkins Hospital. Report of the Photographic Department. General Improvements.

Donations.

VOLUME XIX. 358 pages with 29 plates.

The Structure of the Normal Fibers of Purkinje In the .Adult Human Heart and Their Pathological Alteration in Syphilitic .Myocarditis. By O. Van Der Stricht and T. Wingate Todd. M. D.

The Operative Story of Goitre. The Author's Operation. By William S. Halsted. M. D.

Study of Arterlo- Venous Fistula with an Analysis of H' Cases. By CuRLK L. Callander. M. D.

VOLUME XX. 314 pages with 82 plates.

The Pathologv of the Pneumonia In the Inited States Army Camps During the Winter of 1917-18. By William G. MacCallum. M. D.

Pathological .Anatomv of Pneumonia Associated with Infiuenza. By William G. Mac'Callu.m, M. D.

Lymphosarcoma. Lymphatic Leukiemia. Leucosarcoma. Hodgklns Disease. Leslie T. Webster, M. D.


CONTENTS

  • Generalized Megalocaryocytic Reaction to Saponin Poisoning. (Illustrated.) By J. FiRKET and E. S. Campos 271
  • The Survival of Cells after the Death of the Organism. (Illustrated.) By Warren H. Lewis and Chari-es C. McCoy . . 284
  • A Study of the Austin-Stillman-Van Slyke Index of Urea Excretion. By Charles H. Hitchcock, M. D., and Ruth LoveLAXD, A. B. 294
  • Studies on Experimental Rickets. XXII. Conditions Which llust Be Fulfilled in Preparing Animals for Testing the Anti-Rachitic Effect of Individual Foodstuffs. By E. V. McCoi.r.UM, Nixa Simmonds, P. G. Shipley and E. A. Park 296
  • Notes on Books.

Books Received 304


GENERALIZED MEGALOCARYOCYTIC REACTION TO SAPONIN POISONING

By J. FiRKET and E. S. Campos

(From the Department of Pathology, The Johns Hopkins University)


Our interest in the subject of this investigation was aroused by the occurrence of a peculiar case of extreme anaemia in which there was complete aplasia of the bone marrow together with a new formation, in the liver and spleen, of myeloid tissue in which megalocarycK-ytes predominated. This case, in itself worthy of ])ul)lication, we could not explain on the basis of anything that could be learned of tlie man's hi.story, but in attempting to imitate it bj' any means in our power, we found that a verj' similar condition could be produced by saponin jioisoning. Bunting, as we found later, had shown that sajioniii poisoning destroys many of the blood i)latelets and produces a multiplication in the bone nuarrow of the megalocaryocytes; these observations we have been able to confirm.

The case of an;T»mia was as follows: Clinical History: J. W., a healthy man, aged 40 years, began to feel weak during December, 1020. He continued working until


January 11th, li)21, but with increasing fatigue, weakness, dyspnoea and pain in the lumbar region. At about that time he developed oedema of the ankles and puttiness of the face. On January i:Uh the blood examination showed 1,744,000 E. B. C, 5,:J00 W. B. C, and 27)% haemoglobin. Two days later, during which he had been semicomatose, these figures were, 1,272,000 K. B. C, 4,200 W. B. C, and 17% luemoglobin. The color index was 0.71. The blood picture showed especially anisocytosis, poikilocytosis, 48% neutrophilic polymorphonuclear leucocytes, 2.5% myelocytes and a very few normoblasts. Platelets were practically absent. Ha-molysis began at .48% sodium chloride and was complete at .32% sodium chloride. There were no other positive symptoms. Notwithstanding two transfusions of 500 c.c. each of citrated blood, which raised the red-cell count to 2,000,000 with 34% of haMuoglobin and 3,3(50 white cells, the patient


[Xo. .'$78


sank deeper into coma, the pulse became weaker and death followed on January 20th. Autopsy ten hours after death by Dr. Athens.

Audtoinical diagnosis: Extreme amcmia, aplastic bone marrow; splenic tumor; focal necrosis of liver and spleen; extensive myeloid changes in liver and spleen; acute serofibrinous pericarditis and pleurisy; tuberculosis of mediastinal glands; caseous encapsulated nodule in left lung; catarrlial bronchitis.

Deposits of pigment were found in the liver cells, a slight hiemorrhage in the fundus of the eye and a complete aphisia of the marrow of the long bones, which was entirely fatty. Throughout the red pulp of the spleen and between the hepatic cells myeloid elements were present. It is very striking that, although in this myeloid tissue one finds normoblasts and myelocytes iu fairly large abundance, proportionally the most numerous elements are megaloearyocytes. In other words, the number of megalocaryocytes is relatively larger than in any other myeloid tissue: this is especially in contrast with what is seen in other myeloid metaplasias of sj)leeu and liver, as, for example, iu cases of myeloid leuka'mia. Some of these megalocaryocytes show a large, swollen, clear nucleus, with very little chromatin; others have a shrunken, deeply stained, almost i)yknotic nucleus.

In the spleen tliere were some megalocaryocytes which had gone through the capillary system of the liver into the pulmonary circulation, where they were found in the sections of the lung.

As far as the diagnosis is coucerned, the myeloid, or better, the megalocaryocytic reaction of the spleen and liver, the presence of focal necroses in the liver and spleen, the presence of iron pigmentation in the tissues and the absence of mai-ked haemorrhages, lead us to abandon the diagnosis of pui-e aplastic anaemia.

It seems probable that there was a jjoisoning of some sort and indeed the history records the fact that the patient was exposed four years before (but not since then) for about a year to benzol vapors and that the room in which he had worked lately was filled with fumes from soldering. It is obvious that the late effects of exposure to benzol vapor must be considered in our ettort to explain the existence of comitlete aplasia of the bone nmrrow with the presence of myeloid tissue in other organs. Selling found that myeloid metaplasia occurred in the spleen after the cessation of the myelotoxic action of the ])oison and at a time when the ai)lastic bone marrow was regenerating itself. It was not possible to enter upon experiments to decide whether a stage in the recovery from benzol poisoning might be readied at which myeloid metaplasia of distant organs could coexist with a still completely aplastic bone marrow. This, as well as the question of the possible production of abundant megalocaryocytes in distant organs, remains to be determined.


Our interest took a different turn toward an effort to find some poison which would produce this condition.

Saponin is well known as a powerful lutmolytic agent, producing, as Foa states, a severe au:emia with complete aplasia of the bone marrow and simultaneously a myeloid metaplasia of the spleen. This then seemed the most s\iitable substance for our experiments.

Chemically the term saponin is applied to a number of glucosides which, according to Cushny, have many features in common both iu their chemical properties and their pharmacological action. While the saponin bodies seem to have a localized destructive action upon the cells with which they come in contact, their effect is practically restricted to the blood and ha-mopoictic organs when injected into the body.

Saponin had been used in tlie experimental production of auicmia iu rabbits by Buntiug in lOOG. Later, as mentioned, in his paper of 11)0!> he records the fact that saponin destroj's blood platelets as well as red corpuscles and stirs up in the bone marrow an excess production of megalocaryocytes. Isaac and Mockel in 1910 state that saponin destroys the bone marrow, leaving it aplastic and producing a myeloid metaplasia of the spleen resembling the condition to be found in tlie hypothetic "acute aleukiemic myeloid leuk.emia."

The results of the.se experimenters led us to believe that by using saponin we might produce in rabbits a condition resembling that described in the fatal case in the human being.

The first experiments showed a conspicuous new formation of myeloid tissue in the liver and spleen with one feature which has not been observed by the other investigators, namely, the predominant new formation of megalocaryocytes in those organs. This becomes the chief poiut in our results, because on account of their function in forming blood platelets these cells assume a great importance in connection with the destructive action of saponin upon the platelets. Bunting liad mentioned tliis rather specialized destructive action and we proceeded to study it in further detail and to consider the subsequent regenerative processes.

Before relating our experiments and observations, it is a pleasure to acknowledge our indebtedness for the opportunity for work afforded us in the laboratory of Dr. W. G. MacCallum.

Material and Methods

Saponin: The sample used was the same for all the experiments. It came from The Heyl Laboratories, Inc., Chicago. All the doses were weighed with the same balance and, on account of the instability of solutions of this glucoside, fresh saponin w;is dissolved for every experiment.

The pliysiological salt solution, used as solvent, was also made up with tlie same sample of chemically pure sodium chloride (Merck I previously dehydrated by one


August, 1922]


273


hour's exposure at 150° C. The solution used was always at the strength of O.O^c.

All the animals used were of the same variety of Belgian hare, and none of them were very young. They were all submitted to the same conditions of food (wheat, water and occasionally fresh lettuce) and kept in the .same room at a fairly constant temperature.

Each of the forty-five rabbits was given a number as soon as it was used once and this number was not changed afterwards.

All the injections were made intravenously after shaving and cleaning the region of the marginal vein of the ear. As the localized necrotic action of saponin, when used in relatively strong concentrations, was very marked, there usually occurred thrombosis of the veins of the ear with very often oedema, and later on, fibrosis of the cellular tissue surrounding the vein. This, in a few instance's of long repeated injections obliged us to inject into the veins of the abdominal wall, without any other trouble than the same localized action just mentioned.

Autopsies. Each dead rabbit was submitted to autopsy. These w^ere made most frequently immediately after death, as the rabbits were usually killed. 8ome rabbits found dead in their cages the next morning were examined as soon as possible, so that the tissues were not in any case exposed to post-mortem changes for a period of more than ten hours. All autopsies were performed according to the same method. The liver, spleen, bone marrow of the femur, tibia and humerus, adrenals, lungs and kidney were fixed. In many autopsies the lymph glands were also preserved.

Zenker's formol fluid (Helly's formula) was used as fixing agent; occasionally 10% formalin was used.

The organs, embedded in paraffin, were then sectioned, the sections being from 5 to 7.5 in thickness.

Longitudinal sections of the whole organs, as far as jtossible, and transverse sections of the bone marrow of each specimen, were made. This is important, as only a thorough examination of the marrow of all the long bones can give a true impression of the condition of this hemoIioietic apparatus. A longitudinal section of the complete spleen was also cut in most cases.

The staining methods used were: Hsematoxylin (Griibler's) and eosin; Wright's stain and the Poulenc "Panchrome de Laverau."

For blood examination and blood counting the usual routine methods were employed. The same pipette and counting-chamber (Thoma-Zeiss) were used for all the counts of one animal. The diluting fluid for red corpuscles was 0.9% salt solution and the special fluid used for platelets (see below) ; for the white cells, Ttirck's


fluid. As soon as the sample of blood was diluted, the pipette was shaken in an electric shaker for from five to ten minutes to avoid any agglutination and precipitation. The haemoglobin estimation was made always with the same Gowers-Sahli htemoglobinometer.

In order to make the report of our results clear, we shall proceed in the following manner: the experiments will be selected according to the problem to which they are related ; by that means we shall deal first with the question of blood destruction which has led us to study (1| the resistance of the red cells to saponin; (2) the specific action of saponin on blood platelets in vivo and in vitro; after this the process of regeneration will be described and with it the most appropriate protocols of experiments and autopsy records.

Finally, we shall have to consider the question of the mechanism which leads to the so-called "aplasia" of the bone marrow after injections of saponin.

Studies of the Resistance of Red Cells to Saponin

"When we began .systematically our study of the action of saponin, we realized that in order to come to an understanding of the blood curve, we had to study not only the process of regeneration but also the process of destruction. Indeed, there was a possibility that the resistance of the erythrocytes of normal rabbits might change after saponin injection, making difficult our understanding of the blood curve.

Realizing this, in a first series of experiments we investigated the resistance of the red blood cells of the normal rabbits to our sample of saponin. After we had found this we tested the resistance of the red blood cells of the same rabbits after they had been injected with saponin.

In a third series of experiments, we investigated the same property of the blood in splenectomised rabbits. This was done, because it had been shown by Pearce and others, in their experiments on splenectomised dogs, that there is, after splenectomy, an increased resistance of the red cells to hypotonic salt-solutions, hsemolytic serum, saponin, and other injurious substances.

Technique. — Only washed blood-cells and not the blood in toto were used for the hemolytic test. The blood was taken through a puncture of the marginal vein of the ear and allowed to drop freely into a 1 per cent solution of sodium citrate. The suspension was then centrifuged, the plasma removed by means of a pipette, and the red corpuscles washed three times with a 0.9% NaCl solution. A suspension of wasjied red cells of about 3% was then made in the same physiological solution.

Saponin was prepared in the same salt solution at the strength of .25 mmgr. saponin for 1 cc. of physiological salt solution. This mother solution was diluted with physiological salt solution as follows:


274


[No. 378


Tube No.


Sol. of Saponin .25 mmgr. for 1 cc. salt eol. .9%


0.9% Sal


solution


10


.10


cc.


.90


CC.


11


.09


cc.


.91


cc.


12


.08


cc.


.92


cc.


13


.07


cc.


.93


cc.


14


.06


cc.


.94


cc.


15


.05


cc.


.95


cc.


16


.04


cc.


.96


cc.


17


.03


cc.


.97


cc.


18


.02


cc.


.98


cc.


19


.01


cc.


.99


cc.


20


0.00


cc.


1.00


cc.


To each tube of this system were added, with the same pipette, two drops of the suspension of red coi-puscles. The heniolvtic sj'stem was placed in the water-bath at 38° C. for four hours, during which it was shaken several times. When taken out of the water-bath, the readings were made after half an hour of sedimentation. In describing our results, we shall refer to the tube numbers given in the above table.

Results of the Hwmolyfic Tests irith Saponin in Normal Rabbits

By normal, we mean the rabbits having not yet received an injection of saponin and those which did not present at autopsy any lesions of coccidiosis or other parasitic infection.


No. of rabbit


Dat


"


Hieni


ol. begins n tube


Hsro. complete in tube


23


May


21



13


12


24


May


21



13


12


30


May


21



13


12


30


May


28



13


12


30 38


May June


These results indicate that the erythrocytes of different rabbits differ only slightly as regards their resistance to saponin. It also shows that resistance remains fairly constant in the same rabbit {Rabbit No. 30).

Results of the hfemolytic tests with saponin in rabbits having received intravenous injections of saponin.


No. of rabbit


Before Injection 1


After Injection


No. of inieccions received


Ha:m. begins in tube


Hasm. com- 1 plete in tube


Hsem. begins in tube


Hsm. complete in tube


21


13


12


13


12


6 inj. 4.2 mg. in 7 days


24


13


12


13


12


3 inj. 5.8 mg.

in 4 days


33




13


11


11 inj. 1 mg. in 15 days


25




13


12


10 inj. 5.8 mg. in 10 days


Hwmolytic Tests with Saponin in Splenectomized Rabbits

The rabbits of the following table had not received, when the test was made, any injection of saponin.


No. of Rabbits


Condition


Hsmolysis begins in tube


Hxmolyais complete in tube


24


Normal


13


12


29


Splenectomized 2 days ago


14


12


27


7 days ago


13


12


8


7 days ago


13


12


19


" 11 days ago


13


11


29


Splenectomized 12 days ago


13


11


27


15 days ago


13


11


19


18 days ago


14


12


19


Splenectomized 48 days ago


14


11


38


Normal


13


12


Although Rabbit No. 19 did not show any increased resistance to saponin, it had shown, six days before the last test, an increased resistance of its red cells to hypotonic salt solutions, as can be seen from the following table. This system was prepared so that — Tube 1 contained 20 drops of physiological salt-solution, Tube 2 contained 19 drops of physiological salt-solution

plus 1 drop of distilled water, Tube 3 contained 18 drops of physiological salt solution

plus 2 drops of distilled water, Tube 20 contained 1 drop of physiological salt solution

plus 19 drops of distilled water. To each tube of this system two drops of a S'/o suspension

of the washed red cells were added.

Ilwmolytic Test icith Hypotonic Salt Solution in a Splenecfoniizcd Rabbit


No. of rabbits


Hamolysis j Haemolysis Condition begins 1 complete at tube ' in tube


38 37 19


Normal 9 14 Normal J 9 14 Splenectomized 4 2 days ago' 11 15


In summing up these results we are i)erniitted to draw only the following conclusions :

(1) After intravenous injections of saponin in rabbits, the resistance of red cells to this drug does not show any marked change.

(2) The red cells of splenectomized rabbits are not different in their resistance to saponin from those of nornuil rabbits, although they are more resistant to hypotonic salt solution.

This is not completely in accord with what I'earce found in his splenectomized dogs in which increased resistance of red corjjuscles to saponin as well as to hypotonic solutions and other hemolytic substances occurred. They do not differ, however, from the observations of McNeil in man.

Although it is not our jnirpose to discuss at length the question of hivniolysis by saponin, this part of our results


August, 1922]


275


convinced us that, as far as the corpuscles alone were concerned, the destructive power of saponin remains fairly constant when used in the same amounts and the same dilutions. We know, however, that the action of saponin ill vivo is far more complex than that shown in a testtube with washed corpuscles. Ranson found that cholesterin and lecithins, in salt solution, have a protective power against hemolysis by saponin. Clark and Evans have recently shown that normal human serum shows a protective power against hemolysis by saponin far more active than the protective power of cholesterin and lecithin ; they^ were, however, unable to tell to what mechanism or substance this normally |)resent protective power of serum against hemolysis by saponin was due.

We have not made any study along these lines and have not investigated the chemistry of the blood of our rabbits before injection. We had the impression, however, that in our series of rabbits two pregnant animals showed a greater resistance to saponin. Receiving the same amount of saponin intravenously, they became less rapidly auiBmic, and this was due certainly to a diminished destruction of corpuscles, because the hemopoietic reaction, found at autop.sy, was less marked in these animals than in the controls. For this reason, and to maintain more constant conditions, we used later only male rabbits which, being all fed in the same manner, would have probably a more constant amount of lipoids in their blood.

Action of Saponin on Blood Platelets

As we found, in our first rabbits injected with sajionin, the liver and spleen filled with megalocaryocytes, we were naturally forced to consider the action of this drug on platelets. Indeed, most of the hematologists in Europe as in this country, agree at present with Wright's doctrine of the megalocaryocj'tic origin of blood platelets (Naegeli, Schridde, Cesaris-Demel, Ferrata, Aschofif, Ogata, Downey, Klein, Foa, Brown, Lesourd and I'agniez, Di (iuglielmo, and others), although some Italian investigators, for example, I'ianese and I'erroncito, have rejected it recently.

Consequently we were inclined to think that a careful investigation of the platelet curve of our injected rabbits would be of some use.

Teehniqup. In order to avoid, as far as possible, any causes of error, we were always careful to draw the sample of blood from the first drop of blood arriving freelj' after the puncture of the marginal vein of the ear of the rabbit. In drawing the blood into the red counting pipette, the tip of the pipette was held in the middle of this drop and did not touch the lips of the vascular wound. Then it was rapidly diluted in the pipette with physiological salt solution or more often with the following fluid: 30 cc. of a 3% solution of sodium citrate in distilled water and 1 cc. of a 1 :150 brilliant-cresyl-blue solution in


distilled water, the two fluids being mixed and filtered before use.

As soon as the pipette was filled with blood, it was shaken in the electrical shaker. If this is done rapidly, one succeeds almost always in avoiding any agglutination of platelets. The very few samples in which agglutination was observed were discarded as not reliable for accurate counting. After ten minutes' shaking, the drop for examination was allowed to deposit for tAventy minutes, which is the time required for a complete sedimentation of the platelets. Let us add that all counts have been made with the same pipette and same chamber, by the same observer, and were often controlled by the other.

The following table shows the effect upon the platelets of the injection of large doses of saponin.

TABLE I. Reduction of platelet-count after injection of saponin.




June 29 — 9 a.m. 9:45 a.m.

10:15 a.m. 2 p.m. 4: 15 p.m.


15 mgm. in 15 cc. phys. salt solution


7,776,000


6,400,000 5,850,000 5,800,000


496,000 200,000 160,000


On June 30th the animal was found dead.


41


July 1—

9 a.m. 10:30 a.m.

12:30 p.m. 2:30 p.m. 4:30 p.m.


15 mgm. in 7.5 cc. phys. salt solution


6,100,000


4,800,000 4,936,000 5,008,000


320,000 290,000 272,000


On July 2nd the animal was found dead.

Similar observations were frequently made during our experiments. They show plainly that there is a diminution of the number of platelets in the circulating blood, following an injection of saponin.

We had to keep in mind, however, that several substances have been used to diminish temporarily the blood platelets of the circulating blood. Quite recently Roskain succeeded in doing so in dogs, with injections of gelatin. While one does not know exactly what becomes of these platelets, one might wonder if they were not stored up in the visceral circulation.

Two rabbits were used to answer i)artly this question as regards saponin. After injection of saponin they were anesthetized with ether, and laparotomized; simultaneously samples of blood were taken by puncture of the vein of the ear and of the portal or splenic vein.

These experiments, while very concordant, show that the number of platelets circulating in the blood of the portal system is not higher than the number of platelets


276


[No. 378


TABLK II. Platelet counts in peripheral and visceral blood.


39 June 28 — a.m. 1:15 p.m.


2:45 p.m. 4:45 p.m.


July 11 — 8:30 a.m. 9:15 a.m.

11:20 a.m, 1:15 p.m.

4 p.m.

5 p.m.


Peripheral blood


6,576,000


6,070,000 4,800,000


6,626,000


6,400,000! 342,000 6,144,000 280,000 6,648,000 220,000


670,000


360,000 328,000


Portal Vein


4,832,000 304,000


Splenic Vein


4,996,000 200,000


in the peripheral blood. They do not allow us, however, to discard the hypothesis that blood platelets are retained iu tlie capillaries of an organ or even of the whole body. A real proof that we are dealing with a destruction of platelets would be to obtain this destruction in vitro, and

TABLE III. Action of saponin in vitro.



1.

Saponin solution:

0.25 mgm. per 1 cc. 0.9% NaCl


II.

0.9% NaCl solution


Corresponding tube in hKmolylic system


Platelets per cu.mm.




cc.


cc.





42



0.035


1

0.965



428,000 262,000





0.04


0.96


16


260,000





0.045


0.955



240,000





0.05


0.95


15


104,000





0.055


0.945



100,000





0.06


0.94


14


36,000





0.07


0.93


13


48,000





0.075


0.925



40,000



44




II.

Sodium citrate and brilliant cresyl blue



Platelets 420,000


Erylhrocylea



1 cc.


7,936,000




0.06 cc.


0.94 cc.


14


148,000


7,736,000




0.07 cc.


0.93 cc.


13


172,000


6,584,000


Another







sample



1 cc.



488,000


6,080,000


of blood.







other


0.06 cc.


0.94 cc.


14


196,000


5,584,000


pipette







and







chamber







Another







sample of



1 cc.



496,000


6,968,000


blood



0.08 cc.


0.92 cc.


12


168,000


6,112,000


Another






sample of


1 cc.



364,000


5.824,000


blood 0.09 cc.


0.91 cc.


11


176,000


5,808,000


a series of experiments was planned to see if this could be done.

Technique: Several samples of blood, drawn from the same drop, were taken in several pipettes ; one with ordintii-y 0.9% salt solution or with the fluid used for platelets (see above) ; others with physiological salt solution to which had been added a certain amount of a solution of saponin containing .25 mg. to 1 cc. of salt solution. These last diluting fluids were measured so that hiemolysis could not be obtained at all or could be obtained only after a long exposure. The strength of these dilutions were known by our previous h;pmolytic tests. When all the pipettes were filled with these special diluting fluids, they were shaken in the electrical shaker. As twenty minutes were required for the sedimentation of the drop in the counting chamber before any counts of platelets were made, the platelets were expo.sed to the action of saponin, in tntro, for about 30 minutes.

Of course, none of the rabbits u.«ed in these experiments had received previously any injection of saponin.

This demonstrates that saponin in physiological salt solution, in a dilution which hcemolyses red cells slowly or not at all. destroys many of the platelets. It is probable, therefore, that the platelets do not all show iu vitro the same resistance to hemolytic agents, because some survive while others are destroyed just as in the case of the red corpuscles, for it is well known that young red cells, more especially nucleated red cells, have a higher power of resistance than mature erythrocytes.

While Bunting deserves entire credit for the discovery of this action of saponin, we think that a report of our experiments may be useful, because they seem to meet, more clearly than Bunting's, the objections which may be raised against the statement that saponin destroys the platelets. Several investigators have attempted to withdraw all the platelets from the circulating blood by means of injections of such substances as gelatin. This is effective in causing their disappearance, but we do not know how it acts nor whether the ])latelets are really destroyed. The actual destruction of platelets would stimulate their specific regeneration in the ha>mopoietic organs, while this would probably not follow the action of any agent which only temporarily withdraws platelets from the peripheral blood.

Procrss of Rerfcncratinii Our rabbits injected with saponin did not all sliow the same clinical picture. It is well known that besides a toxic action, not always measurable and leaving no lesions, saponin destroys the corpuscles in the circulating blood, and, also, as Bunting and the writers have sliown, destroys tlie blood platelets. Thus the effect of saponin, like that of any other poison, is subjected to many factors which are probably not all known and not measurable, depending on the individual resistance, the concentration of the dose, its excreti(ni from the bodv, ct cetera.


August, 1922]


277


But as far as its action on the blood curve is concerned, we had an impression that in general our rabbits behaved in two different ways ; some receiving doses of saponin became rapidlj- anemic, lost weight and appetite and died after a rather short time with a high degree of anemia (rabbits 3, 5, li), 3S) ; others, receiving somewhat smaller doses (per body weight) or the same doses but less often repeated, showed in the beginning also anemia, loss of weight and appetite. They then entered a period where the same doses of poison did not seem to have the same effect any more on their blood picture; the red blood cell count became higlier again ; the rabbits, more lively, seemed to have come to a stage of equilibrium in which their hsemopoietic organs were able to restore daily the blood elements destroyed (rabbits 11, 12, and others). On the contrary, the rabbits of the first categoiy, while their htemopoietic organs showed a tremendous attempt to repair their losses, were unable to reach before death this stage of equilibrium.

In the rabbits which had received injections of saponin during long periods, there was an adaptation of the activity of the hiemopoietic organs to the losses of blood, and this was made still more clear in the rabbits which did not receive regularly the same dose. These, while running a long course, became irregularly anemic, but never were able to reach the stage of equilibrium above mentioned (rabbits 1 and 4).

The rabbits of another group received a dose strong enough to kill them in less than twenty-four hours, so that their hiemopoietic organs had not had time to show any tendency to repair the loss of blood (rabbits 39, 40, 41). Consequently, we shall present separately the records of these three groups.

Rabbits 39, JfO and ^1: All these rabbits were injected with large doses of saponin from 15 mgr. to 30 mgr. All of them died in less than twenty-four hours after the injection. The autoi)sy findings were all alike: liver and spleen normal; hyperemia of the bone marrow with numerous diffuse and small ha?morrhages ; no increase of cellular activity in the bone nuirrow but a large u\imber of megalocaryocytes. While the spleen, the liver and other organs show no pathological changes, the bone marrow even so shortly after the injection (four or five hours for rabbit ;!9) shows a marked dilatation of its capillaries filled with blood and diffuse haemorrhages.

We shall describe the protocols of three rabbits of the second group which had shown tremendous restorative efforts of the ha?mopoietic organs.

Autopsy (performed immediately: Bone marrow dark red. Microscopically: Hplcen. Tremendous myeloid metaplasia with normoblasts, myelocytes and especially megalocan-ocytes (see Fig. No. 1). These are proportionally more abundant than in any other myeloid tissue ; this is so well mai-ked that one seems to deal with a genuine megalocarj'ocytic reaction. These megalocaryocytes


PROTOCOL OF RABBIT No. 3 (Old, long hair)


Date Time 1921


Saponin injected intravenously


Erylhrocytesj Leucocytes


Hemoglobin


observations


Mar. 10 —







3 p.m.



4,084,000


11,400


104%



4 p.m.


5 mgm. in 2.0 cc.







phys. salt sol.






Mar. 11 —







3 p.m.



2,416,000


44,000




4 p.m.


5 mgm. in 2.5 cc.







phys. salt sol.






Mar. 12 —







1 p.m.


4 mgm. in 2 cc.






Mar. 14 —


phys. salt sol.





ruraplegiu .if liiiij lees




400,00


10,200


85%






Mar. 14 —







12 a.m.







The animal dies


show numerous figures of Wright and marked phagocytosis of polymorphonuclear leucocytes with pscudoeosinophilic granules and small lymphocytes. All the myelocytes found have pseudo-eosiuophilic granulations; no basoi)hilic myelocytes are seen. Many macrophages containing granules of pigment exist throughout the splenic pulp. Many free megalocaryocytes, myelocytes and normoblasts are in the large splenic sinuses and splenic veins.

Liver: Many megalocaryocytes in the capillaries (see Figs. 2 and 3). Most of them probably come from the spleen and stop in the hepatic circulation. Some may be formed in the liver itself. Also, in the hepatic circulation there are myelocytes and normoblasts which have surely the same origin as the megalocan-ocytes.

Lung: Myelocytes and megalocaryocytes in the capillaries and in the pulmonary vessels. The megalocaryocytes are almost devoid of cytoplasm.

Adrenal: A few megalocaryocytes in the capillaries (see Fig. 4).

Kidney: Normal.

Bone marrmv: Hyperplasia with many normoblasts and erjthrocytes and many megalocaryocytes, which show a great tendency to phagocytosis. Hyperaemia and hemorrhages.

Rabbit Xo. 5. This rabbit showed exactly the same clinical picture; became rapidly ana?mic, lost 300 grams and (lied after five days.

Two and three days after the beginning of the injection, the number of normoblasts in the circulating blood was very high. The autopsy findings are exactly the same as for Rabbit No. 3. Abundant haemorrhages in the bone marrow.

These two tyjiical autopsies show how rapidly the rabbits become anaemic during the first days after injection. They died without having reached any equilibrium be


278


[No. 378


tween the losses of blood-elements and the repairing process, although there is a great activity in the hismopoietic organs (bone-marrow, spleen and liver). Megalocaryocytes are very abundant in all these organs. Hajmorrhages of the bone-marrow are more numerous than they were in the first group described.

It is interesting to notice that, even when the anjemia occurs less rapidly, the hemopoietic reaction to saponin injections seems, in animals killed at the same period, quite as marked. This is shown by the following record:

PROTOCOL OF RABBIT No. 24 (Weight 1940 grams; female, pregnant)


May 17 — May 22—


Saponin injected

4 mgm. per kilogran of body weight


Erythrocytes I Leucocytes


7,168,000 13,200


10 a.m.


5.8 mgm. in 1.41 cc. phys. salt sol.





May 23 —

10 a.m.


Similar injection





May 24 —

10 a.m.


Similar injection





May 25—

10 a.m.



5,568,000


15,200


71


The rabbit is killed at 12 m., being in excellent condition.

Autopsy : Spleen — numerous megalocaryocytes and several myelocytes.

Liver: Megalocaryocytes, showing phagocytosis, and very few myelocytes in the capillaries.

Lung: Megalocaryocytes, in the capillaries, almost deprived of their cytoplasm.

Bone marrow: Hyperaemia; hyperplasia with many megalocaryocytes.

Lymph gland: Normal; no megalocaryocytes or myelocytes.

Adrenal: No megalocaryocytes.

While the autopsy findings of the three last records are almost the same, it must be noticed that Rabbit No. 24 became much less ansemic than Rabbits 3 and 5 ; this may have occurred because it was pregnant and in this condition, on account of the increased cholesterina^mia, the protective power of the blood serum against haemolysis by saponin may be increased.

Before we describe the case of a rabbit, in which an equilibrium between the destructive and repairing processes is reached, we shall present a case where, although the animal remained living for about twenty days, the antemia was irregularly progressive and no equilibrium


was ever reached. This was due to the irregularity of the amount injected.

PROTOCOL OF RABBIT No. 4 (Weight 2375 grams on Jlarch 16th)


Dale Time 1921


Saponin injected intravenously


Erythrocytes per cu.mm.


Normoblasts


Leucocytes per cu.mm.


glut, in per cent *


March 16



5.600,000



12,000



10 a.m.


6 mgm. in 2 cc.







phys. salt sol.






March 17



3,348,000


376


37,600


85


10 a.m.


Similar injection






March 18



2,357,000



43,000


78


10 a.m.


Similar injection






March 19



2,048,000


2480


24,800


70


10 a.m.


Similar injection






March 21



816,000


327


10,900


60


10 a.m.


7.5 mgm. in 2.5 cc.







phys. salt sol.






March 22



720,000


792


13,200


60


12 a.m.


Similar injection






March 2 3



690,000


1026


18,000


52


11 a.m.


Similar injection






March 26



708,000


790


15.000


40


10 a.m.


Similar injection






March 29



972,000


455


9.100


50


10 a.m.


6 mgm. in 2 cc.







phys. salt sol.






March 30



786,000


315


10.500


48


11 a.m.


8 mgm. in 2 cc.







phys. salt sol.






Mar. 31-12


10 mgm. in 2.5 cc.







phys. salt sol.



131




April 1



600,000


82


8.200


45


Weight 1790 gms.

11 a.m.


Similar injection







April 2



436,000


968


8.600


45


10 a.m.


Similar injection






April 4



760,000


396


13,200


47


11 a.m.


Similar injection






On April 5th the animal was found dead.

AuTorsY : Weight — 1320 grams. The inferior part of the bone marrow of the long bones is red and the superior part is pinkish gray. Liver and spleen enlarged. Some lymph glands also show a slight increase in size.

Mwroscopioally: Liver — necrotic foci; slight myeloid reaction and exaggeration of the connective tissue.

Spleen: A greater myeloid reaction with numerous megalocaryocytes.

Adrenals: Some megalocaryocytes situated between the cortical and chromaffine tissue.

Bone marrow: Well defined focus of necrosis resembling a fresh infarct. Hyperplasia of moderate degree.

We shall describe now two rabbits presenting the typical history of tho.se where an equilibrium between the effects of blood destruction and blood repair is reached. The second rabbit described was splenectomised ami shows a verv interest iiiir blood regeneration.


AUGUST, 19iil!J


279


PROTOCOL OF RABBIT No. 12 (Weight on April 29th — 2340 grams)


Date Time 1921


Saponin injected

intravenously

4 mgm. for body

weight


Platelets per cu.mm.


Erythrocytes per cu.mm.


Leucocytes per


globin per cent


April 23




6,384,000


5,900' 102


April 29




5,632,000


9,200 93


April 30



580,000'


— 1 —


2 p.m.


5.6 mgm. in 1.4 cc.





phys. salt sol.








May 2


112,000 2,567,000 30,000 71


10 a.m.


Similar injection






May 3,







4 p.m.


Similar injection






May 4



250,000


1,392,000


8,000


65


May 5


Similar injection






May 6




2,400,000


6,200


50


12 a.m.


Similar injection






May 9



396,000


3,328,000


4,400


50


12 a.m.


Similar injection



.




May 10







12 a.m.


Similar injection





65


May 11



386,000


3,936,000


10,000


65


12 a.m.


Similar injection






On May 11, at 4 p.m., the animal died.

AuTOi'SY : The bone marrow is firm, pinkish-gray in some phices and grayisli-white in others.

Microsoopicalljj : The spleen shows nnmcrous megalocaryocytes and numerons macropliages loaded with pigment.

The liver sliows pigmentation of the hejiatic cells and a few megalocaryocytes.

The hone-marrow shows hyperplasia, hypera;mia, hiiemorrhages (see Fig. No. 5) and foci of necrosis where there is a beginning of organization.

In the lungs there are some megalocaryocytes almost without cytoplasm.

PROTOCOL OP RABBIT No. 11

At 3 p. m. the animal was in very good condition. Then it was killed as a control for Rabbit No. 12 whose spleen had not been removed.

Autopsy: Liver: A few megalocaryocytes, in the capillaries; almost no myelocytes. I'ignient in the hepatic cells.

Adrenal: Normal.

Lymph nodes: Several lymph nodes show myeloid reaction with numerous megalocaryocytes, several showing phagocytoses. (See Fig. 6.)

Lung: A few foci of haemorrhages.

Bone niarroir: Hyperplastic almost everywhere, with all kinds of myeloid cells (myelocytes, normoblasts, but more especially, many megalocaryocytes). Several megalocaryocytes show figures of Wright and phagocytosis. Other portions of the bone marrow are not very hyperplastic and remain fatty; even here, however, the most


1921

April 22 April 23 April 27 April 30 2 p.m.


injected


4 mgm. 1 1 cc. phys. »h solution


May 2 i

May 3 |

4 p.m.' Sim. inj. May 4

3 p.m. Sim. inj. May 5 May 6


globin per cent


105


Platelets Erythrocyte


6,992,000' 11,000


8.096,000 6,90o!


Sim. inj. Sim. inj.


2 p.m. May 9 I

2 p.m. Sim. inj. May 10 I Sim. inj. M ay 1 1 I

2 p.m.' Sim. inj.

May 12

May 13 I

2 p.m. Sim. inj. May 14 Sim. inj. May 15 Sim. inj. May 16 Sim. inj. May 17

1 p.m. Sim. inj.


103 840,000 7,752,000


400,000 4,256,000 12,000


192,000 3,200,000 24,000


wgt.nOOgnis


3,232,000 16,000 4,800,000


5,560,000


6,008,000


7,392,000


11,600


6,000


rgt.1600 gms


numerous cells, among the fat cells, are megalocaryocytes. Scattered throughout the organ are numerous foci of haemorrhages; other portions show young granulation tissue with fibroblasts beginning to elaborate connective fibers.

Summary and Discussion

We have studied rabbits which had been injected with large doses of saponin, others with small doses, the doses remaining the same or being changed at each injection. We have studied rabbits, so treated, during a few hours, others during a few days, still others during a few weeks (Rabbit No. 33, one month). Some rabbits were splenectomized. We have never found an aplasia of the bonemarroiv.

On the contrary, the preceding protocols demonstrate that while on the day of the first injection there is no appreciable haimopoietic activity, a few days later (3, 4 or 6 days) the bone-marrow is hyperplastic and that also in the spleen and the liver a metaplasia of myeloid elements takes place. Let us insist on the fact that in adult rabbits the presence of myeloid elements between the fat cells does not mean necessarily hyperplasia, as it would in man. Normally the adult rabbit has a bone marrow with a fairly large amount of myeloid cells, as Roger and Josu6 had shown some time ago and as evei-yone familiar with blood formation in rabbits knows. It is necessary to compare almost the entire bone marrow of tlie ex])erimental rabbit with that of the normal to reach a decision as to the presence or absence of hyperplasia.


280


[No. 378


This being known, we observed, however, on the third, fourth and fifth daj's following the injection, a definite hyperplasia of the marrow, but a disturbing factor to this hyperplasia was always present: hiBmorrhages occurred following a marked hyperaemia of all the capillaries. (See Fig. 7). These ha*niorrhages in the bone marrow are the only lesion found during the first day after injection of saponin and this leads us to believe that at the start there is an impainnent in the circulation and consequently in the function of the bone marrow. This is probably the reason why myeloid metaplasia occurs so soon in other organs (spleen, liver, and in certain conditions in lymph-nodes). In order to repair the losses of bloodcells, a complementary blood-forming tissue is needed, as the normal myeloid tissue is unable to develop with full strength.

Isaac and Meckel raised the question whether such a condition, which they also saw in expei'imental poisoning by saponin, could not be compared to myeloid aleukaemic leukaemia. In fact, when we saw the tremendous metaplasia described in Kabbit Xo. 3, for example, without niucli evidence of myeloid elements in the circulating blood, tlie same comparison came into our minds. This seems, however, less reasonable than the explanation of a vicarious blood formation due to functionally impaired bone marrow. Indeed, in using saponin we produce above all an erythropathia. As saponin has practically no action on leucocytes, it seems improbable that the reaction to such a poison would re.semble one of the diseases involving especially white cells.

The myeloid metaplasia, taking place outside of the bone marrow, is strikingly a reaction to the loss of blood platelets and red cells which are the elements destroyed. Megalocar5-ocyte.s and normoblasts are propoi-tioually the most numerous elements of that niyeloiilastic tissue. Of course, there are some myelocytes, but their number is conspicuously low.

Megalocaryocytic reaction. Such a statement cannot be 'made without considering somewhat carefully the question of megalocaryocytic reaction. Dominici, Pianese and others, studying many sections of the spleen and liver of several normal animals, found in all of them a few megalocaryocytes. Some of the sections of these organs, in our normal rabbits, show also occasionally a megalocaryocyte. We speak of megalocaryocytic reaction only when this number is markedly increased (see figures). That this reaction is markedly increased in relation to the destruction of platelets is probable, although we do not find ourselves able to say that no other platelet-repairing mechanism than this exists. Brown and Ferrata have shown that monocytes or cells resembling them could form platelets (monocytoid cells of Ferrata). Bunting is of the oi)inion that even lymphocytes give rise to blood platelets.


Although we have not in mind to study especially the morphology of megalocaryocytes, we have observed during our experiments the following types of these cells :

Types of inefiulocarj/ocyten : The megalocaryocytes found in the bone marrow, spleen, liver and other organs of the rabbits injected with saixmin, vary in appearance with respect to their size, sliape, structure of the cytoplasm and of the nucleus (see Figs. 8 and 9). However, megalocaryocytes described as belonging to dift'erent types of cells, may well have been really the result of sections of the same kind of cell made at diB:'erent levels. This fact must be kept in mind, especially when we are dealing with megalocaryocytes of rabbits, which are far larger than the human megalocaryocytes and in which the nuclei are more comi)licated. Carnegie Dickson, who made a careful cytological study of the bone marrow, shows a set of drawings made from serial sections of the same megalocaryocyte, where one can see the diversity of aspect of the cytoplasm and nucleus, and may receive the impression that there are various types of cells. Nevertlieless, in the saponin-injected rabbits we have seen megalocaryocytes strikingly dift'erent in structure. These we nuiy describe before separating them into groups.

These cells vary in size. The smallest are slightly larger than circulating monocytes, while the largest measure about i!00/i in diameter, trenerally, the cells are round or oval, but they may be reniform, crescentshaped, fusiform or almost triangular in outline. Some cells show pseudopod-like processes of varying size, form and number. Tliese amoeboid changes can be very well observed in sections of the liver where the megalocaryocytes inside the capillaries are greatly elongated and cylindrical in order to permit their passing through. The outline of the megalocaryocytes is either sharp, coarse or dift'use ; sometimes there is a bay-like depression.

Many authors have described the structure of the cytoplasm of the megalocaryocytes and, since the classical work of Wright on blood-platelet formation, this question has been thoroughly and carefully studied. Our findings are closely similar to what has been described. Indeed, there are cells in which the cytoplasm is apparently homogeneous and basophilic. Frequently the cytoplasm of these cells shows one or more homogeneous eosinophilic areas of dift'erent size and shape, which may have been produced by the fusion of several phagocyted erythrocytes. Some cells have a more abundant cytoplasm, apparently homogeneous, but less basophilic, often witli the same oxyphilic areas above described ; while in others the protoj)lasm is slightly acidoj)hilic. Many cells, however, with a basophilic or slightly eosinophilic cytoplasm, show a gi'anulated structure with the differentiation of zones as described by Wriglit, Foa, Sdiridde, Cesaris, Demel, Di Guglielmo, Ferrata and many others. These cells show a cytoplasm generally formed by two zones, usually sharply distinct : the large, granulateii,


August, 1922]


281


perinuclear zone and the narrow, apparently homogeneous, peripheral zone, showing a delicate basophilic or a light acidophilic reaction. The granules, very small, round or elongated, are stained lilac or violet, and are generally arranged in more or less definite parallel rows, circumscribing the nucleus. Some cells show an irregular granulation, with small and large granules, the latter generally lying near the periphery of the cell. As these large granules are separated one from another and embedded in a basophilic protoplasm, each granule is surrounded by a delicate zone, bluish in color. The cytoplasm of the megalocaryocytes frequently shows its phagocytic activity by containing several embedded cells; sometimes there are cells lying in small depressions of the cytoplasm, showing the act of engulfing.

The nuclei also present a great variety of shape and size, often due to projection of their sectioned portion, as mentioned above. According to their strikingly ditferent structure, it is easy to di.stiuguish several types :

1. Large nuclei, vei"y complicated in form, unevenly globular and hollow, with some irregular openings that pemiit the communication of the cytoplasm, situated inside and outside. These nuclei have been compared to a "basket-work," composed of threads twisted in the most complex way. They vary greatly in aspect, according to the level through which the section passes. The sections near the periphery show the nucleus as fomied by more or less numerous small pieces grouped togetlier, each one seeming to be limited by a definite membrane. The sections near the centre, through the equator, for instance, show a ring or wreath-shaped nucleus, formed by many nuclear pieces, irregularly joined by their extremities.

The chromatin is dispersed in more or less numerous granules of various size, probably supported by a reticulum of Unin. These nuclei are full of nuclear juice, or paralmin, so that they appear as though formed by deeply stained basophilic granules embedded in a colorless or slightly basophilic substance.

2. Small round nucleus of the same fine structure.

3. Large or small nuclei of various and very irregular forms, such as to defy any description. The.se nuclei are so rich in chromatin that the entire nuclear body is homogeneously very deeply stained, almost black, giving the idea of irregularly spread ink spots. These are verjpyknotic nuclei.

4. A nucleus that appears (•omi)osed of many short segments of a very dense, deeply stained chromatin, giving the impression of numerous chromosomes. Sometimes the thread of dense chromatin seems to be continuous, like a loose spircm — Itito spireme. The nucleoli in these types of nuclei are sometimes very conspicuous and acidophilic.

These different conditions of the cytoplasm and nucleus of the megalocaryocytes perhaps are the result of different evolutional phases of the same kind of cell.


In resuming, we have seen the following types of cells or probably the various successive evolutional phases of the same type of megalocaryocyte :

1. Small cells with a deeplj^ stained basophilic cytoplasm, apparently homogeneous, and a small round, oval or reniform nucleus, rich in nuclear juice (megalocaryoblast of Ferrata ?).

2. Large cells with an apparently homogeneous and either basophilic (lymphoid megalocaryocyte of Ferrata) or acidophilic cytoplasm, and a complex "basket-work" nucleus, rich in paraliniu.

3. Large cells with a granulated cytoplasm and a complex "basket-work" nucleus, rich in nuclear juice (granulated megalocaryocyte of Ferrata).

4. Cells of various sizes with an apparently homogeneous protoplasm and an irregular pyknotic nucleus (degenerate megalocaryocyte of Carnegie Dickson ?).

5. Cells of various sizes, the nucleus of which has the appearance of a mitotic figure — Into spireme — or chromosomes in formation (megalocaryocyte with nucleus in act of dividing or of rearrangement?).

G. Free nuclei, apparently without protoplasm at all. These nuclei are frequently pyknotic but sometimes vesicular.

Our cytological studies have been made with paraffin sections cut at 4.5/n and stained with Wright's stain.

That the megalocars'ocytic reaction is a specific reaction to the most urgent needs of the blood, was shown by our Rabbit No. ll. This rabbit, which had been splenectomized, after having received many injections of saponin had reached a state of equilibrium between the destructive and repairing processes of the blood. When killed, the bone-marrow of this animal presented the usual lesions which impair partially its functional capacity ; there was a slight myeloid metaplasia in the liver; but — and this is the interesting point — there was a megalocaryocytic reaction in almost all the lymph nodes studied.

We had never found megalocaryocytes in the lymph nodes of our other rabbits (Rabbits 21, 22, 23, 24, 25). What happened in this case would be explained by those who admit the unicist theory of the secondary blood formation, in the following nmnner: In the lymph nodes there are cells which cannot be considered morphologically different from the hannocytoblast (Ferrata) or lymphoidocyte (Pappenheim) of the bone marrow, as has been admitted by Dominici, Maximow, Pappenheim, Ferrata, and others, l^sually in the lymph nodes these cells give rise to lymphoid elements and are, as Ferrata clearly expresses it, hjemocytoblasts in lymphoid function ; but, like the medullary ha»mocytoblasts which produce megalocaryocytes and other myeloid cells in the bone marrow, in special conditions the ha-mocytoblasts of the lymphnodes can produce these megalocaryocytes or other myeloid elements. The myeloid potentiality of the lymph


282


[No. 378


nodes has been stimulated, in the case of Rabbit 11 by the absence of the spleen. That the myeloid potentiality of the lymph nodes has been specifically stimulated to form almost only megalocaryocytes, seems to show that the need of these cells or of the elements deriving from them, was the greatest.

Mechanism of the Disturbance in the Bone Marrow During the first hours following an injection of saponin, we noticed an extreme dilatation of all the capillaries of the bone marrow, which are packed with blood. This occurs, however, when other blood poisons are injected into rabbits, as, for example, benzol. We confirm this observation on the effect of benzol injected intravenously, on two rabbits (No. 11 and No. 16). After the intravenous injection of saponin, however, this hyperaemia is almost immediately followed by h;T?morrhages due to rupture of the overfilled capillary. This does not occur, so far as we know, in benzol poisoning. The question arises whether the rupture of the capillaries may not be due to au injury and weakening of the capillary wall.

It is, however, not easy to understand why this action of saponin is localized only in the capillaries of the bone marrow, since we do not find any hiemorrhages in other organs. Whatever the cause of these haemorrhages, it may be that all the other changes are consequences or a response to them. Necroses of some portion of the bone marrow are easily understood as a consequence of an impairment of its normal blood-circulation.

Organization of the necrotic foci or the foci of hiemorrhages is also a natural reaction. The general result of this process will be a partial incapacity of the bone marrow, which, in the areas in which the normal circulation is maintained, shows a high degree of blood formation. This is certainly different from a complete aplasia of bone marrow, such as saponin is classically said to produce.

Eeturning to the history of the case which determined our experimental study of anajmia following saponin poisoning, we are forced to admit that although saponin gives a clinical picture and autopsy findings in rabbits comparable to those given by the above described patient, there are very important differences which have to be emphasized. Like the spleen and liver of the patient, these organs in our rabbits showed an intense megalocaryocytic reaction; like his blood, their blood contained few red cells, numerous normoblasts, very few myelocytes in rare cases, and very few platelets. There are, however, striking differences: whereas the patient had a low color index and a fatty bone marrow, the experimental animals always had a high color index. There was a high grade of haemoglobinaemia and hajmoglobinuria (as, for instance. Rabbit No. 4). All our rabbits at autopsy had a hyperplastic bone-marrow, although with its normal capacity very much diminished by diffuse haemorrhages. The pathological problem of the case described concerns the one


sided blood-formation, but cannot be explained by our findings with saponin poisoning.

We have to admit that we were dealing in this case with another etiological factor and another mechanism. If any suggestion might be made, we would say this: it is improbable that any toxic factor in the body would prevent the formation of myeloid tissue where it normally exists, while at the same time it would allow the building up of myeloid tissue in organs where it is not found in the normal condition. Whatever the etiological factor in such a case may be, it is probable that it acts indirectly on the myeloid tissue of the bone marrow. Perhaps a careful study of the vessels and the nerves of the bone marrow, the functions of which are not well known and have not been thoroughly studied, would be of a greater help.

Conclusions

1. Single or repeated intravenous injections of saponin in rabbits do not change the resistance of the red blood cells to this toxic substance.

2. The erythrocytes of splenectomized rabbits are more resistant to hypotonic salt solution but not to saponin.

3. In addition to its hsemolytic action saponin is a highly destructive agent for blood platelets, both in vivo and ill vitro.

4. The intravenous injection of saponin into rabbits produces in the bone marrow hyperplasia and, simultaneously, numerous diffuse or circumscribed foci of hiemorrhage; there is not a true aplasia.

5. As the function of the bone marrow is impaired by haemorrhages and their effects, the spleen, liver and other organs show a vicarious myeloid formation. This newly grown myeloid tissue shows an unusually large number of nucleated red blood cells but princijially megalocaryocytes — megalocaryocytic reliction.

C. A megalocaryocytic reaction was observed in the lymph glands, after intravenous injection of saponin, in splenectomized but not in non-si)lenectomized rabbits. This may be explained as a vicarioiis function.

7. The megalocaryocytes found in the bone marrow, spleen, liver and other organs of the rabbits injected with saponin are not all identical in appearance. While presenting differences in morphology and structure of the nuclei and cytoplasm, they may be different stages of the same type of cell.

8. For a good understanding of the pathological changes in the bone marrow, it is very important to study large longitudinal sections (of almost the whole organ in rabbits) and cross-sections of the marrow of several bones. This is absolutely necessary in the study of the bone marrow of saponin-injected rabbits.

LITERATURE Aynaud: Les globulins des mammifferes. Thdse de Paris. 1909.

Betanc^s: La granulation azurophile. Paris. 1918. Bunting: Jour. Exper. Med., 1906.


THE JOHNS HOPKINS HOSPITAL BULLETIN. AUGUST. 1922


PLATE XXXVl


a-^



Fig. No. 1

Spleen of rabbit No. 3.

Myeloid reaction witlr numerous megaloliaryocytes.


Fk. No. 2

Liver of rabbit No. 3.

Myeloid reaction with numerous megalokaryocytes.



Fio. No. 3

Liver of rabbit No. 3

showing a megalokaryocyte in a capillary



Fig. No. 4

Adrenal of rabbit No. 3

showing a megalokaryocyte in the cortical tissue.


PLATE XXXVII


THE JOHNS HOPKINS HOSPITAL BULLETIN. AUGUST. 1922








Fh.. Xci. ,-,

Bone marrow of rabbit No. 12.

Diffuse liaemorrhage and vicarious hyperplasia.


Fig. No. 6 Lymph gland of rabbit No. 11.

Myeloid reaction; a megalokaryocyte is seen in the lymph sinus.





Fig. No. 7

Bone marrow of rabbit No. 22

showing hyperplasia and a large zone of hs>morrhage.


THE JOHNS HOPKINS HOSPITAL BULLETIN, AUGUST, 1922


PLATE XXXVIII


Q


^ ^




Fig. No. 8

Megalokaryocytes from the spleen of rabbit Xo. 3

(above) and of rabbit No. 11 (below).



Fii.. Xo. 'J

Megalokaryocytes from the spleen (above)

and liver (below) of rabbits No. 5 and No. 3.


August, 1922]


283


Idem: Blood platelets and megalokaryocytic reaction. Ibid., 1909, vol. XI.

Idem: Blood platelets and megalokaryocytes in Hodgkin's Disease. Johns Hopkins Hosp. Bull., 1912, p. 114.

Idem: Vicarious blood-platelets formation. Ibid., 1920, XXX. Johns Hopkins Hosp. Bull., 1920, XXXI.

Burnett: The clinical pathology of the blood of domesticated animals. New York. 1917.

Braun: Jour, of Exper. Med., 1913, XVIII.

Castronuovo: Piastrine e pseudopiastrine nel sangue circolante. Hjematologica. Vol. 1, Fasc. IV, 1920.

Carnegie Dickson: Bone marrow. A cytological study. 1908.

Clark H. M. and Evans F. A.: One factor in the mechanism of hemolysis in hemolytic anemia. Johns Hopkins Hosp. Bull., 1920, XXXI. No. 356.

Clark H. M. and Evans F. A.: Studies on lecithin and cholesterin in relation to the antihemolytic property of human serum. Johns Hopkins Hosp. Bull., 1921.

Cesaris Demel: SuH'origine delle piastrine dei megacariociti. Archivio per le Scienze Mediche, 1914, XXXVIII.

Cesaris Demel: Sulla funzione piastrinocinetica dei megacariociti e sulle modalita della loro penetrazione in circolo. Pathologica, 191.5.

Campos E. S. : Estudo das cellulas myelomatosas em un caso de myeloma. Doenga de Rustizky Kahler. Thfese de S. Paulo (Brazil), 1919.

Downey H.: The origin of blood platelets. Folia haematologica. 1913, Bd. XV, Heft 1, p. 25.

Di Guglielmo G.: Megacariociti e piastrine negli organi emopoietici e nel sangue circolante. Atti della R. Academia Medico-Chirurgica di Napoli, Anno LXXIII, 1919.

Di Guglielmo G.: Eritroleucemia e piastrinemia. Folia Medica, Nos. 1, 2, 3, 4, 5, 1920.

Di Guglielmo G.: Megacariociti e piastrine. Hsematologica, Vol. I. Ease III, 1920.

Eppinger: Die hepatolienalen Erkrankungen. Berlin. 1920. Ferrata: Le emopatie. 1918.

Fuzari: Contributo alio studio delle piastrine del sangue alio stato normale e patologico. Archivio per le Scienze Mediche. Bizzozero. Vol. 10, No. 12, 1886.

Foa,: Trattato di Anatomia Pathologica. Torino. 1920. Foti: Contributo sperimentale alia genesi delle piastrine del sangue nell'avvelenamento acuto da pirodina. Archivio de Fisiologia, 1913.

Gorke H.: Das Verhalten der Milz und des Knochenmarks und die Aussicht der Spleneclomie bei der aplatischen Anaemie. Deutsch. Arch, fiir Klinische Medizin, 1921, Bd. Ill, p. 143. Isaac S. und Mockel K.: Ueber Experimentelle schwere Ansmien. Verhandl. d. d. Congress, fiir innere Medizin, Wiesbaden, 1910, XXVII, 471-473.


Jordan: The histogenesis of blood-platelets in the yolk-sac of the pig embryo. Anat. Rec. 1919, XV, No. 7.

Lavenson: The nature of aplastic anaemia, etc. Amer. Jour. Med. Sci., 1907.

MacCallum, W. G.: Pathology, 1920.

Marino F. : Sur la non-existence des plaquettes de Bizzozero comme elements constants normaux et independant du sang des vertebras. Folia hsematologica, 1912, XIII, p. 89.

Marino F. : Remarques sur le travail de Bizzozero relative aux plaquettes. Fol. hsematologica, 1912, XIII, p. 93.

Morris S. R.: Blood formation in the liver and spleen in

experimental ansemia. Johns Hopkins Hosp. Bull., 1907, p. 200.

McNeil: The resistance of human red blood cells in health

and disease to hemolysis by saponin, etc. Jour. Path, and

Bact, 1910.

Moynihan B.: The spleen and some of its diseases. 1921. Perroncito A.: Sulla derivazione delle piastrine. Hsematologica. Vol. I, Fasc. Ill, 1920.

Perroncito A.: Sulla derivazione delle piastrine dei megacariociti. Haematologica, Vol. I, Fasc. I, 1920.

Pianese G.: Per una miglior conoscenza del megacariociti. Hsmatologica, Vol. I, Fasc. I, 1920.

Pearce M. R., Krumbhaar E. B., Frazier C. H.: The Spleen and Anaemia. Experimental and clinical studies. 1918. Rieux J.: Precis d'hematologie et cytologie. 1911. Roger et Josu^: La moelle osseuse a I'etat normal et dans les infections. Oeuvre medico-legale, 189 9.

Ramson: Saponin und sein Gegengift. Deutsche med. Wochenschr., 1901, XXVII, 1914.

Roskan J.: Globulins et temps de saignement. Soc. Beige de biologie. Mai 1921, p. 8.

Selling L. : Benzol als Leukotoxine. Studien iiber die Degeneration und Regeneration des Blutes und des hemopoietlschen Organe. Ziegler's Beitrage z. path. Anatomie. 1911, LI, 516.

Thiel and Downey: The development of the mammalian spleen, with special reference to its hematopoietic activity. Amer. Jour, of Anat., 1921, XXVIII, No. 2, 279.

Wright: Die Entstehung der Blutplattchen. Virchows Arch., 1906, CLXXXVI.

Wright: The histogenesis of blood platelets. Jour, of Morph. 1910, XXI.

Weinberg et Gilbert: Traite du sang. 1913, t. 1. Warthin A. S. : The pathology of pernicious anaemia, with special reference to changes occurring in the haemolymph nodes (eight autopsy cases). Am. Jour. Med. Sci. 1902, CXXIV, 674.


THE SURVIVAL OF CELLS AFTER THE DEATH OF THE ORGANISM

By Warren H. Lewis and Charles C. McCoy (From the Carnegie Laboratory of Embryology, and the Department of Pathology, Johns Hopkins Medical School)

Introduction


It is now a matter of common knowledge that cells of certain types can survive for a limited period after removal from the body. The ectoderm, especially noteworthy in this regard, appears to be able to live several days if kept under proper conditions. The test of viability is the "take" of the skin graft. Skeletal, heart, and smooth-muscle fiber-s can be made to contract and relax for a considerable period of time outside the body. The muscle-fibers in the physiological preparations used for such experiments are supposed to remain alive; the criterion here is the ability of the muscle to contract and relax in a proper artificial environment with suitable stimuli. In muscle-nerve specimens the isolated nervefibers are able to conduct stimuli to the muscles for varying periods. Do the axones continue to live, after their separation from the cell bodies, as long as the nerves are able to conduct stimuli to the muscles?

Carrel {'07) succeeded in getting good results from the heterotransplantation of blood-vessels that had been kept in cold storage for 4 days, while those kept 7, 17, and 20 days, respectively, were not so successfully transplanted. It is not clear from his data whether the cells in the transplanted 4-day blood-vessel remained alive or whether a process of substitution made the transplant a success. In 1910 he succeeded in replanting spleen 44 minutes, and kidney 50 minutes, after removal from the body. The criterion for life in such experiments is the success of the tran.splantation and the resumption of function of the organ.

Tissue-cultures have shown that most cells not only survive but move about and even multiply for many days after removal from the body. Fischer ('21) cultivated epithelial cells for over two months, while the connectivetissue cells may live and multiply for years (Ebeling, '21). It is probable, therefore, that many types of cells are potentially immortal, given the proper environment. Tissue-culture affords the most advantageous environ ment that has yet been devi.sed.

In the present study we have endeavored to determine the periods of survival of several difl'erent types of cells after the death of the animal, under various conditions other than those of tissue-culture. Our criterion for life was the presence within the cells of certain granules and vacuoles that have a great affinity for neutral red. The value of neutral red as a testing agent was suggested by


the following considerations. Experience with tissueculture has shown that all types of living cells either have or can develop granules and vacuoles with a marked aflSnity for neutral red. This dye does not become concentrated within the living cells unless there is some pre-existing body or substance to which it can become attached. In the ordinary strengths used it does not stain the cytoplasm or nucleus, nor does it seem to injure the cells. It is a well-known fact that in dead cells of tissuecultui-es the granules and vacuoles do not take up neutral red as they do in living cells, although the entire dead cell may stain diffusely if the dye is strong enough. Dead cells, but not living ones, are stained diffusely with various other dyes, such as toluidin bine (Gross, '11; Steckelmacher, '14), and benzidine dyes (Evans and Schulemann, '14), Nile blue B extra and brilliant cresyl blue (Lewis and Lewis, '15).

Granules and vacuoles that have been colored with neutral red in the living cell lose their color when the cell dies (W. H. Lewis, '21). We have noted repeatedly that when the cells of a spread are killed rapidly by heating, the red vacuoles lose their color and the cells become more or less diffusely pink. Much the same results were obtained when the spreads were killed with acetic acid.

As a rule, the dead cells in the various spreads, except those from the intestine, retained their form both as to cytoplasm and nucleus. It was possible to distinguish the dead from the living cells even without the use of neutral red. The main differences are in the texture of the cytoplasm and nucleus and in the character of the nuclear membrane, which becomes more marked after death.

Most cells, under abnormal conditions, develop within their cj'toplasm these granules and vacuoles that have an affinity for neutral red. The process takes place not only under the usual conditions of tissue culture, but even more rapidly under special conditions, such as the introduction of typhoid bacilli into the medium (M. R. Lewis, '20) or the exclusion of dextrose (M. R. Lewis, '21). Similar granules and vacuoles accumulate rapidly in connective-tissue cells and blood-cells, which normally possess few if any, when their supply of oxygen is cut off by sealing the spread under a coverglass (Prigoseu, '21). They are also jiroduced in the cells of living animals under the action of numerous acid-azo dyestuft's (Evans


August, 1922]


285


and Scott, '21). We have found that these ueutralied bodies also accumulate in various tj'pes of cells that remain alive in the dead animal, or in organs or parts of organs that have been removed, even when they do not appear to be present in the normal, healthy cell. At the death of the animal all the cells of the body are subject to great functional disturbances of one kind or another. With the cessation of heart action all the tissues are immediately deprived of their chief source of oxygen, their food supply, and the usual pathway for the elimination of waste products. Lack of oxygen and food and the accumulation of waste products are probably all concerned in the production of the granules and vacuoles.

In these experiments most of the observations were made on the adult white rat, with a few additional ones on the guinea-pig, rabbit, and man. There were four series of experiments, with numerous repetitions of the individual observations. The rats were killed with ether, chloroform, or a knock on the head. The method of killing caused no variation in the subsequent formation of granules and vacuoles in the cells.

Series I. Fourteen rats were used in this series. Various organs were cut out aseptically immediately after death, put into a moist chamber (sterile petri dishes) and kept at 37° F. in an automatically regulated refrigerator for varying periods of time (21, 3G, 18, 60, 72, 9G, 120, 111, 1G8, 192, and 210 hours). Small pieces of the organs were then cut up at room temperature in about 5 c.c. of Locke's solution (NaCl .9, CaCl .021, KCl 0.12) without the sodium bicarbonate, plus 1 or 2 drops of a 0.5 per cent solution of neutral red in distilled water. After allowing a few minutes for the dye to penetrate, spreads were examined during the next few hours.

Series II (22 rats). Small pieces of tissue were taken from the various organs immediately after death, cut up into minute fragments in the neutral-red solution mentioned above and kept at room temperature (about 70° F.) in the small petri dishes in which they were cut up. In some of the experiments the tissues were handled aseptically. Spreads were examined every few minutes during the first hour or two, then at about one-hour intervals during the remainder of the first 21 hours. The intervals were then lengthened and spreads were examined at about 30, 36, 18, 60, 72, 96, 120, and 111 hours after death.

Series III (11 rats). The entire animal was kept at 37° F. for varying periods— 6, 12, 21, 36, 18, 72, 96, and 120 hours. Small pieces were then cut up in the neutralred solution and examined as spreads.

Series IV (8 rats). The entire animal was kept at a temperature of about 70° F. for varying periods (1, 2, 6, 9, 12, 18, 21, and 18 hours) after death. Small pieces were then removed and cut up for s])reads in the neutral-red solution in the usual manner.


After allowing a few minutes for the dye to diffuse into the minute pieces in each of the above seines, the fragments were more or less flattened out under a coverslip in a drop of the fluid, thus forming the spreads for examination. Such spreads can be examined with the oilimmersion; when the granules are first beginning to form, high powers are necessary to recognize them, since they are exceedingly minute. Later, as the granules and vacuoles become larger, they can be recognized with the dry lenses or even with the high power Greenough binocular. The latter type of microscope is very useful for examination of entire organs and large pieces in the dishes. The mechanical injury with resulting death of the cells, produced by cutting the organs with scissors, extends to varying depths in the ditt'erent organs and tissues composing them, so that along the cut edge there is often a ditfu.se pink stain. The neutral red does not penetrate veiy rapidly into the larger pieces; thus the surface may be brilliantly stained while the cells just beneath show scarcely any trace of color. The epithelial lining of the bladder, trachea, and uterus, and the surface of the liver and kidney are very beautiful when the cells become loaded with red vacuoles.

Observations ox Rats

In the following table are given the maximum intervals after the death of the animal in which red granules and vacuoles were observed in the various types of cells. These figures approach closely the extreme limit of survival under these several conditions. It will be seen that cells survived longest when the organs were cut out and kept at 37° F. (Series I). The cells of tissues cut up into minute fragments and kept at room temperature in Locke's solution without sodium bicarbonate (Series II) survived about as long as when the entire animal was kept at 37° F. (Series III). When the entire animal was kept at room temperature the cells did not survive nearly as long as those in the other three series. It must be borne in mind that there were, as a rule, but few surviving cells in any of the spreads at the extreme time limit and often spreads were observed in which none of the cells contained red vacuoles. It is more than likely that renewed observation and more diligent search would reveal a slightly longer life for most of the cell types mentioned in the table and might considerably increase that for some of the others, as for example, the endothelium in series III.

The table also shows under series II the earliest period at which red granules and vacuoles were observed in the various tj'pes of cells. The observations are not complete, but thej' serve to show that most types of cells begin very quickly after death of the animal to develop granules and vacuoles that have an aflSnitj- for neutral red. In smooth muscle, endothelium, and red blood-corpuscles there was sometimes a considerable delay before


286


[No. 378


any red granules or vacuoles appeared. In the macrophages they were seen at once and appeared very rapidly


TABLE


in most of the other types of cells.

In series I, III and IV, when the organs or animals were kept for some time before examination, the various types of cells probably developed the granules and vacuoles before the tissues were cut up in the neutral-red solution, with the exception of the smooth muscle and endothelium of the animals kept only one or two hours in series IV. Otherwise, in these series it was foun<l that the cells when first examined contained about the maximum number of granules and vacuoles. In series II, how

SERIES


I.


II.


III.


IV.


Large macrophages

Tracheal cartilage

Kidney epithelium

Smooth muscle


Hrs.

240 240 240 240


Hrs.

5'-144 20'-144 10'-120 90'-120


Hrs.

120 120

120 12


Hr,.

24

24

6

3


Salivary gland epithelium .

Bladder epithelium

Tracheal epithelium

Tongue epithelium


192 192 192 192


16'-120 12'-120 10'-120 45'- 48


96

72

120

96


3 3 6


ever, where the tissues were examined as rapidly as



168 168 168 168


35'- 72 25'- 48 25'- 48 25'- 48


12 60 60


3


possible after death, there was a gradual increase in the number of red granules and vacuoles in most types of cells during the first few hours until a maximum was


Small lymphocytes

Large lymphocytes

Microcytes


6 6 3




144 120 120


35'- 52 45'- 24 20'- 49


48 3


attained. This remained more or less stationary for vary


36 1


ing periods, according to the type of cell, and then decreased until all color was lost from the granules and vacuoles. Near the end some spreads showed a few surviving cells among thousands of dead ones, while others of the same group contained no living cells at all. No spreads, even those earliest observed, were made up


Kupffer cells


60 12


Brain macrophages

Pancreatic epithelium

Red Blood-corpuscles

Liver cells

Sertoli cells


96 96 72 96 96


10'- 48 30'- 49 53'- 24 20'- 15 7'- 12


96 12 96 3 24 1 12

3


entirely of living cells. Many cells were undoubtedly killed by mechanical injury; most spreads of the various tissues showed areas in which all or most of the cells were dead and other areas in which many cells were alive, that is, contained red granules and vacuoles. There is a comparatively short period near the end when it is



72 72 72 72


17'- 72 30

20'- 53 24


48 1 3


Ovarian follicular cells.. . .

Uterine epithelium

Uterine gland cells


43 1 1 12 1 12 1


Epididymis epithelium....

Adrenal cells

Fat


48

24



55'- 18

15'- 32

18

60'- 18



3 3 1


difficult to decide whether a cell is alive or dead, but this


Intestinal epithelium




does not materially influence our results, as we have





10'- 1





utilized only the more positive data from the many


Skeletal muscle






ob.servations.




Large macrophages, crowded with red vacuoles, were observed from 2J: to 240 hours in series I ; in series II, from 5 minutes to 1-14 hours; in series III, from 6 to 120 hours, and in series IV, from 1 to 24 hours after death. The vacuoles, apparently normal inclusions of these cells, take up the neutral red as rapidly as it penetrates to them. They seem to have the maximum number of vacuoles from the very outset of the ob.servations. The vacuoles do not seem to be of quite the same character as those which develop in most of the other types of cells after the death of the animal, and it is very probable that they do not indicate such a rapid degeneration or injury as do the vacuoles in other types of cells. In series I, the red vacuoles were still present at 240 hours in the macrol)hages of the diaphragm, tongue, lungs, bladder, trachea, and fat; in the salivary glands at 192 hours; in the heart, thymus, and uterus at 168 hours ; in the spleen at 120 hours; and in the epididymis and pancreas at 96 hours. They usually survive as long or longer than most of the other types of cells in the organ. Near the end of their period of survival the number of macrophages with colored vacuoles often decreases, the color changes to a


reddish brown, and the distinction between living and dead cells becomes uncertain. The large macrophages were very abundant in the lungs, loose mesenchyme, skeletal muscle, organ capsules, and fat (Figs. 1, 2, and 3). They were fairly numerous in the uterus, bladder, spleen, lymph nodes, thymus, and blood clots, and less so, as a rule, in the heart, ovary, and epididymis. In the kidney, pancreas, salivaiy glands, testis, and liver they are comparatively rare and probably occur only in the connective septa, so that they were not often seen in spreads. They were not seen in the cortex, cerebellum, cartilage, ectoderm, or adrenal. The macrophages of the cortex and cerebellum and the Kupffer cells of the liver are of a somewhat different character.

Tracheal cartilage is one of the most persistent of tissues. In series I red vacuoles were seen in its cells from 24 to 240 hours, in series II from 20 minutes to 144 hours, in series III from 6 to 120 hours, and in series IV at 1 to 24 hours after death. There was a considerable increase in the size of the vacuoles as the period after death lengthened. Macrophages were very abundant in


August, 1922]


287


the peritracheal inesenchTiiie and in these the red granules and vacuoles usually persisted longer than those in the other cells of the trachea, with the exception of the cartilage.

The epithelial cells of the kidney tuiules developed many granules and vacuoles in each series (Fig. 6). In series II, in which the development of these granules and vacuoles was followed, there was at first little or no red color, but during the next 3 or -I hours red granules and vacuoles appeared, gradually increasing in number and size until the cells were crowded with them. This condition remained more or less stationary for varying periods. Red granules and vacuoles were observed in tlie cells from 24 to 240 hours in series I, from 10 minutes to 120 hours in series II, from 6 to 120 hours in series III, and from 1 to G hours in series IV. Most of the spreads showed dead cells diffusely colored, as well as cells containing red vacuoles. The number of cells containing red vacuoles gradually decreased as the period after death lengthened, until only a few could be seen, and finally the color disappeared from these also. The neutral red did not penetrate the larger pieces very rapidly, so that for a time only the uninjured cells on the surface of the larger pieces contained the red vacuoles. In many specimens every cell in the tubules on the normal, uncut, uninjured surface contained vacuoles, while only a part of those on the cut surface showed them, the injured cells being diffuse pink in color. At first only those cells on the superficial side of the tubules on the uncut surface contained red vacuoles, while the deeper ones, to which the neutral red had not penetrated, contained very little color. ArVhen, after two or three hours, fresh cuts were made across the larger pieces, the cells in the newly exposed tubules usually showed little or no red color. Vacuoles were undoubtedly present, for the uninjured cells along this surface accumulated neutral red quite rapidly, much more rapidly than when the tissue was first removed.

In the glomeruli red granules were seen, but whether they were in the epithelium, in the endothelium, or in both types of cells could not be determined with certainty. Macrophages are very rare in the kidney.

Smooth muscle was noted in the bladder and uterus and occasionally in blood-vessels. This tissue, like endothelium, does not appear to develop vacuoles as rapidly as uMst of the other tissues. The vacuoles have a characteristic linear arrangement (Fig. 7). In series I, I'ed vacuoles were seen from 24 to 240 hours, in series II from 114 to 72 hours, and in series III from 6 to 12 hours. Further observations would probably show a longer persistence of vacuoles in series III. In series IV tlie smooth muscle was examined at 1 and at 3 hours, but no vacuoles could be seen ; later, however, spreads from the same dishes did show numerous red vacuoles


in the smooth muscle. Either the neutral red had not penetrated or the vacuoles had not developed at the time the first observations were made.

Salivary-gland epithelium became heavily loaded with red granules and vacuoles (Fig. ISj. There was usually a clear peripheral zone that varied in width in different glands. It was not so pronounced as in the pancreas. The colored vacuoles were crowded in among the secretion granules, which did not take up the neutral red. The number of colored vacuoles seemed to vary in direct proportion to the number of secretion granules. Red granules and vacuoles were found from 24 to 192 hours in series I, from 16 minutes to 120 hours in series II, from 6 to 96 hours in series III, and from 1 to 3 houra in series IV. In series II the duct epithelium was occasionally observed (120 hours) to be loaded with red granules and vacuoles, while very few of the gland cells contained them, most of the latter being dead. Macrophages were not common.

The bladder epithelium appears to consist of two rather distinct types of cells. When first examined from 10 to 60 minutes after death in .series II, the cells contained only a few small granules and vacuoles, but during the course of the next 3 or 4 hours these increased greatly in both number and size. At this time a marked difference in their reaction to neutral red becomes apparent in the two types of cells. The larger type, often binucleated or multinucleated, developed many granules and vacuoles that became very heavily laden with the neutral red (Figs. 13 and 14). These cells, less numerous but much larger than the second type, are scattered in more or less irregular patches throughout the entire epithelial lining. When viewed with the binocular dissecting microscope, they stand out veiy conspicuously, especially the many large giant cells. The second type, smaller in size, has fewer and less deeply staining granules, arranged in a circle around a relatively large nucleus. Since the epithelial cells are on the surface, the neutral red has easy access and probably diffuses into the cells as rapidly as the granules and vacuoles form, and the slow increase during the first 3 or 4 hours probably is a fair indication of the rate at which the vacuoles and granules develop. The larger cells seem to develop vacuoles at a faster rate than the smaller ones and to reach a maximum earlier, maintaining this maximum for a considerable period of time. In large pieces of the bladder one can see the brilliantly stained epithelial surface in marked contrast to the pale muscular layers, for only after several hours does the latter accumulate enough red vacuoles to become conspicuous. This is due partly to the slowness of penetration by the dye. When the epithelial wall of the bladder is thrown into folds, as it usually is in the larger pieces, the neutral red appears first in the liigh spots of the folds, often long before the cells in the depths show any appreciable color. This is probably


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merely a question of ditifusiou of the dye iu the solution itself. Red granules and vacuoles were obsened in the cells from 24 to 192 hours iu series 1, from 12 minutes to 120 hours in series II, from 6 to 72 hours in series III, and from 1 to 3 hours in series IV. Macrophages are fairly abundant in the bladder wall and live longer than most of the other types of cells.

The tracheal epithelium was about as persistent as the cartilage but was not always present in the spreads. In series I, red granules and vacuoles were seen from 24 to 192 hours, in series II from 10 minutes to 120 hours, and in series III from 6 to 120 hours. In series IV the epithelium was observed at 1 and 6 hours; it probably lived longer, but unfortunatelj' our spreads did not contain these cells at later periods. It was possible to watch with the low power the gradual accumulation of red color in the epithelial cells of large pieces, until finally the whole lining of the trachea was deep red. Ciliary movement was observed in the bronchi 120 hours after death in series I and in series III.

Observations on the tongue ectoderm were unsatisfactory. In series I, red granules were seen at 24, 48, and 192 hours but not at 72, 96, 120, 168, or 240 hours. In series II, red granules were seen in some of the cells at 45 minutes, 4 hours, and a very few at 48 hours, but none at 13, 18, 36, 96, 120, or 144 hours. In series III, granules were found at 48, 72, and 96 hours, but none at 12, 24, or 60 hours. In series IV, although ob.servations were made from 1 to 24 houre after death, no red granules were seen in the cells. On the base of each papilla there developed a superficial area of pink granules or small vacuoles. These differed so markedly from the other vacuoles and granules that we have not considered them as indicators of the condition of the cells. Other superficial cells did not develop granules or vacuoles and they probably prevented the penetration of the neutral red to the deeper layers. At the edge of the cuts the cells were killed and the deeper layers of the epithelium were stained a diffuse pink. The granules and vacuoles occasionally seen in the cells of the deeper laj'ers were observed only in spreads from exceptionalh' favorable pieces, and we doubt if our data give an adequate idea of the period of survival of these cells. Macrophages were very abundant in the connective-tissue spaces beneath the ectoderm and retained their red gi-annles for 240 hours in series I, 72 hours in series II, 96 hours in series III, and 12 hours in series IV.

Endothelium was observed in the capillaries of various organs. The granules and vacuoles that take up the neutral red did not always appear to develop as rapidly as in most other types of cells. In series I, endothelial cells with red vacuoles were seen from 24 to 108 hours; in series II from 35 minutes to 72 hours; iu series III, red vacuoles were seen at 12 hours; in series IV observations at 1 and 2 hours did not reveal red granules, but


the same material kept for 24 hours longer in the neutralred solution showed cells containing red vacuoles, indicating that they were alive at the time of the first observations. The size and number of vacuoles varied greatly in different spreads and in different parts of the same spread. The endothelial cells in some capillaries were filled with red vacuoles, whereas others were entirely without them (Figs. 16 and 17). At the first observation in series II there were a very few scattered vacuoles in the capillary walls, the number slowly increasing until the cells became quite full of them.

In the thymus, small lymphocytes, with from one to four small red vacuoles or granules, were seen from 24 to 168 hours iu series 1, from 25 minutes to 48 hours in series II, at 60 hours in series III, and at 1 and 3 hours in series IV. Large lymphocytes, containing a greater number of red granules and vacuoles, were seen at the same periods. The microcytcs, with one or two rather large red vacuoles, were usually seen at the same time. Large macrophages were fairly abundant and usually survived as long or longer than the other cells. Similar cells from the spleen and lymph nodes were obsened. They lived longer in the thymus than in the lymph nodes and longer in the lymph nodes than in the spleen. When the lymphocj'tes were dying, it was not uncommon to see cells, especially the large lymphocytes, with a few large vacuoles of a brownish red color and a dead nucleus having a sharp nuclear membrane.

In the lung, epithelial cells, with red granules and vacuoles, were seen from 24 to 144 hours in series I, from 35 minutes to 52 hours in series II, from 6 to 48 hours in series III, and from 1 to 3 hours in series IV. In series III there were possibly a few at 60 and 96 hours but none at 72 hours. The epithelial cells do not contain many vacuoles at any time and it was not always easy to determine whether the vacuoles were in these cells or in the endothelium. The macrophages, which occurred in great numbers, were the most characteristic objects iu the lungs. They were found, loaded with red granules and vacuoles (Fig. 1), in series I from 24 to 240 hours, in series II from 30 minutes to 120 hours, in series III from 6 to 96 hours, and in series IV from 1 to 24 hours after death.

Blood-cells were examined in various organs and in blood clots. Leucocytes containing numerous red granules and small vacuoles were found from 24 to 120 hours in series I, from 45 minutes to 30 hours iu series II, from 6 to 36 hours iu series III, and at 1 hour in series IV. Red blood corpuscles, with one to three or four granules or small vacuoles, were seen at 72 hours in series I, from 1 to 24 hours in series II, at 6 and at 24 hours in series III, and at 1 hour in series IV. Usually, very few if any of the corpuscles examined in a spread contained red vacuoles. Large macrophages were very abundant in


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blood clots that had formed iu the body cavities after the organs were removed for exaniiuatiou.

The Kupffer cells were usually seen without much difticulty and when first observed contained the maximum number of red vacuoles. They were often noticed in spreads in which the liver cells were dead or without vacuoles, and hence are apparently not killed as easily by the manipulations as the latter type. The vacuoles in the Kupffer cells were somewhat different in character from those formed in the liver cells, and they also either had a greater affinity for neutral red or were more easily penetrated by it. The fact that the Kupft'er cells always contained the maximum number of vacuoles at the first observation indicates that the vacuoles are normal inclusions of these cells. They were seen from 24 to 120 hours in series I, from 20 minutes to 19 hours in series II, from 6 to GO hours in series III, and from 1 to 12 hours iu series IV.

Brain macrophages were observed in both the cortex and cerebellum (Fig. 21). They were not numerous and were usually scattered along the small blood-vessels to which they sometimes seemed to cling closely. They were often elongated, especially when attached to a blood-vessel. The vacuoles did not, as a rule, stain as deeply nor were they as numerous as those in the tissue macrophages. Like those in the tissue macrophages and in the Kupffer cells, they appeared to be different in character from the vacuoles formed in the tissue cells and are probably normal inclusions of these cells, as there was practically no increase in their number. The brain macrophages are not as large as the tissue macrophages. They were observed from 21 to 96 hours in series I, from 10 minutes to 48 hours in series II, from 6 to 96 hours in series III, and from 1 to 12 hours in series IV.

The jxincreatic gland cells developed many small red granules that were always definitely located in the lumen end of the cell among the seci-etion granules. At the distal end of the cell there was usually a clear zone, free from red granules and secretion granules, which varied iu width in different animals according, apparently, to the state of secretory activity as already noted in the cells of the salivary gland (Figs. 8 and 9). The red granules wei-e observed from 21 to 96 hours iu series I, from ^O minutes to 49 hours in series II, from 6 to 96 hours iu series III, and at 3 hours in series IV. Macrophages were very rare.

Liver cells usually developed granules and vacuoles rather slowly and the cells on the surface, especially the uncut surface, showed the red color long before the more deeply placed ones. The liver cells are apparently easily killed and in many of the spreads no living ones, or at most only small patches of them, could be seen, whereas practically all the cells on the uninjured uncut surface of the larger pieces were filled with vacuoles. The vacu


oles varied iu size and were usually scattered through the cell (Figs. 10 and 11). Occasionally, red granules were seen only in that end of the cell bordering the bile capillary (Fig. 12). The vacuoles were found from 24 to 96 hours in series I, from 20 minutes to 15 hours in series II, and from 1 to 12 hours in series III. Although numerous observations were made in .series IV, no cells containing red granules or vacuoles were seen. Further tests, with due appreciation of the susceptibility of the cells to mechanical injury, would probably have revealed living cells in series IV and for longer periods in series II. The large tissue macrophages are rare in the liver.

Testis. — The Sertoli cells were very conspicuous objects in spreads from pieces of the testis cut up in the neutral red solution. They formed regular patterns in the walls of the tubules (Fig. 20). The long axis of these cells is normally perpendicular to the surface of the tubule, but under pressure of the coverglass they became bent parallel to the surface, especially at the cut end of the tubule (Fig. 19), where they often became detached and floated off with a group of attached spermatogonia. Red vacuoles were observed in series I from 48 to 96 hours, iu series II from 7 minutes to 16 hours, and iu series IV at 3 hours after death. No observations were made in series III. It seems probable that the vacuoles are normally present iu these cells. Macrophages were rarely seen. Interstitial cells with red granules and vacuoles were occasionally ob.served as early as 45 minutes in series II. Male germ cells and moving spermatozoa with one or more red granules were occasionally seen in series II from 40 minutes to 13 hours after death.

Mesenchyme cells were observed in various places but they did not often contain red granules or vacuoles. These cells are apparently rather sensitive; the nuclei were usually stained pink, indicating that the cells were dead. We have not attempted to distinguish between the various types of cells which constitute the connective tissues, except the tissue macrophages. It is rather surprising that the me.senchyme cells did not show up better. Red granules and vacuoles were observed iu the cells from 24 to 72 hours in series I, from 17 minutes to 72 hours in series II, from 6 to 48 hours iu series III, and at 3 hours in series IV. These cells did not develop as many vacuoles as some of the other types.

The follicular cells of the ovary were rather difficult to observe in spreads and we have comparatively few observations on the granules and vacuoles. Dense tissues like the ovary and adrenal are difficult to handle by our methods. Red vacuoles and granules were observed at 24 and at 72 hours in series I, from 24 to 30 hours in series II, at 12 and at 48 hours in series III, and at 1 hour in series IV. Observations on the ova were very difficult and unsatisfactory. In series III many fine granules were seen in the ovum at 12 hours but none at 24 or at 48 hours. In the other series we did not sue


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ceed in getting satisfactory observations. The yerminul epithelium contained red granules and vacuoles from 3 to 5 hours in series II. No special attempt was made to observe it in the other series.

The uterine epithelium developed rather small granules and vacuoles. They were first seen 20 minutes after death in series II and gradually increased in number during the next 3 or 4 hours. Large areas of the epithelium observed at this time were deep red in color under the Greenough binocular, but quite different in character from the bladder or tracheal epithelium. In series 1, granules and vacuoles were observed from 2i to 72 hours, in series II from 20 minutes to 53 hours, in series III from to 12 hours, and in series IV at 1 hour. The uterine gland cells often became loaded with red granules and vacuoles which retained their color about as long as did tho.se in the epithelial cells.

The epithelial cells lining the tubules of the epididymis contained red granules and vacuoles at 48 hours in series I, from 1 to 18 hours in series II, and at 3 hours in series 1\'. There were no observations in series III.

The adrenal cells are difficult to examine by this method ; few of the essential ones containing red granules could be seen, in spite of many observations. In series I, many cells appeared to contain red granules at 24 hours but none were observed from 3C to 210 hours. In series II, cells were occasionally found with red granules from 15 minutes to 32 hours, and in series IV at 1 hour. None were seen in spi'eads from series III. Macrophages loaded with red were found in the capsule, but none in the body of the organ.

Very rarely were jat cells with red granules and vacuoles observed. In series II they were found at 4 to 18 hours. None were seen in series I and III. In series IV, fat-cells examined 1 hour after death were clear, but after standing for 24 hours in the neutral-red solution red vacuoles were found in some of them. They were not seen at any other time in this series. Large nuicrophages were abundant (Fig. 3).

Epithelial cells from the intestine showed red granules and vacuoles from 1 to 18 hours in series II, but not from 24 to 144 hours. None were seen in series I, III, or IV. The fact that the intestine is always infected probably explains why its cells did not live long. Macrophages were not often observed.

The nerve cells in the cortex and cerebellum rarely gave any indication of red granules and vacuoles. A few cells with pink vacuoles and granules were seen 15 minutes to one-half hour after death in series II. Most of the nerve cells at the periphery began to show a diffuse pink at this time, indicating that they were dead. An hour later practically all the cells were a diffuse pink in color in fresh spreads. The presence of brain macrophages and endothelium with red granules and vacuoles indicated that the environment was not especially un


friendly to life (Fig. 21). Death of nerve cells probably proceeds so rapidly that the granules and vacuoles do not have a chance to form.

Observations of skeletal mwscle were made on the diaphragm, tongue, and abdominal muscles, but in no case were we able to find red granules or vacuoles in the fibers. Many observations were made in each series. Striated muscle behaved in a peculiar manner toward neutral red. The fibers rapidly became a diffuse deep pink, more marked than in other types of cells after death. We are unable to explain this diffuse coloring of muscle, as it is hardly to be expected that the fibers should die as rapidly as the diffuse coloration seemed to indicate. The contractile tissue evidently contains some diffuse substance that has a marked affinity for neutral red. Large macrophages, always abundant between the fibers and especially in the larger connective-tissue spaces between muscle bundles, were loaded with red granules and vacuoles.

Heart muscle behaved in a manner similar to skeletal muscle and always showed a dift'use pink. The large macrophages were not as common as in skeletal muscle, but they took up the neutral red in exactly the same manner.

A few scattered observations on the mesothelium of the body cavities, the epithelial cells of the choroid i)lexus (Fig. 23), the retina, iris, and cornea, showed that there is a similar accumulation of granules and vacuoles in these cells.

Observations on Other Animals

Human tissues from three autopsies were examined in the usual manner as spreads from small pieces cut up in the neutral-red solution. Tissues from the first autopsy (the patient having died of carcinoma of the stomach) were examined G to 7 houre after death and, although the cadaver was in the ice-box at 37°F. for an hour during this time, only a few macrophages in the lungs and capsule of a lymph node and a few Kupft'er cells contained red vacuoles. The other cells from the lung, lymph-node and liver, and all the cells of the testis, kidney, spleen, and skeletal muscle that were examined, were stained a dift'use pink by the neutral-red solution in which small pieces were cut up. In the second case (a child autopsied 31 hours after death) a few macrophages containing red vacuoles were found in the diai)hragm, pancreas, intestine, capsule of a lymph-node, blood clot, and thymus. The body was in the ice box at 37°F. for 15 hours of this period. None of the other tissues examined, striated muscle, epididymis epithelium, adrenal, pancreas, liver, lymphocytes, tracheal cartilage, mesenchyme, endothelium, or smooth muscle, contained red vacuoles. Some of the tissues from the third autopsy (a ly^ months" fetus) were examined about 24 hours after death. The fetus had been kept in the ice-box for about IS hours. Lung, diaphragm, kidney, pancreas, lymph node, liver,


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spleeu, trachea, thj'imis, adrenal, and retroperitoneal tissue were examined. Macrophages with red vacuoles were found in the lymph node, spleen, thymus, retroperitoneal tissue, and capsule of the adrenal, but no other types of cells containing red vacuoles were seen.

Cells from various organs of the rabbit were examined as in series II from 15 minutes to 5 hours after death. About the same accumulation of granules and vacuoles occurred as in the cells of the rat (Fig. 15). The lungs contained many large macrophages ; the adrenal cells showed a more pronounced accumulation of red vacuoles than in the rat. A fetus G.5 cm. long, from the same rabbit, was examined especially for the macrophages in the lungs, but none were found, although these cells were numerous in the subcutaneous tissue. The epithelium of the intestine and kidney contained manj- red vacu oles 5 hours after death.

Cells from various tissues of a guinea-pig were examined, as in series II, from 45 minutes to 30 hours after death. The accumulation of red vacuoles and granules presented very much the same picture as seen in the rat under similar conditions (Figs. 22 and 21). The macrophages were numerous in the lungs.

Discussion

Other methods might perhaps be employed which would preserve the life cells for longer periods, but these observations establish the fact that the different types of cells survive for different periods of time after death, and that the manner in which the tissues are treated makes a very great difference in the length of survival of all tj'pes of cells.

It is desirable to consider the nature of the granules and vacuoles and their probable origin before we attempt to explain why cells lived longer under some conditions than under others, and why certain cells always lived longer than others under the same conditions. With the exception of the macrophages, Kupffer cells, Sertoli cells, and leucocytes, probably none of the cells examined contained, under normal conditions in the body, more than a few, if any, grannies and vacuoles having an aftinitj- for neutral red. Such granules and vacuoles developed at different rates in the various types of cells, but we have not followed this particular phase with sufficient care to present here any definite data. Endothelium and smooth muscle often seemed to develop the vacuoles more slowly than most other types of cells. The number and size of the granules and vacuoles sooner or later attained a maximum state, which was different for each type and which was maintained for varying periods; then, as tho cells approached the last phases of life, there was a diminution in the number of the red granules and vacuoles, until finally all the red color was lost. The color sometimes did not disappear from all the vacuoles until after the nuclear changes indicating the death of the nucleus


had taken place. Increased refractivity and visibility, accompanied by the appearance of a marked nuclear membrane, were the most characteristic death changes in the nucleus. Dead nuclei were easily distinguishable from living ones and were very similar to those seen in cells after fixation, before dehydration and staining. The red color that sometimes lingered in the granules and vacuoles after nuclear death indicated that not all parts of the cell died at the same time; that the nucleus might have died before the cytoplasm and that some regions of the cytoplasm died before others.

The factors involved in the production of the.se granules and vacuoles is not altogether clear. There is, of course, first of all, the question whether they are to be regarded as food vacuoles, as an accumulation of waste products, or as autolytic products. It is possible, though not probable, that the condition of functional stress of the cells after the death of the animal might produce such alterations in the surface layer and in the body of the cytoplasm as would permit the rapid diffusion of dissolved food and other substances from the fluids still bathing the cells and their segregation as granules and vacuoles; to these something must be added from the cell itself to give to the resulting granules and vacuoles an aflBnity for neutral red. Such an explanation, however, does not seem vers^ tenable. As the cells remained alive, they must have been undergoing metabolic processes, utilizing their own substance or substances in the fluid surroiinding them ; in either case the difficulty of eliminating waste products would seem to be obvious and so the granules and vacuoles might be looked ui)on as accumulated waste products. It is very doubtful if food substances surrounding the cells are present in sufficient quantity to play any role, in view of the greatly reduced supply of oxygen. So for the present, at least, it seems more reasonable to consider the granules and vacuoles as the waste products of autolytic or self-digestive processes and their affinity for neutral red as a coincidence. We can hardly regard them as a segregation apparatus, as Evans and Scott (1921) have interpreted somewhat similar vacuoles found in cells after the injection of acidazo dyes into living animals; they are much more like the degeneration granules and vacuoles described by W. H. Lewis (1919) in the cells of tissue-cultures. If, then, they are to be considered, for the present at least, as the accumulated products of autolysis, starvation granules and vacuoles would be perhaps as appropriate a term as degeneration granules and vacuoles.

Mrs. M. R. Lewis has recently shown that a trace of ammonia added to the medium of tissue cultures produces a prompt formation of vacuoles which disay^pear if the cultures are subsequently washed with a fresh nutrient medium. This would appear to indicate that the vacuoles are produced by the accumulation of the


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waste products of uitiogeuous metabolism ; iu other words, that they are a sign of degeneration or autolysis produced by the breaking down of the cytoplasm and au accumulation of the resulting waste products.

Wells ('06) examined histologically from time to time various tissues undergoing autolysis, noting the relative rates of alteration, especially in the nucleus. The autolytic rate in such supposedly dead tissues did not correspond to the periods of survival as determined in our experiments. Brain cells, which we found to die rapidly, according to Wells retain their essential morphological chai-acter longer than most of the body cells.

In series IV, where the entire animal was kept at I'oom temperature, the factors contributing to the rapid death of the cells would seem to be the temperature, the spread of infection from the alimeutaiy canal, the absence of a pathway for the elimination of waste products, and the lack of oxygen and food. The first two factors would differentiate it from series III, where the entire animal was kept on ice at 37°F. There was a very great difference in the length of survival of the cells of the two series, as can be seen readily iu the table. Temperature aloue is not so directly important in producing rapid death, since in series II the cells in the small fragments which were kept at room temperature lived as long on the average as when tlie whole rat was kept in the ice-box at 37°F. (series III). Infection may play a very important r61e, for the intestinal cells rarely lived more than a few hours in any of the series and often no liviug cells with vacuoles were seen at any time.

In series II (small fragments of tissue kept at room temperature in the neutral-red solution) and III (the entire animal kept at 37°F.) the survival period was approximately the same. The retardation of metabolic processes in series III was counterbalanced by more rapid metabolic processes with increased opportunity for the elimination of waste by diffusion. Infection was not entirely eliminated in series II, as only part of the tests were made under strictly aseptic conditions.

In series I infection was practically eliminated except for a few of the organs. Metabolism was retarded by the cold and the few cells that survived beyond the period for series II and III were probably favorably located.

We are hardly in a position to go farther into an explanation of the causes which bring about cell death under the conditions of our experiments. They are undoubtedly closely related to the factors which produce the granules and vacuoles. Most of the cells were probably going down hill from the hour the animal was killed.

The various types of cells must differ from each other in many ways, in chemical and physical constitution, in the rate of metabolism, in the amount of oxygen, various salts, and foodstuffs used, and in the chemical composi


tion of the waste products. Each cell is a complex mechanism which acts effectively only in the normal environment in the animal and this environment varies more or less for each type. AVhen the animal dies the environment is changed, whether the cells are left in the body or the tissues are removed and placed in the neutral-reil solution. The period of survival in the changed environment depends on how well each type of cell is naturally fitted to live there. Now, when we consider the natural environment together with the known function of each type, we can perhaps see how this corresponds in a way with the varying periods of survival.

The tissue macrophages may owe their ability to survive for relative!}' long periods to the fact that they are constructed to live, under normal conditions, in the tissue spaces without close contact to either oxygen supply or special elimination paths, and that they normally contain vacuoles which are probably, in part at least, composed of waste products.

The cartilage cells likewise, though fixed in position, are naturally rather far removed from the capillaries, so that neither of these types of cells would respond to the cutting off of the blood supply as quickly as cells that have perhaps a higher rate of metabolism. Although the kidney cells are accustomed to a rich blood supplj", they are also constructed to deal with waste products, so that the accumulation of waste products in the fonn of granules and vacuoles within them is perhaps not so detrimental as to other types of cells.

The epithelial cells from the bladder, tongue, and trachea might also be expected to survive for a relatively long period, since their peculiar location on the surfaces would indicate that they were adapted for living some distance from the blood supply.

The list of cells given in the table might thus be followed in the order of their survival and reasons suggested for their relative length of survival, without, however, bringing us much nearer to the actual reasons, which are still obscure. Since in each series the environment is approxinuitely the same for all cells, the differences in length of survival must depend on the make-uj) of the cells themselves.


Summary

Small pieces of tissue from various organs of the rat, examined at varying periods after death, showed that most types of cells survived for a time and certain types of cells survived longer than others, in approximately the following order: tissue macrophages, cartilage cells, kidney-tubule cells, smooth-muscle cells, salivary-gland cells, bladder epithelium, tracheal epithelium, tongue epithel ium, endothelium, lymphocytes, large lymphocytes, micro


THE JOHNS HOPKINS HOSPITAL BULLETIN, AUGUST. 192^


PLATE XXXIX Ck



PLATE XL


THE JOHNS HOPKINS HOSPITAL BULLETIN. AUGUST. 1922




X. ^


t^


^* ^£mW3


18


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cytes, lung epithelium, leucocytes, Kupffer cells, braiu macrophages, pancreas cells, reil blood-corpuscles, liver cells, Sertoli cells, mesenchyme, ovarian follicular cells, epididymis epithelium, adrenal cells, intestinal epithelium, and nerve cells. Macrophages survived for about 24:0 hours; brain cells for less than an hour; striated muscle developed no granules or vacuoles.

The criteria for life were: (1) the presence in cells of granules and vacuoles that had an affinity for neutral red while the cells were alive; almost all types of cells either had such granules or developed them after the death of the animal; (2) a homogeneous nucleus without trace of a nuclear membrane. The criteria for death were: (Ij loss of color from the granules and vacuoles; (2) diffuse pink staining of the cytoplasm and nucleus; (3) the appearance of a sharp and distinct nuclear membrane and a change in the texture of the cytoplasm and nucleus.

Four series of experiments were conducted : In series I organs were kept at 37°F. ; in series II tissues were cut up into minute fragments in a neutral-red solution and kept at room temperature; in series III the entire -animal was kept at 37°F. ; and in series IV the dead rat was kept at room temperature. Cells lived longest in series I, about half as long in series II and III, and a very much shorter time in series IV.


BIBLIOGRAPHY

Carrel, A., 1907: Heterotransplantation of blood vessels preserved in cold storage. J. Exp. Med., vol. 9, p. 226.

Idem., 1910: Remote results of the replantation of the kidney and the spleen. J. Exp. Med., vol. 12.

EJbeling, A. H., 1921: Measurement of the growth of tissues in vitro. J. Exp. Med., vol. 34, p. 231-243.

Evans, H. M., and K. J. Scott, 1921: On the differential reaction to vital dyes of the two great groups of connective tissue cell. Contributions to Embryology, vol. 10. Carnegie Inst. Wash., Pub. 273.

Evans and Schulemann, 1914: The action of vital stains belonging to the benzidine group. Science N. S., vol. 39, p. 443-4.54.

Fischer, Albert, 1922: A strain of epithelial cells in pure culture. Anat. Rec, vol. 23.

Gross, W., 1911: Experimentelle Untersuchungen iiber den Zusammenhang zwischen histologischen Veranderung und Funktionsstorungen der Nieren. Ziegler's Beitr., Bd. 51, 528.

Lewis, M. R., 1920: The formation of vacuoles due to bacillus typhosus in the cells of tissue cultures of the intestine of the chick embryo. J. Exp. Med., vol. 31.

Lewis, W. H., 1919: Degeneration granules and vacuoles in the fibroblasts of chick embryos cultivated in vitro. Johns Hopkins Hosp. Bull., vol. 30, p. 86.

Idem., 1921: The effect of potassium permanganate on the mesenchyme cells of tissue cultures. Amer. J. Anat., vol. 28, p. 442.


Lewis and Lewis, 1915: Mitochondria (and other cytoplasmic structures) in tissue cultures. Amer. J. Anat., vol. 17, p. 339-401.

Prigosen, R. E., 19 21: The formation of vacuoles and neutralred granules in connective-tissue cells and blood-cells observed under abnormal conditions. Johns Hopkins Hosp., Bull., vol. 32.

Steckelmacher, S., 1914: EJxperimentelle Nekrose und Degeneration der Leber. Ziegler's Beitr., Bd. 57, 314.

Wells, H. G., 1906: The relation of autolysis to the histological changes occurring in necrotic areas. J. Med. Res., vol. XV, 149.


LEGENDS FOR FIGURES

Fig. 1. — Macrophages in the lung heavily laden with neutral red granules and vacuoles. Series II, 5 hours after death. X 480.

Fig. 2. — Macrophages in the lung; much of the red color has gone. Series I, 192 hours after death. X 480.

Fig. 3. — Macrophages in fat. Series I, 36 hours after death. X480.

Fig. 4. — Macrophages in renal capsule. Series II, 51 hours after death. X 146.

Fig. 5. — Tracheal cartilage. Granules and vacuoles near cell nuclei. Series I, 72 hours after death. X 480.

Fig. 6. — Kidney tubules. Most of the tubule cells have large irregular vacuoles. Series II, 6 hours after death. X 480.

Fig. 7. — Smooth muscle from bladder. Note linear arrangement of vacuoles. Series III, 37 hours after death; ice box 12 hours, room temperature 25 hours. X 480.

Fig. 8. — Pancreatic gland. Series I, 5 hours after death. X 480.

Fig. 9. — Pancreas. Series III; ice box 37°F. 12 hours after death. X 480.

Fig. 10. — Liver cells. Series I, 72 hours after death. X 146.

Fig. 11. — Liver cells with vacuoles. Series I, 72 hours after death. X480.

Fig. 12. — Liver cells. Series II, 5 hours after death. X 480.

Fig. 13. — ^Bladder epithelium, small mononuclear, binucleate and giant cells with vacuoles. Series III, 37 hours after death; ice box 12 hours and room temperature 25 hours. X 480.

Fig. 14. — Bladder epithelial giant cells. Series II, 4 hours after death. X 480.

Fig. 15. — Bladder epithelium from rabbit, examined as in series II, 51/2 hours after death. X 480.

Fig. 16. — Capillary net from ovary, vacuoles in endothelial cells. Series I, 36 hours after death. X 480.

Fig. 17. — Capillary net from bladder, endothelial cells with large vacuoles. Series II, 48 hours after death. X 480.

Fig. 18. — Salivary gland cells crowded with vacuoles. Series I, 24 hours after death. X 480.

Fig. 19. — Sertoli cells in testis tubule under pressure. Series II, 41/2 hours after death. X 480.

Fig. 20. — Sertoli cells in testis tubule. Series II, 16 hours after death. X 480.

Fig. 21. — Brain macrophages. Series I. 72 hours after death. X4S0.

Fig. 22. — Peritoneum from diaphragm of guinea-pig examined as in series II, 26 hours after death. X480.

Fig. 23. — Epithelial cells of choroid plexus. Series II, 4i/' hours after death. X 480.

Fig. 24. — Macrophage from lung of guinea-pig examined as in series II, 23 hours after death. X 1450.


294


[No. 378


A STUDY OF THE AUSTIN- STILLM AN -VAN SLYKE INDEX OF THE

UREA EXCRETION


By Charles H. Hitchcock, M.D.,

and

Ruth Loveland, A.B.

(From the Chemical Division of the Medical Clinic, The Johns Hopkins Hospital)


It is uot within the scope of this short paper to attempt any review of the literatnre on urea excretion, and it is with considerable trepidation that one ventures to add to its bulk. Since the original work of Anibard in 1909, niudi evidence, clinical and experimental, has been accumulated to demonstrate that the rate of urea excretion may be used as a rough indicator of the functional capacity of the kidneys, although the mathematical formulations of the "laws" governing this rate have vaiied from time to time and from person to person. A detailed discussion of the subject is presented in the recent paper of Austin, Stillman, and Van Slyke, to which the reader is referred.^ Their formulation of the relationships of the factors involved is expressed as K= bvW ° ' "' which D is the total urea excretion in grams for 24 hours, V is the urine volume in liters for 24 hours, B is the concentration of the blood urea in grams per liter, W is the patient's weight in kilos, and K a constant which in normal individuals has been found to vary between the limits 4.-5 and 10.5. The data upon which their calculations are based were derived from repeated observations upon a few healthy subjects. It is naturally a matter of no little interest to study the behavior of the Austin-StillmanA'an Slyke constant under varying conditions of disease.

The subjects, all of whom were males, were chosen more or less at random from the public wards of the Johns Hopkins Hospital. With one exception, one observation only was made on each patient. The urine was collected usually for a period of seventy minutes; the blood for urea determination was drawn as nearly as possible at the middle of the period. There was no preceding starvation of the patients, although they were requested to abstain from taking anything by mouth for the duration of the test. In each instance, furthermore, the morning meal preceded the first voiding by at least two hours. The blood urea was determined by the Van Slyke-Cullen method. Urea of the urine was determined by the Van Slyke-Cullen modification of Marshall's urease method.- The results are presented in tabular form below.

Discussion

Little comment is necessary upon Cases 1 to 9, with the exception of No. 6. Here definite urinary findings


and a decreased phenolsulphonephthalein excretion are associated with a normal index. However, the urine volume and the total urea excretion are certainly excessive, particularly as there was no polyuria, and it is reasonable to presume that the patient's slight prostatic hypertrophy interfered with the complete emptying of the bladder at the first voiding.

It is noteworthy that two cases of myocardial failure, in each of which there was evidence of renal congestion, yield indices well within the normal range. In each case, observations were made before complete recovery of compensation had occurred. In neither ca.se was there final evidence of permanent renal damage, as judged by the usual criteria.

Case 10 was that of a patient suffering from lobar pneumonia, in cultures from whose sputa a type 1 pneumococcus had been recovered in large numbers. Following the therapeutic administration of 700 cc. of homologous immune serum, a well-marked urticarial rash developed, and coincidently urinalysis demonstrated marked renal irritation. In the face of this, a normal urea excretory index was found. This result is of interest when compared with the observations of Rackemanu, Longcope, and Peters on the chloride and water excretion in .serum sickness.^

Two cases of pernicious anemia yielded definitely low results, while a third gave a low normal. Case 15 is of j)articular interest. The first estimation was made during a period of comparative quiescence, in which tlie red cell count had remained practically stationary for two weeks at a level just below 2,000,000, and the hemoglobin had been at 45% (Sahli). On the day of the determination, the actual count was 1,804,000, and the hemoglobin 43%. Shortly thereafter an active relapse ensued, the patient's red cell count dropping from 2,200,000 to 1,200 000 in seven days. On the fourth day of this relajjse two further determinations were made, the second following the ingestion of a high nitrogen test meal, which had been given for quite another purpose. It is of great interest to note that the coefficient, which had stood at 4.33 during the remission, dropped to 3.55 during the course of the relapse, and reached the still lower level of 3.07 following the ingestion of a meal very rich in protein. The actual red cell count on the day of this experiment


August, 1922]


295



CtlNICAL

Diagnosis


Ace


Uhine Picture


B. P. Range


Eye Gbounds


Abterio.




P„r„A,.E,.


NPN Wi


is






Name


Size of Heart (cm.)

Rt. Lt.


i

1





70 m. 24 hrs.! 70 m. 24 hrs. Liters j Grams




1— Chalk


Pneumonia Convalescent


33


Sp. gr. 10101028 Casts 0. W.B.C. 0. Blood 0. Alb.O.


100/75.90/55


Negative


Slight


2.5


9


2 hours = 60


30.1


64


16.6


0.3


6.0


1.124


22.48


6.9.



2— Applebaum


Arteriosclerosis


65


Sp. gr. 1015-1030 Casts 0. W.B.C. 0. Blood 0. Alb. trace


140/72


Vascular changes


Extreme


2


9


18-fl0 = 28


46.2


56.5


24.4


0.053


1.06


0.7925


15.85


8.39



3— Keith


Tabes dorsalis


42


Sp. gr. 1018-1025 Casts 0. W.B.C. 0. Blood 0. Alb. 0.


165/90117/84


Negative


Slight


2.5


8.5



34.3


59


14.8


0.077


1.54


0.4898


9.796


6.94



4— Talkington


Neurasthenia


43


Sp. gr. 1018 Casts 0. W.B.C. 0. Blood 0. Alb. 0.


118/82


Negative


None


3


8.5



32.9


60.5


15.6


0.045


0.9


0.49156 0.70661


9.83118.54 1



S— SoBnoski


Emphysema Myocardial insufficiency


57


Sp. gr. 10161040 Casts 0. W.B.C. 0. Blood 0. Alb. -f


150/90.100/57


Negative


Moderate


5 9


65-f20 = 85


40.0


69.5


25.2


0.05


1.0


14.132


6.73



6— Henderson


Elephantiasis Chronic nephritis


56


Sp. gr. 1010-1016 Casts + W.B.C. + Blood + Alb. trace


120/66


Negative


Slight


3.5 13


30+5 = 35


36.2


103


14.0


0.18


3.6


1.10


22.0


8.17



7— Block


Sub-acute endocarditis. Streptococcus viridans


45


Sp. gr. 1018.1024 Casts 0. W.B.C. 0. Blood once. Alb. 0.


128/75-100/57


Vascular changes


Moderate


3


10.5


38+15 = 53


36.4


52


21.5


0.095


1.9


0.5355


10.71


5.02 5.01 5.91



8— Lesslie


Peritoneal carcinomatosis


58


Sp. gr. I0I8.I021 Casts 0. W.B.C. 0. Blood 0. Alb. 0.


190/95


Negative


Moderate


3.5


12


35+18 = 53


33.0


69.5


15.3


0.028


0.56


0.239


4.78



9— Milchell


Mitral stenosis Myocardial insuf. Chr. bronchitis


36


Sp. gr. I0I8.I010 Casts 0. W.B.C. 0. Blood 0. Alb. trace


115/75-90/65


Negative


None


4


9.5


Unsuccessful


33.3


70.5


14.3


0.095


1.9 0.489


9.78



10— Erdman


Lobar

pneumonia

Serum sickness


44


Sp. gr. 1010-1020 Casts + W.B.C. + Blood + Alb. +


124/75-90/60


Negative


None


3


10.5


40+?



68


11.9


(SOm) 0.258


(SOm) 4.644 0.646


11.628


5.50



11 — Groeninger


Chr. bronchitis Bronchial asthma


51


Sp. gr. 1026 Casts 0. W.B.C. 0. Blood D. Alb. 0.


142/50


Negative


Moderate


2.5


9.5


25+10 = 35


33.3


49


16.4


0.238


4.76 0.894


17.88 8.928


7.16 4.24 4.74



12— Cosgrove


Diabetes insipidus


52


Sp.gr. 1003-1009 Casts 0. W.B.C. 0. Blood 0. Alb. 0.


175/95-149/80


Negative


Slight


3


10


35+35 = 70


28.6


73


11.5


0.23


4.6 0.44642



13 — Heinicke


^Vn^tT


41


Sp. gr. 1011 Casts 0. W.B.C. 0. Blood 0. Alb.O.


114/64


Negative


None


4


9.5


35+20 = 55


23.3


73


16.7


feom)

0.109


1.962


(SOm) 0.528

0.1975


9.504


SUtionary low count.


14— Gibbons


Pernicious



Sp. gr. 1015.1026 Casts 0. W.B.C. 0. Blood 0. Alb. 0.


90/50


Negative


Slight


3.5


9


45+20 = 65


33.3


66.5


15.1


0.0355


0.71


3.95


3.80

4:33 3.55

3^7

3.79

3.60

2.90

2.02


Stationary fairly


15 — Lucas


Pernicious

Chronic cardiorenal disease. Indolent ulcers

Chronic nephritis

Hypertension

Duodenal

ulcer


36 Sp. gr. 1010-1018 Casts 0. W.B.C. 0. Blood 0. Alb. 0.


110/50


Negative


None


3


8


2 hours = 30


36.2


59.5 60

60


18.2 21.1

17.5


0.122 0.212 (60m) 0.248


2.44 0.474 4.24 0.597

(60m) 5.95 0.422


9.48 11.94

10.128

8.136

5.873


Stationary active

relapse.


16 — Wilson


53 30


Sp. gr. 1015-1030 Casts 0. W.B.C. few Blood 0. Alb. trace


190/115-140/85


Slight vascular • changes


Moderate


3


13.5


38+15 = 53


38.7


75.5


24.7


0.05


1.0 0.4068 0.62 0.29365



17 — Scheminant


Sp. gr. 10101020 Casts ++ W.B.C. 0. Blood 0. Alb. +4

135/90


Negative


None


3


8.5


22+18 = 40


42.8


45.5


30.7


0.031



18— Stain


35 Sp. gr. 1018-1030 Casts 0. W.B.C. 0. R.B.C. 0. Alb. trace


200/135-180/110


Slight vascular changes


Moderate


3


12


20+40 = 60


39.8


54


19.9


0.076


1.52


0.26251


5.24



19— Wickers


Hypertension


37 li5 53


Sp. gr. 1008.1024 Casts 0. W.B.C. 0. Blood 0. Alb. 0.


180/124-130/94


Negative


Moderate


2.5


8


10+35 = 45


40.6


61


16.8


0.244


4.88


0.2918


5.836



20— Stollar


Chronic hypertrophic arthritis /Vneriosclcrosis Chronic cardiovascular-renal

disease_

Arteriosclerosis.

Arteriosclerotic

ulcers


Sp. gr. 1020 Casts 0. W.B.C. 0. Blood 0. Alb. 0.


180/105-138/78


Negative


Marked


2.5 10


2 hours = 28


26.7


74


12.4


0086 1.72


0.1745


3.49


2.49



21— Hynson


Sp. gr. 1012-1030 Casts ++ W.B.C. + Blood 0. Alb. -H-+


235/136-208/78


Retinal arteriosclerosis. Exudate. Few haemorrhages.


Marked


5 16


21+14 = 35


34.3


59


19.3


0.062


1.34


0.1894


3.788


2.21



22— Levy


70 Sp.gr. 1022-1036 Casts occ. W.B.C. occ. Blood 0. Alb. 0.


155/78


Vascular changes


Extreme


3.5


10.5


14+18 = 32


29.5


72.5


17.8


0.042


0.84 0.2875 2.96 0.4855


5.75


4.14



23— Stewart


Chronic nephritis


43 Sp. gr. 1010-1018 Casts 0. W.B.C. 0. Blood 0. Alb. -f +


165/105-158/98


Negative


Slight


3 12.5


43+18 = 61


44.5


70


18.1


0.148


9.71 3.73



24— Meti


Sub -acute

nephritis.

Death


32 Sp. gr. 1029

Casts -t-f+ W.B.C.++ Blood++ Alb.+-f+-f


170/90-178/86


Minimal retinal edema


None 3.5 9.5


Traces


341.0 79


150.0


0.005


0.1


0.0041


0.082 0.02


Dr. Darner*


25— Webb


Sub-acute nephritis

Chr. cardio-rena disease. Broncho pneumonia. Death


18 Sp. gr. 10161021 Casts +-f W.B.C. + Blood + Alb. +++


140/78-107/50


Negligible


None 2.5 8


Traces


95.3 28 59.9


0.0265


0.53


0.0762


1.524 0.66



26— Bazzle


52 Sp. gr. 1015

Casts + W.B.C. + Blood + Alb. ++


150/80117/70


Retinal arteriosclerosis. Massive exudate Many haemorrhages.

Negative


Extreme 3.5 12.5


Traces


95.2


1 55 j 59.0


0.076


1.52


0.428


8.56


1.59

0.81

_


Dr. Oppen heimer**


27— Leahy


Obstructive jaundice


61


Sp. gr. 1020 Casts + W.B.C. -fBlood 0. Alb. trace


195/98


None 3.5


10.5


304-5 = 35


35.7


90 1 21.1

1


0.011


0.22


0.0379


0.758



• Necropsy

glands. Phleboli

Necropsy fluent lobular p


— Acute and chr. diffuse nephritis. Anasarca, ths of spleen and liver.

— Chronic diffuse nephritis; arteriosclerosis; e neumonia. Acute splenic tumor. "Milk plaquci


Bilateral hydrothorax. Pulmonary edema. Broncho


sion and calcification of aortic valve with insufficiency an^ on heart. Old pleuritic adhesions.


pneumonia. Acute splenic tumor. Scarred rt. apex. Caseous mediastinal d stenosis. Cardiac hypertrophy and dilatation. Acute bronchitis. Con


296


[Xo.378


was 1,780,000 aud the hemoglobin reading was 37% (Sahli). Evidence of impaired renal function in severe anemias has been advanced by Moseuthal, and Christian has presented observations suggesting the same conclusion in a series of cases of pernicious anemia.^ However, as in Case 13, with a red cell count of 1,200,000, and a hemoglobin reading 25%, an index of 1.71 was found, the possibility of the participation of factors other than the severe anemia alone must be considered. This latter was a case in which the count had hovered just above one million for two weeks, with corresponding slight fluctuations in the hemoglobin readings. Case 11 at the time of observation presented a red cell count of 2,200,000, with hemoglobin of 38%, and was slowly losing ground. Coincidently with the gradual decline in the count, a definitely low index was found. The inference is attractive that during the active relapses of pernicious anemia there is a definite impairment in the function of urea excretion, which shows definite improvement during the remissions of the di.sease, and that perhaps this impairment is not dependent solely upon the severity of the anemia.

The early detection of failing kidney function in hypertension is an absorbing problem. It is noteworthy in that connection that Cases 18 and 19, who present little evidence of functional impairment by the usual tests, yield indices comparable to those obtained from outspoken cases of chronic nephritis.

Except for Case 27, little comment upon the renuiinder is necessary. Even cursory scrutiny of the data will suffice to demonstrate the constancy with which marked renal damage is associated with a lowered excretory


index. In the case of obstructive jaundice, there is definite evidence of renal insufficiency, but the calculated coefficient is so low as to leave the impression that the collection of specimens was faulty. Yet a liberal allowance for error still gives a low index upon recalculation, and it seems quite likely that in this case also there was a grave degree of kidney damage.

Summary Twenty-seven cases are presented in which the AustinStillman-Van Slyke urea excretory coefficient was determined. In borderline conditions, as well as in cases presenting marked evidence of renal damage, and also during the relapses of pernicious anemia, there was found a definite lowering of the value of the coefficient.

Conclusions The Austin-Stillman-Van Slyke coefficient of urea excretion may be of value in demonstrating renal insufficiency, and, particularly in borderline cases, deserves further studv.


The authors desire to acknowledge their indebtedness to Dr. William S. McCann for helpful interest and valuable suggestions.

REFERENCES

1. Austin, J. H., Stillman, E., and Van Slyke, D. D.: J. Biol. Chem., 1921, XLVI, 91.

2. Van Slyke. D. D. and CuUen, G. E.: J. Biol. Chem., 1914. XIX, 211.

3. Rackemann. F. M., Longcope, W. T., and Peters, J. P.: Arch. Int. Med., 1916, XVIII, 496.

4. Mosenthal, H. O.: Arch. Int. Med., 1915, XVI, 733.

5. Christian, H. A.: Arch. Int. Med., 1916, XVIII, 429.

Fig. 2. — Camera-lucida drawing of a cell containing pigment.


STUDIES ON EXPERIMENTAL RICKETS. XXII.

CONDITIONS WHICH MUST BE FULFILLED IN PREPARING ANIMALS FOR TESTING THE ANTI-RACHITIC EFFECT OF INDIVIDUAL FOODSTUFFS

By E. V. McCoLLUM and Nina Simmonds

(From the Department of Chemical Hygiene, School of Hygiene and Public Health, Johns Hopkins University, Baltimore)

and

P.. G. Shipley and E. A. Park

(From the Department of Pediatrics, Johns Hopkins University, Baltimore)


During the past two years we have carried out a study of the effects of faulty diets of various types on bone development. The results of this study have in part been reported in earlier papers.^ Our observations have indicated that three dietary components are especially concerned in influencing the behavior of the several anatomic elements in the bones. These are calcium and phosphorus, and an organic substance which has as yet not been definitely characterized. AVe have recently


demonstrated that this substance is not identical with fat-soluble A. The relative proportions of these factors determine whether the animal will develop bones which are normal or bones which are pathological ; whether the animal will have rickets, or osteoporosis, or an osteosclerosis.

Mellanby and we ourselves have shown that an animal must grow in order to have rickets. Given a diet which induces rickets, the more rapidly the animals grow the


August, 1922]


297


more extensive are the lesions which are characteristic of the disease. When growth is but slight the rachitic metaphysis will be narrow, and the other characteristics of rickets will be poorly developed. This we have described in the eighth paper of this series.- The diet must be excellent except as regards calcium, phosphorus and the calcium-depositing substance, in order that maximal ' growth may be secured. The proteins of the food mixture should be of good quality. There should be a moderate amount of fat-soluble A (anti-xerophthalmic substance) available, and the diet should contain a satisfactory amount of water-soluble B, and the inorganic elements other than calcium and j)hosphorus. The calcium-depositing substance should be distinctly limited. Furthermore, as we ourselves, and Sherman and Pappenheimer,^ have shown, the quantitative relations between the calcium and phosphorus in the food supply is, within certain limits of concentration, of very great significance in influencing the development of the bones. The present paper deals especially with the effects produced on the anatomic structure of the bones by different ratios between the calcium and phosphate in the diet, other factors remaining essentially constant.

In a preceding paper of this series * we described in detail a method for preparing young rats for the demonstration of the antirachitic effect of various substances in the diet.

In order to make this test in a satisfactory manner we have found it necessary to have the diet on which the animals are fed in the preparatory period of 25-35 days very nicely adjusted with respect to several factors. It is our purpose in the present communication to define more clearly the dietary conditions under which this test can be satisfactorily carried out, and to describe the lesions which are likely to be observed provided the composition of the diet used to prepare the animals for the test deviates, even to a relatively slight degree, from those which we have laid down.

Table I shows the composition of the diets discussed in this paper, together with their sources of additional calcium and phosphorus. In only two diets was the amount of phosphorus increased over that furnished by the organic components of the food mixtures. The table also shows the absolute amounts of calcium and of phosphorus in the diets, and the atomic and weight ratios between these elements. The effects of the several diets on the histological structure of the bones of young rats which were confined to them were characteristic. A description of the bones of animals confined to this series of diets makes clear how sensitive the rat is to changes in the composition of its diet of a magnitude which would have been considered as of no significance a few months ago. This series of experiments makes it clear that extremely careful work is required in studies on bone growth. A full appreciation of this fact will go far


toward preventing faulty interpretations by investigators who may undertake unsuccessfully to duplicate or extend observations in this direction.

Methods of Selecting and Preparing Foodstuffs

There are several details of importance in experimental studies in nutrition wiiich, it appears, some experimenters are likely not to appreciate, which we take this opportunity to emphasize. These relate especially to (1) the selection of vegetable food substances for feeding purposes; (2) the purification of proteins to be employed in supplementing the proteins from natural foods, or for supplying the total protein moiety in diets consisting essentially of purified food substances; (3) the determination of the composition of the mineral salts to be employed in feeding experiments; (4) the proper preparation of fats for supplying in experimental diets the desired amount of fat-soluble A; and (5) the proper evaluation of the vitamin content of certain extracts of natural foods employed as additions to food mixtures for experimental purposes.

1. We have always been very careful to select for feeding purposes only such grains as are free from disease. Any one familiar with seed grains knows that it is frequently found that the germinating power of grain is low or lacking. There are a number of causes for this, the chief ones being damage to the grains by unfavorable climatic conditions during the last stages of development, and infection of the kernels by bacteria or moulds. An inspection of many samples of maize, for example, will show that the germ or "heart" is darkened, owing to invasion by bacteria. In such seeds extensive decomposition of the food substances may have taken place, and even toxic products may have been formed. In other grains, badly developed kernels with low germinating power are the rule rather than the exception. While in the Wisconsin Experiment Station, it was easy to select grains which were sound. Since transferring our work to Baltimore we have for the above reasons secured our grains only from reputable seed dealers who handle grains which are known to be healthy and to have the power to germinate. Grains for nutritive studies should never be purchased from feed stores, since they will in most instances be inferior, and to some extent contaminated with other seeds, and great variation of quality is to be expected.

The history of leafy structures which are to be used for experimental purposes should be known, and they should be practically free from stems. They should not have been cured in the field unless the weather was exceptionally favorable for rapid desiccation. Leaching and the growth of bacteria and moulds must be avoided in their preparation. It is well known that sufficient salts leach from shocks of grains standing in the field to fertilize the soil to an extent that may greatly stimulate the growth of the next year's crop on these spots.

2. In the purification of casein two methods have been almost universally employed, and it seems to us that considerable confusion is likely to arise unless certain standards are adopted in the preparation of food materials. This is especially true In the study of experimental rickets. It is well known that when casein is precipitated from milk it takes with it very considerable amounts of inorganic salts. Whey, which is formed in cheese making through the action of rennet, is very poor in calcium, since this element is in the main carried down with the curd. It is not possible by any simple and easy method to purify casein in considerable amounts so as to make it suitable for experimental purposes.

During the last few years it has become a matter of some importance to use casein for feeding experiments which was free from vitamins, and a number of investigators have subjected casein, the purity of which was not stated, to continuous extrac


298


[No. 378


tion with hot alcohol for the purpose of freeing it from fatsoluble A and water-soluble B. One is led by the comments of certain workers to conclude that they are of the opinion that exhaustion with alcohol is the most important treatment to which casein should be subjected in order to make a preparation of high quality for feeding purposes. It is difiBcult to see how this idea gained credence. Dry casein is impervious to alcohol, and extraction with alcohol is not likely to be effective in removing impurities incorporated within the casein granules. It is next to Impossible to grind casein to an impalpable powder, and even if it is so ground, it is diflScult to extract it effectively in a continuous extractor, because the material packs so tightly that the solvent does not permeate it uniformly, but finds paths of little resistance and tends to follow them, leaving much of the material incompletely acted upon. It is not to be expected that inorganic salts will be removed to any great extent by alcohol, so a casein preparation may be very laboriously extracted, and yet be little more suitable for use in nutrition experiments than before It was treated.

Our own method of purifying casein appears to have been little used by others except at the University of Wisconsin. The procedure has been described in a previous paper.'^ It involves the handling of commercial casein in the following manner. The finely ground casein is placed in a tub and treated with a large amount of distilled water, to which acetic acid to the extent of about 0.2 per cent is added. About five pounds are handled at a time, and from eight to ten gallons of water are used. The first day the casein is washed by putting it on cheese cloth three times, and each time the greater part of the water is removed by moderate pressure on the cloth, after which the protein is returned to the tub and fresh water and acid are added. Thereafter for seven days more the change is made once a day. The repeated washing at first rapidly removes most ot the sugar, soluble salts. and other contaminating substances on which bacteria would grow. The long continued washing with dilute acetic acid offers an opportunity for the soluble constituents to be extracted or to dialyse out of the granules. The acetic acid sets free the inorganic elements with which the acid groups of the casein molecule were combined, and these soluble acetates diffuse out of the granules and are washed away. Finally, the ..-asein is soaked in water, drained, and then dried at about 75° C, and ground. This procedure results in a casein which is all but ash-free, and contains no demonstrable amounts of any vitamins. This method is, we are confident, much superior to that of extracting commercial casein with alcohol.

In selecting gelatin for experimental feeding, it is essential that only the highest grade be used, since the cheaper kinds contain, or are likely to contain, very considerable amounts of phosphorus and other inorganic elements. We have found Bactogelatin of the Digestive Ferments Company suitable, its only drawback being the high cost, which becomes a serious matter in extensive feeding studies. This we have analyzed and found to contain on an average about 0.120 per cent of phosphorus, and to be essentially free from other elements which might be of importance in influencing our results. Chemical analysis of gelatin should never be omitted in experimental work relating to rickets, and the small amounts of the significant elements which it furnishes should be taken into account in stating the content of any of these in the food mixtures.

3. Owing to the low ethical standards ot certain manufacturers of inorganic salts which are widely used in chemical laboratories in this country, it is not safe to rely upon the label as evidence of chemical purity, or of the content of certain contaminating substances when these are stated, and purported to be based upon analysis by the manufacturer. There is much evidence to show that no labels are wasted, and that an analysis made at some time


in the past may be still serving as a basis of guarantee today. Salts should be purchased in large quantities if possible and a chemical examination of each purchase should be made before use.

4. For the preparation of butter fat we have regularly placea about eight pounds of first class creamery butter In a double boiler and allowed it to melt as quickly as possible. The water, salts, casein, etc., separate and settle to form a lower layer, while the fat forms an upper layer. This is decanted off upon a large folded filter supported in a hot-water funnel, the water in which is maintained at boiling temperature. The filtered fat is collected in pint Mason jars which are closed, and are stored away from direct sunlight.

5. It should not be necessary to point out that extracts of wheat germ, now so widely employed as a source of water-soluble B, may likewise contain considerable amounts of fat-soluble A. It is not warranted to introduce liberal amounts of such extracts into diets used to study the fat-soluble A content of fats, for the results may be entirely vitiated by so doing. Thus, an alcoholic extract of ten grams of wheat germ per 100 grams of food may add a considerable amount of fat-soluble A to a diet in which lard or cottonseed oil are being studied, and erroneous conclusions may be drawn from the results of feeding the mixture.

Discussion of Experimental Work

In a foot-note to Table I the quality of the basal food mixture which served in these experiments has been sufficiently discussed. It should be borne in mind, however, that although the diets employed in these studies are fairly complex in their composition, each component has been the object of careful special study in our earlier work, and that the specific dietary properties of each is well understood. It is possible to predict from these special studies the results which will be secured from feeding mixtures of natural foods, with or without supplementary additions of purified food substances.

Although the most significant features of the results of feeding the experimental diets described have been noted under remarks in Table I, there are certain points which need further consideration.

The diet of Lot 3143 gives exaggerated rickets when administered to young rats. The histological structure of the bones induced by this diet has been described in another paper.^ We have made a special study of the extent to which slight modifications of this diet (our "line test" diet) interferes with the successful conduct of our "line test" for the calcium-depositing eft'ect of any natural food. The following description of the histological characteristics of the bones of animals fed the series of diets described in Table I serves admirably to illustrate how very refined the experimental conditions must be maintained in order to avoid error in the conduct of this test.

If the diet of a young rat is modified so as to deviate from the composition of that of Lot 3143 in certain respects, distinct eft'ects are observed in the histological picture of the bones (See Table I, Lot 3211). Under such conditions, even though the changes in the diet may


August, 1922]


299


be of small magnitude, inconstancy of effects will be likely to occur and the data from a series of animals will appear confusing. To illustrate this the following modilications of diet 31-43 will be described and their effects on the bones noted.

Diet 3241 produces changes which are identical with those seen in rachitic children. The epiphyseal disc of cartilage is much wider than normal and a rachitic metaphysis is formed between the cartilage and the shaft. The metaphysis is made up of a disorderly mass of osteoid tissue, connective tissue, blood vessels, and bone-marrow cells from tne shaft of the bone. The trabeculae of osteoid are separated from each other by dilated blood vessels and delicate reticular tissue, which may or may not contain a few marrow cells. Where cartilage cells are in contact with metaphyseal blood vessels, the cartilage may be in all stages of metaplasia into osteoid. Many of the cartilage cells degenerate. On the other hand, in places the cartilage shows a much greater tendency to retain its individuality and staining characteristics than it does in the bones of rats fed on diet 3143. Long tongues of cartilage persist in the metaphysis, which retain the morphology and staining characteristics of the parent tissue. In other words, the tendency of the cartilage cells to metaplasia and to degeneration is less pronounced than it is in the bones of animals on diet 3143. The metaphyses are not quite so wide, nor are the ends of the bones as greatly enlarged as those of animals on the latter diet. The bones of many of these animals show traces of incomplete healing. These take the form in sections of more or less complete or fragmentary lines of calcified intracellular substance crossing the metaphyses of the bones. This calcified intracellular substance results from the reformation of the zone of provisional calcification which is commonly the first sign of healing in the rachitic bone. When relapses occur after remissions, the metaphysis goes on growing and the new proliferative zone is not immediately resorbed, but remains buried in the new osteoid tissue. Some bones show traces of as many as three of these lines of calcified cartilage. Bones which show this picture are apparently identical with those human bones which show relapsing or healing rickets. The trabeculae of the shaft are surrounded by broad zones of osteoid, but they are not more numerous than normal, and apparently little or no resorption of bone or osteoid occurs. The cortex is largely made up of osteoid tissue, and may be greatly thickened on one side. The marrow shows no change from normal, and there is no encroachment on the medullary cavity. The osteoid may or may not be lamellated, and the osteoid corpuscles are small.

These peculiarities of growth are due to the change in ratio between calcium and jjhosphorus in the diet. The ratio between these elements was nearer the optimum in this group (Lot 3241) than it was in that of Lot 3113, and the conditions were, therefore, more favorable to the production of normal bone, notwithstanding the shortage in the diet of the organic factor which favors the normal deposition of calcium. In Lot 3211, as in Lot 3113, the failure of the calcification was brought about by an excessive amount of calcium in the diet rather than by a deficit. The pliosphorus content of both diets was essentially the same.

We may next consider the effects of reducing the calcium carbonate content of diet 3241 from 2 per cent to 1.5 per cent (See Table I, Lot 3228). This diet contained as nearly the optimal content of calcium as we can determine it at present. The phosphorus and the


organic factor concerned in calcium deposition were contained in the diet in amounts almost exactly corresponding to those of Lots 3113 and 3211 discussed above.

The bones of animals on this diet in general differed only very slightly from normal. The cartilage was narrow and well calcified. The cortex and trabeculae were slender, but were almost completely calcified. Only occasionally In tlie shaft was the border of osteoid tissue slightly wider than the physiological osteoid. There was some connective-tissue replacement of medullary tissue between the trabeculie of the spongiosa, and a few large cells with basophilic granules were found in the immediate neighborhood of the trabeculfe. The cortex had a tendency to porosity in the neighborhood of the spongiosa. One of the animals on this diet showed the remains of calcified intercellular substance in the cortex as far down as the middle of the shaft of the bone. The trabecuhe were not as straight as those of normal bones, and the trabeculie in the nucleus of ossification showed rims of osteoid tissue which were wider than those found in the normal animal. The bones of one animal. No. 1186, showed somewhat greater exaggeration of osteoid tissue than the rest of the series.

When we restricted young rats to diet 3227, which is the basal diet employed in .this series, with no calcium additions, the bones were quite uniform in different animals.

This diet produced very imperfectly calcified bones. The cartilage in general was narrow and completely or nearly completely calcified. The trabeculae were small, convoluted and very numerous, as they are in the bones which show osteosclerosis. The trabeculae were incompletely calcified, and the calcium deposits in them had a granular appearance as though they were being rapidly disintegrated and rebuilt. They were partially or completely surrounded by zones of osteoid. A metaphysis was formed which had an orderly arrangement. The marrow was replaced between the trabeculae by young connective tissue containing many basophilic cells. The osteoblasts varied greatly in size and shape. The majority had a deeply stained uniform cytoplasm. The bone corpuscles were often surrounded by a relatively clear area which was bordered by a granular deposit of calcium. There were abundant signs of resorption present in the spongiosa. The cortex of the shaft was a lattice-like condensation of spongiosa. In the nucleus of ossification signs of resorption were wanting, and the osteoid tissue was broader than the osteoid borders of the trabeculae in the shaft.

The effect of modifying the ratio between calcium and phosphorus in our basal diet was further shown by certain experiments in which we have employed the same food mixture. Lot 3113 (see Table I), but with dicalcium phosphate added. This effect we nuiy illustrate by Lots 3229 and 3173. Diet 3229 contained 2 per cent of calcium phosphate, which furnished the same amount of calcium as 1.5 per cent of calcium carbonate. Diet 3173 contained calcium phosphate etpiivalent to the calcium in 3 per cent of calcium carbonate.

The bones of animals in Lot 3229 were very well formed. The cartilage was somewhat narrow and was calcified throughout. The trabeculae were not quite so straight as those in a normal bone, and tended to be thicker. They were completely calcified, had no osteoid tissue about them, and were surrounded by a layer of flat osteoblasts. The space between the trabeculae was greater than normal and was filled with a delicate cellular connective tissue, which contained many basophilic cells and a few marrow elements. The cortex of the shaft was solid and very thick.


300


[No. 378


The center of ossification showed distinct porosis. The trabeculse ot the center were few, thick and completely calcified.

The bones in Lot 3173 differed very little from nonnal. Trabeculae were more numerous and were straighter than in the bones of animals on diet 3229. Calcification was complete, save for the physiological osteoid tissue, which is evidence of normal growth.

We deemed it of iniportance to test the question whether several other calcium salts would yield comparable results iu experiments of the character discussed. For this reason we tested the chloride and the lactate of calcium with our basal diet (See Table I). A rather surprising result was obtained with diet 3240, which contained calcium lactate 4.G per cent (equivalent in its calcium content to calcium carbonate 1.5 per cent). Although this diet contained the same calcium: phosphate ratio as did diet 3228, the bones were not identical.

The bones of animals on diet 3240 were not abnormally thickened or deformed, but the histological picture was that of very mild rickets of low calcium type. The cartilage was abnormally broad, though the disc was not as wide as that which was produced by feeding diet 3142. It wUs incompletely calcified and had been invaded in places by blood vessels from the diaphysis, which had spared the calcified cartilage but invaded the rest of the disc. There was no definite wide rachitic metaphysis. The trabecula" of the shaft abutted directly in many places on the cartilage. In this situation they consisted of little more than spicules of calcified intercellular matrix or bits of calcified cartilage surrounded by narrow borders of structureless osteoid. The latter might contain very many large-sized osteoblasts crowded together almost to the limit of the capacity of the osteoid; or the osteoid might contain almost no cellular elements. The large osteoblasts were oval or polyhedral and their cytoplasm was uniform in shape and was not so deeply stained. The osteoid corpuscles lay in lacuns in the osteoid. In places the tissue in their neighborhood was Infiltrated with sparse granular deposits of lime salts which did not enter the lacunae or encroach on the cytoplasm of the cell. Even at the epiphyseo-diaphyseal border of these bones the trabeculae showed granular deix)sits of calcium salts in their centers, which were the beginnings of ossification. The trabeculae farther down in the shaft were largely made up of mature bone. The osteoid tissue in the growing region was either lamellated or structureless. It was covered in places with a single or multiple layer of very large osteoblasts, or it was quite baie, or surrounded partially or completely with fiattened osteoblasts of an endothelioid form. The trabeculae were separated by delicate reticular tissues, which supported numerous thin-walled blood vessels filled to capacity with blood cells. The connective tissues contained very few haematopoietic cells. A large number of osteoblasts lay free in it, and in some cases they were so numerous as to almost fill the space between the trabeculae. The trabeoulie of the shaft were few in number. They were irregularly arranged and were surrounded by zones of lamellated osteoid tissue containing very few corpuscles. The osteoblasts which surrounded the osteoid in this region were of a flattened endothelioid type. The cortex was merely a lattice-like condensation of the spongiosa. The histology of the center of ossification corresponded to that of the shaft. In the growing region of the bone there were many signs of resorption as well as of active bone formation. Osteoblasts were fairly numerous but were not present in extraordinary numbers. The newly formed trabeculje showed many signs which can only be interpreted as evidence of bone destruction. Large numbers of perforating blood vessels were to be seen in these trabeculae in many places. In others


the osteoid tissue and the bone were being eroded by vascular loops from the diaphyseal circulatory system. In still other places newly built bone and calcified cartilage, partly stripped of its osteoid, seemed to be crumbling away under the influence of cellular and vascular activity. Here and there, large cells filled with basophilic granulations were to be found in approximation to the trabeculae. They were most numerous in the cortex and the older parts of the spongiosa. The bone marrow was apparently normal.

It will be .seen that, in spite of the fact that the ratio between calcium and phosphorus was precisely the same in diet 3240 as in diet 3228, which with one exception produced animals with approximately normal bones, the bones of animals on diet 3240 were affected with a mibl grade of atypical rickets. Moreover, the histology of the lesion would suggest that this rickets was a mild grade of the low calcium type. The sole difference between the two rations was that the calcium iu diet 3240 was supplied in the form of the lactate, whereas that in diet 3228 was in the fonn of the carbonate. It is not possible at present to offer a satisfactory explanation for this difference in the relative effectiveness of the carbonate and lactate of calcium in promoting the development of the bones.

Lot 3230 contained 9.2 per cent of calcium lactate. This amount was sufficient to make the calcium content of the food eqtial to that of Ix)t 3143, which had 3 per cent of added calcium carbonate.

The bones of these animals were affected with very severe rickets, which corresponded exactly with the severest type commonly seen in children. At the same time, although the calciumphosphate ratio in this diet was exactly the same as that in diet 3143, the bones bore a closer resemblance to those which resulted from the administration of diet 3241 (which contained 2.0 per cent of calcium carbonate) than to those which were produced as the result of feeding the former diet. The bones of animals of lots 3236 and 3241 differed from those of 3143 in that there was a greater tendency for the cartilage cell to retain its individuality and primitive characteristics in the former two. Moreover, on diet 3236, as on diet 3241, abortive attempts at healing with reformation of the provisional zone of calcification occurred, which left traces in the form of more or less complete lines of calcified intercellular substance in the cartilage and metaphysis of the bone.

Lot 3235 received calcium chloride 3.4 per cent (equivalent in calcium content to 3.0 per cent calcium carbonate). Animals on this diet developed very severe rickets.

The pathological picture of these bones was comparable to that seen in rats on diet 3143, except that the epiphyseal disc was slightly wider than that found in the bones of animals on the latter formula. There was a slightly greater tendency on the part of the cartilage cells to maintain their individuality and staining characteristics on diet 3235 than on 3143. Therefore, since the epiphyseal disc was invaded by numerous capillaries from the shaft, the epiphyseal disc had a very ragged margin on the metaphyseal side. Although the metaphysis of these bones was wider than the metaphysis of bones of animals on diet 3241, they were alike in that traces of abortive attempts at healing were found in the metaphysis. These were in the form of broken lines of calcified intercellular substance which were embedded in the osteoid of the metaphysis at right angles to the long axis of the bones.


August, 1922]


301


TABLE SHOWING COMPOSITION OF DIETS AND THE NATURE OF THEIR DEFICIENCIES


.s

2


1



.a

1 o


1


G 8

I 3


u 3


ll


o


U


1


"a.


1"


REMARKS



Cms.


Gms.


Gms.


Cms.


Cms.


Cms.


Cms.


Gms.


Cms.


Cms.


Cms.





3143


33


33


15


15



3





1.2316


0.3019


1:0.2531


1:0.3163


This diet produces a very exaggerated form of rickets.


3241


34


33


15


15



2





0.8320


0.3059


1:0.367


1:0.472


This diet produces bones which are identical with those seen in rachitic children.


3228


43.5


33


15


15



1.5t



1


0.6320


0.3059


1:0.4840


These bones in general differ only slightly from l:0.6245i normal.


3227


36


33


15


15






0.0322


0.3101


1:9.630


These bones would seem to indicate that in this 1:12.430' ration the phosphate tended to over-balance the calcium in its effects.


3229


34


33


15


15





'r


0.497


0.6668


1:1.3416


1 Bones well formed. Trabeculae not so straight as 1:1.298 \ those of normal bone.


3173 33


32


15


15





4** 0.962


1.0277


1:1.0683


1:1.3718


Bones of these animals differ very little from the normal.


3240 31.4


33


15


15





4.6t



0.6303


0.2938


1:0.4661


1:0.6015


Histological picture is that of mild rickets of the low calcium type.


3236 26.8 33


15


15





9.2t1


1.2305


0.2700


1:0.2194


1:0.2831


Very severe rickets corresponding to human type.


3235


1 1 32. 6| 33 15| 15


1



3.4



il.2303


0.3019


1 1 Very severe rickets. l:0.2454|l:0.3166|


Furnished calcium equivalent to that in 1.5 grams CaCOj, and phosphorus 0.3609 grams. •« Furnished calcium equivalent to that in 3.0 grams CaCO,. and phosphorus 0.7218 grams.

t Furnished calcium equivalent to that in 1.5 grams CaCO,. tt Furnished calcium equivalent to that in 3.0 grams CaCOj.

t Contained 0.600 grams of calcium.

The quality of all the diets listed in the table was essentially identical as respected protein, fat soluble A, water-soluble B, and the uncharacterized substance which plays a role in directing the growth processes in the bones. None of these diets were capable of inducing growth at a rate faster than half the optimal for a period of a few weeks. This was due to deficiency of fat-soluble A, and except in the cases of diets 3229 and 3173 to lack of sufRcient phosphorus. When these faults are corrected these food mixtures are all capable of inducing good nutrition in rats through successive generations.


We could not detect any inflnence of the chloride, which contains a non-oxidizable acid radical, in contrast with the carbonate or lactate, which are both potential nentralizers of acid in the body.

The results reported in this paper serve to extend and confirm our previous studies on the effect of deviating from the optimal ratios between calcium and phosphorus in the diet when the organic factor is low. They leave no room for doubt as to the importance of the relation between these two elements. The primary purpose in presenting these data was, however, to emphasize how one may readily fall into error in studying the antirachitic effect of any substance, unless the greatest care is taken to fulfill the conditions which we have so carefully standardized.

Conclusions

Dietary conditions are discussed which must be fulfilled in conducting our "line test" for the calcium-depositing substance (anti-rachitic effect) of foodstuffs.

The importance of selecting the highest quality of food grains; of using a rational system of protein purification; the use of salts of proven quality, and of taking into account the content of fat-.soluble A in alcoholic extracts of wheat germ or other substance which may be employed


as sources of water-soluble B, are discussed and emphasized.

The pronounced anatomic changes which are brought about by relatively small variations in the composition of the diet, with respect to those factors which play a role in bone growth, are illustrated by specific examples from our experimental data.

The lactate of calcium proved a less satisfactory source of this element for promoting growth of the bones than did the carbonate. The cause of this difference must await further investigation.

BIBLIOGRAPHY

1. Shipley, P. G.. Park. E. A., McCollum, E. V., and Sinimonds. Nina. Studies on Experimental Rickets. VII. The relative effectiveness of cod liver oil as contrasted with butter fat for protecting the body against insufficient calcium in the presence of a normal phosphorus supply. Amer. Jour, of Hygiene. Vol. 1, p. 512, .July, 1921.

McCollum, Simmonds, Shipley, and Park. Studies on Experimental Rickets. VIII. The production of rickets by diets low in phosphorus and fat-soluble A. Jour. Biol. Chem., Vol. XLVII, p. 507, August, 1921.

McCollum. Simmonds, Shipley, and Park. Studies on Experimental Rickets. XVI. A delicate biological test for calciumdepositing substances. Jour. Biol. Chem., Vol. LI. p. 41, March, 1922.

Shipley, Park, McCollum, and Simmonds. Studies on Experi


302


[No. 378


mental Rickets. XVIII. Is there more than one kind of rickets? Amer. Jour. Dis. of Children, Vol. XXIII, p. 91, February, 1922.

2. McCoUum, Simmonds, Becker, J. Ernestine and Shipley. Studies on Experimental Rickets. XXI. An experimental demonstration of the existence of a vitamin which promotes calcium deposition. Johns Hopkins Hospital Bulletin, June, 1922.

3. McCollum, Simmonds, Shipley, and Park. Studies on Experimental Rickets. VIII. The production of rickets by diets low in phosphorus and fat-soluble A. Jour. Biol. Chem., Vol. XLVII, p. 507, December, 1921.

Sherman, H. C, and Pappenheimer, A. M. A dietetic production of rickets in rats and its prevention by an inorganic salt. Proc.


Soc. Exp. Biol, and Med., Vol. XVIII, 193, 1921. Also Jour. Exp. Med., Vol. XXXIV, 189, 1921.

4. McCollum, Simmonds, Shipley, and Park. Studies on Experimental Rickets. VIII. The production of rickets by diets low in phosphorus and fat-soluble A. Jour. Biol. Chem.. Vol. XLVII, p. 507, August, 1921.

McCollum. Simmonds, Shipley, and Park. Studies on Experimental Rickets. XVI. A delicate biological test for calciumdepositing substances. Jour. Biol. Chem., Vol. LI, p. 41, March, 1922.

5. McCollum, and Davis, M. The essential factors in the diet during growth. Jour. Biol. Chem., Vol. XXIII, 231. 1915.


NOTES ON NEW BOOKS


Bibliography of the Wiitings of Sir William Osier, Bart., M.D., F.R.S. Regius Professor of Mediciiie i?i the University of Oxford. By Mi.nnie Wright Blocg, librarian of the Johns Hopkins Hospital. This book is an amplification and virtual perfection of the bibliography of the writings of the late Dr. Osier, which appeared in the Johns Hopkins Hospital Bulletin in 1918. The original publication presented 730 titles, the number being increased later to 773 by titles subsequently sent by the author from England. The indefatigable compiler of the present volume, Miss Blogg, has now brought it up to 1195. The volume before us must, therefore, be considered as the definitive record of the writings of this remarkable man. The task has been an immense one and the thanks of medical men are due to her for her arduous labor. The varied character of Osier's writings is here characteristically shown. There are contributions on physiology, pathology and practical medicine, medical biography, the history of medicine, and reports of hospital practice and general medicine. There are also numerous text-books, the editions of which he so conscientiously revised for their progressive improvement, and monographs upon special subjects. Not the least interesting portion of the titles are the communications, notes and even editorials, which he supplied to the medical journals in which he was interested while a resident of Montreal, Philadelphia, Baltimore and Oxford. The wonderful industry and versatility of Dr. Osier is shown in the many additions which have been made to the original edition of this book. He was interested in every phase of medical knowledge and equally interested in all that pertained to medical education, and the contact between medicine and social service. The present edition of his bibliography is a monument also to the industry and painstaking work of the compiler. It should be on the desk of everyone interested in medical bibliography.

H. M. H.

The Diseases of Infants and Children. By J. P. Crozer Griffith. (W. B. Saunders Company, Philadelphia and London, 101!).)

In presenting these two volumes the author has attempted to bridge the gap between the smaller text-books and a complete system of Pediatrics. The volumes are well edited and contain a moderate number of good illustrations. The author has drawn on his wide clinical experience and in addition shows a comprehensive knowledge of the native and foreign literature. The reader will profit by the nuuierous references which he gives and it is pleasing to note that he has referred to so many of the original articles on different subjects.

In discussing artificial feeding in infancy the author rightly emphasizes the point that there must be no fixed method of feeding. He shows that the "percentage method" and the "caloric method" of feeding are merely the outcome of certain


basic principles of nutrition. The two methods are really complementary and not diametrically opposed to one another, as has so often been imagined.

Except for two short chapters on infantile atrophy and malnutrition, the nutritional disorders of infancy are discussed primarily as diseases of the digestive system. Although it is true that many of the symptoms are referable to the gastrointestinal tract, the primary cause of most of these disorders is not a local lesion or functional disturbance but rather a disturbed tolerance of the whole body for certain food stuffs. Such conditions arise from many causes, as, for example, an improper balance in the food elements, overfeeding, and parenteral infection. In the last named the digestive disturbances are merely secondary complications. As a matter of fact we often see a profound nutritional disorder from improper feeding, with few or no digestive symptoms. If progress is to be made in this important subject a broader view-point than the author's must be adopted.

Apart from this subject the book is well handled. The chapters on systemic diseases are good and due consideration is paid to their peculiar characteristics in childhood. We feel that rheumatic fever might be considered under the infectious diseases, for although its etiology is still in doubt, it is certainly an infectious process. Bacillary dysentery should also be included under the same heading. -Dr. Griffith doubts the value of the agglutination reaction in dysentery, although the majority of workers consider that this test, if properly done, gives a specific reaction in children, showing the presence or absence of infection with the dysentery bacillus.

One must take exception to the statement that B. eoli is unable to grow in an alkaline medium. This has been shown to be incorrect. In his discussion of tetany the author writes: "The outcome is never fatal, although death may occur depending upon the primary disease or upon complications." However, he says that death may occur during attacks of laryngospasm occurring in this disease. As this is an integral part of the disease, we feel that his statement may give rise to misconceptions.

For students and practitioners the book may be recommended; in conjunction with more complete works on special subjects it will fill a place in the library of a specialist.

S. G. R.

The Evolution of Modern Medieine. By Sir Willi.\m Osler. (London, New Haven, Oxford University Press, Yale University Press, 1921.) Throughout the writings of Sir William Osier may be discerned an eager interest in the historical and bibliographic side of medicine. Steeped as he was in the classics and in their modern reincarnations, and endowed with the deepest instincts of the true literateur, it seemed all but impossible for him to write without apt allusion or allegory derived from


August, 1922]


303


older sources. Among his works we find no less than 61 titles which may be said to deal purely with historical matters. It is altogether befitting, therefore, that we should have left to us one larger and reflective piece of historical writing from his pen.

"An airplane flight through the centuries" he calls it — and rightly — for men and events flash by; but there is no blurring, and when we close the volume the whole story is sharply scored on the tables of the mind. The idea of the origin of medicine from vague religious and social impulses is developed, then the first definite crystallization of the art with Asklepios and Hippocrates, and later its expansion under Galen, the dark days of the crash of the Western Empire, the lucky rescue of medicine from oblivion by the curious and indirect route of Arabia, while medisval mysticism held sway in Europe, the Renascence, and finally the rapid and inevitable developments of the modern era. But there is much more than the story of events. The story of ideas is developed with equal keenness. Osier has written not merely a chronicle but a piece of historical reflection of the very highest order.

Few readers in this day and age will read between the lines and grasp the real meaning of the book. It expresses Osier's creed — Grecian from start to finish — more directly stated in "The Old Humanities and the New Science." He realized well that despite material advance in science the road may lead not into Attica but to Boeotia. Never a reformer, he influenced men by precept and by example, and this History more than anything he has done stands for the broad and generous point of view in medicine.

A. L. B.

Acute Epidemic Encephalitis (Lethargic Encephalitis). An Investigation by the Association for Research in Nervous and Mental Diseases. (Neto York; Paul B. Hoeher, 1921.) $2.50. Despite the great importance of epidemic encephalitis the disease has not become common enough for any single observer's experience to be altogether comprehensive and authoritative. One welcomes, therefore, this collective investigation by such men as Barker, Hunt, Amoss, and Griffith, to mention only a few of the names of those concerned. The material of the book includes sound and critical summaries of the various facts which have been collected about encephalitis, each man presenting as it were "the evidence" in his domain of special interest. There is some question in the reviewer's mind as to whether the value of the book is enhanced by the verbal discussions at the end of each section which are reported verbatim apparently from extemporaneous remarks, and which perhaps carry the analogy to court proceedings a little too far. For the time being, however, this book must be regarded as a most valuable storehouse of information aljout an obscure and diflicult disease.

A. L. B.

Oxford Medicine, Vol. V., Edited by Henry A. Christian and Sir James Mackenzie. (Neiv York and London; Oxford University Press. 1022.) Volume V. of the Oxford Medicine continues along the general lines of its predecessors. Interesting summaries of the infectious diseases are presented from a modern and satisfactory point of view.

A. L. B.

The Vitamins, by H. C. Sherman and S. L. S-MIth. American

Chemical Society Monograph Series. (Neio York. Chemical

Catalogue Company. 1922.)

At a time when the lay mind is being actively stimulated to an

interest in "vitamins." both by popular writers and manufacturers

of various commercial vitamin products, it is very desirable to


have a review of the actual scientific knowledge of these substances, particularly of the sources from which they are readily and easily obtainable. To the physician, who is frequently besieged by questions on this subject, the concluding statement of the book is significant: — "We believe it is safe to say that with a dietary selected to make the best use of our ordinary staple foods there will rarely, if ever, be occasion to purchase vitamins in any other form, or to give any greater anxiety to the vitamins than to some other factors which enter into our present conception of nutritive requirements and food values." In the concluding chapter there is a useful table showing the distribution of "vitamins" in investigated food materials.

The bibliography comprises about one thousand titles. It is remarkable and very significant that the authors have been able to condense such an enormous literature into such a small space. To one who has watched the rapidity with which publications in this field have issued from the various laboratories, filled with hastily drawn conclusions and polemics, the task of making such a review is appalling. The authors have handled this difficult task without the introduction of personal bias. One feels that in some respects the review is not sufficiently critical.

It is of value for reference because of its unbiased point of view, and because it assigns to "the vitamins" no more than their proper share of importance in the broad field of the science of nutrition.

W. S. McC.

The Clinical Study of the Early Symptoms and Treatment of Circulatory Disease in General Practice. By R. M. Wilson, {Oxford Medical Publications, London, 1921.)

"You might just as well say, added the Dormouse,

that I breathe when I sleep is the same thing

as I sleep when I breathe."

Alice in Wonderlami.

To those readers at all familiar with the author's views as expressed in his earlier work entitled The Hearts of Man many of the ideas set forth in this volume will come as no surprise, but to the uninitiated there will be much, we fear, that appears obscure iu the elucidation of his unique hypothesis.

We confess at the outset that it was not without great effort that we were able to follow the author's frequently confused text and that our interest flagged tremendously long before the end came in sight. It would be equally false if we conveyed the impression that there are not a number of very sound truths contained in this volume. Unfortunately, many of them are lost In a bewildering maze of paradoxes that are indeed difficult to unravel and too often destructive of the author's purpose.

The raison d'etre of the work is to set forth in great detail the author's theories of what we may call the balanced action met with in every physiological reaction, whether normal or pathological, and an attempt to analyze the effect of a stimulus, and the reaction following, upon the insecure basis of clinical observations alone.

A clue to the author's general attitude toward established physiological truths is offered early in his introduction when he sa,ys: "On the experimental table stimulation of the vagus slows the heart. But in clinical practice methods employed to this end always (italics ours) result in quickening it, even if a temporary slowing may at first occur!" The first five chapters devoted to the subject of Exhaustion constitute an attempt to apply the author's theories to the elucidation of a very complex problem due chiefly, he asserts, to the influence of pathological stimuli affecting primarily either the vagus or the sympathetic nerves. He postulates an extraordinary, one might say speaking paradoxically, almost human relationship between the vagus and sympathetic fibres frequently described as follows: "Thus to


304


[No. 378


borrow a phrase from the commercial world the cheques drawn by the vagus in its responses to stimuli are honored by the sympathetic." On page 39 he says: "Exhaustion, then, in a toxic state is first of all a matter of vagus excitability." A little further on "The toxic patient, then, presents a vagal picture (exhaustion) when and because reciprocal action by the sympathetic fails owing to depletion of that system. The patient with a mechanical disturbance in the viscera is in a different plight. The visceral disturbance causes sympathetic stimulation and so reflex vagal activity, and the latter soon dominates the former just as in response to outside stimuli the sympathetic when healthy soon dominates the vagus."

The chapter devoted to a study of Breathlessness is based upon a further application of the theory of vagal and sympathetic interplay. He divides breathlessness into two main groups, the "reaction type" of toxic origin due to action on the vagus, the "resting type" due to sympathetic exhaustion which In time may give place to a so-called "diaphragmatic type," when the respiratory rate increases and death is at hand. There is no discussion of the physiology of cardiac dyspnoea, although allusion is made to the work of Haldane and Meakins and there is an occasional reference to oxygen want and oxygenation and the importance of the circulation.

The short section dealing with cyanosis is utterly unsatisfactory. All critical discussion of or even reference to the vast amount of work in recent years bearing on the subject is omitted. Referring to the incidence of cyanosis in pneumonia, its occurrence at the time of onset is over-emphasized and explained on the basis of an "invasion in mass of the toxin producing a very great vagus excitability to which the sympathetic could not at once respond."

The remaining chapters of the book are concerned with such topics as the symptoms of the patient's sensations, dealing with hyperalgesia, cardiac pain and headache, observations on blood pressure, the earliest symptoms of heart failure, and concluding with suggested treatment based upon early signs and symptoms; the whole discussed on the basis of the author's theory of the fundamental importance of the nervous system in relation to the subjective and objective evidences of cardiac disease.

The volume is introduced by a foreword from the pen of Sir James Mackenzie, in which he says that "Whether, then, the

hypothesis which Dr. Wilson puts forward is confirmed or

disproved, he has rendered a service to medicine. Those who may cavil at such a work must bear in mind that here is but the beginning of a big undertaking." It is indeed difficult to understand the point of view which seems to have overtaken this distinguished observer in recent years. It is quite true, we admit,


that in clinical medicine two of the most important considerations "are the prognostic significance of a symptom and the mechanism of its production," but above all we need carefully controlled and interpreted observation. We cannot describe in too accurate detail our facts, but we should ever be cautious and over-critical in our conclusion based upon unsupported evidence.

E. P. C.

A Manual of selected Biochemical Methods as Applied to Urine. Blood and Gastric Analysis. By Fkask P. Undebhill. XIV+ 232 pp. (A'eir York: John Wiley d- Sons: London: Chapman .(• Hall. Ltd.. I!).U.)

It is a distressing fact that biochemical methods of analysis are constantly changing and multiplying at a prodigious rate, and that manuals of such methods are short-lived undertakings. Yet Professor Underbill's compact volume presents to the biochemical analyst a more valuable and useful compendium than he has had hitherto. The great variety of the older and more modern methods of urine, blood, and gastric analysis are included, covering more than the ordinary routine procedures of a clinical laboratory, and furnishing ample substance for research work along these lines. A number of new and useful items for a manual of this kind are introduced, such as calibration of apparatus, acidimetry and alkalimetry, standardization of oxidizing solutions, etc. From the standpoint of instruction, the excellent plan of giving the principle involved in every analytical procedure has been faithfully followed. Tlie different methods are mostly quoted verbatim from the original sources, perhaps too faithfully, with little attempt to clarify directions which are not clear or explicit enough in places. No critical appraisal is presented by the compiler on the comparative accuracy or simplicity of the methods, with the obvious presumption that each worker will choose the particular method which happens to suit his needs best. References being given in every instance to the original article, the reader is incited to study the method in which he is interested at greater length. The lack of tables comparing the content of urine and blood in normal and various pathological constituents as approximate guides in interpreting analytical results is also felt. Neither does the reader find any discussion of such debatable problems as the preferability of the use of whole blood or of plasma for the analysis of various constituents. It is hoped that this excellent manual will be revised from time to time so as to include the more valuable of the accretions to the biochemical methodolog>', and that it will have the wide use which it easily merits.

W. A. P.


BOOKS RECEIVED


A Psychiatric Milestone. Bloomingdale Hospital Centenary 18211921. 8°. 220 pages. Privately Printed by the Society of the New York Hospital. 1921.

Arabian Medicine. Being the Fitzpatrick Lectures Delivered at the College of Physicians in November 1919 and November 1920. By Edward G. Browne, M.B., F.R.C.P., 1921. 8°. 138 pages. University Press, Cambridge.

The Harvey Lectures. Delivered Under the Auspices of the Harvey Society of New York, 1919-1920. By Lieut. Colonel George Dreyer, Dr. H. H. Dale, and others. Series XV. 1921. 8°. 157 pages. J. B. Lippincott Company, Philadelphia and London.

Nostrums and Quackery. Articles on the Nostrum Evil. Quackery and Allied Matters Affecting the Public Health; Re


printed, With or Without Modifications, from the Journal of the American Medical Association. Prepared, Compiled or Edited by Arthur J. Cramp, M.D. Volume II. 1921. 8°. 832 pages. Press of the American Medical Association, Chicago.

Menstruation and Its Disorders. By Emil Novak, A.B., M.D., F.A.C.S. With Forty Illustrations. Gynecological and Obstetrical Monographs. 1921. 8°. 357 pages. D. Appleton and Company, New York and London.

Episcopal Hospital, Medical and Surgical Reports. Volume V, 1917-20. 8°. 506 pages. Wm. J. Dorman, Philadelphia.

The Submucous Resection of the Nasal Septum,. By W. Meddaugh Dunning, M.D. 1921. 12°. 97 pages. Surgery Publishing Company, New York.


August, 1922]


305


studies in Influenza and Its Pulmonary Complications. By D.

Barty King, O.B.E., M.A., M.D. (Edin.), M.R.C.P. (Lond.

and Edin.). With 31 illustrations. 1922. 8'. 88 pages. J. & A. Churchill, London.

The Viiaynins. By H. C. Sherman and S. L. Smith. American Chemical Society, Monograph Series. 1922. 8". 273 pages. The Chemical Catalog Company, Inc. New York.

The Treatment of Common Female Ailments. By Frederick John McCann, M.D., Edin., M.R.C.P., London, F.R.C.S., England. 1922. 8^ 152 pages. Edward Arnold & Co., London.

Vice and Health Problems — Solutions. By John Clarence Funk, M.A.. LL.B. 1921. 12°. 174 pages. J. B. Lippineott Company, Philadelphia and London.

Ephraim McDowell "Father of Ovariotomy" and Founder of Abdominal Sii7-gery. With an Appendix on Jane Todd Crawford. By August Schachner, M.D., F.A.C.S. 1921. 8°. 331 pages. J. B. Lippineott Company, Philadelphia and London.

Acute Epidemic Encephalitis (Lethargic Encephalitis) . An Investigation by The Association for Research in Nervous and Mental Diseases. Report of the Papers and Discussions at the Meeting of the Association; New York City, December 2Sth and 29th, 192U. Prepared under the direction of Walter Timme, M.D., Pearce Bailey, Lewellys F. Barker, Sanger Brown, and others. 1921. 12". 258 pages. Paul B. Hoeber, New York.

The Evolution of Modern Medicine. A Series of Lectures Delivered at Yale University on the billiman Foundation in April, 1913. By Sir William Osier, Bart., M.D., F.R.S.

1921. 4". 243 pages. Yale University Press, New Haven. Humphrey Milford, Oxford University Press, London.

Clinical Diagnosis. A Text-Book of Clinical Microscopy and Clinical Chemistry for Medical Students, Laboratory Workers, and Practitioners of Medicine. By Charles Phillips Emerson, A.B., M.D. Fifth edition entirely rewritten and reset. 156 illustrations. 1921. 8°. 276 pages. J. B. Lippineott Company, Philadelphia and London.

An Introduction to Biophysics. By David Burns, M.A., D.Sc. With a foreword by D. Noel Paton, M.D., LL.D., F.R.S. With eighty-five illustrations. 1921. 8°. 435 pages. Macmillan Company, New York.

A Form of Record for Hospital Social Work: Including Suggestions on Organization. By Gertrude L. Farmer. 1921. 8°. 81 pages. J. B. Lippineott Company, Philadelphia, London and Montreal.

Epidemiology arid Public Health. A Text and Reference Book for Physicians, Medical Students and Health Workers. In Three Volumes. By Victor O. Vaughan, M.D., LL.D. Assisted by Henry F. Vaughan, M.S., D.P.H., and George T. Palmer, M.S., D.P.H. Vol. I. Respiratory Infections.

1922. 8°. 688 pages. C. V. Mosby Company, St. Louis. Book on the Physician Himself. From Graduation to Old Age.

By D. W. Cathell, M.D. This is the Vastly Improved Crowning Edition. 1922. 8°. 359 pages. Published by the Author, Baltimore, Maryland.

The Mechanics of the Digestive Tract. By Walter C. Alvarez, M.D. With twenty-two illustrations. 1922. 8'. 192 pages. Paul B. Hoeber, New York.

Neoplastic Diseases; A Treatise on Tumors. By James Ewing, A.M., M.D., Sc.D. Second edition, revised and enlarged with 514 illustrations. 1922. 8°. 1054 pages. W. B. Saunders Company, Philadelphia and London.


Clinical Electrocardiography. By Fredrick A. Willius, B. S., M.D., M.S. in Medicine. With 185 illustrations. 1922. W.

B. Saunders Company, Philadelphia and London.

University of Minnesota. Papers from the Mayo Foundation for Medical Education and Research and the Medical School. Volume I, 1915-1920. 1921. 8°. 659 pages. W. B. Saunders Company, Philadelphia and London.

An Introduction to the Study of the Protozoa: With Special Reference to the Parasitic Forms. By E. A. Minehin, M.A., Ph.D., F.R.S. Second impression, illustrated. 1922. 8°. 517 pages. Edward Arnold, London.

Diseases of the Eye. A Handbook of Ophthalmic Practice. By George E. de Schweinitz, M.D., LL.D. (Univ. of Pa.). Ninth edition, reset, with 415 illustrations and 7 colored plates. 1922. 8°. 832 pages. W. B. Samnders Company, Philadelphia and London.

An Essay on the Physiology of Mind. An Interpretation Based on Biological, Morphological, Physical and Chemical Considerations. By Francis X. Dercum, A.M., M.D., Ph.D. 1922. 12\ 150 pages. W. B. Saunders Company, Philadelphia and London.

Abdominal Pain. By Prof. Dr. Norbert Ortner. Authorized Translation by William A. Brams, M.D., and Dr. Alfred P. Luger. 1922. 12°. 362 pages. Rebman Company, New York.

Protein Therapy and Nonspecific Resistance. By William F. Petersen, M.D. With an introduction by Joseph L. Miller, M.D. 1922. 8°. 314 pages. Macmillan Company, New York.

Clinical Tuberculosis. By Francis Marion Pottenger, A.M., M.D., LL.D. With a Chapter on Laboratory Methods, by Joseph Elbert Pottenger, A.B., M.D. Vol. I. Pathological Anatomy, Pathological Physiology, Diagnosis, and Prognosis. Vol. II. Complications and Treatment. Second edition. 1922. 8°.

C. V. Mosby Company, St. Louis.

Oxford Medical Publications. Publishers: Henry Frowde, London; Hodder & Stoughton, London. The following five volumes:

Arterial Sclerosis. A Consideration of the Prolongation of Life and Efficiency After Forty. By Louis Faugeres Bishop. M.A., M.D.. Sc.D., F.A.C.P. 1921. S". 383 pages.

Hygiene of Women and Children. By Janet E. Lane-Claypon, M.D., D.Sc. (Lond.). 1921. 8°. 354 pages.

Boicel Diseases in the Tropics. Cholera. Dysenteries, Liver Abscess and Sprue. By Sir Leonard Rogers, CLE., M.D., F.R.C.P., P.R.C.S., I. M.S. (Retired). 1921. 8'. 475 pages.

The Clinical Study of the Early Symptoms and Treatment of Circulatory Disease in General Practice. By R. M. Wilson, M.B., Ch.B. With a foreword by Sir James Mackenzie, M.D., F.R.S., F.R.C.P. 1921. 8°. 245 pages.

Medical Electricity for Students. By A. R. L. Browne. 1921. 12°. 231 pages.


306


[No. 378


THE JOHNS HOPKINS HOSPITAL REPORTS


VOLUME I. 423 pagesi, 99 plates.

VOLUME II. 570 pages, nltU 28 plates and figures.

VOLUME III. 766 pages, with 69 plates and figures.

VOLUME IV. 504 pages, 33 charts and Illustrations.

VOLUME V. 480 pages, with 32 charts and illustrations.

VOLUME VI. 414 pages, with 79 plates and figures.

VOLUME VII. 537 pages with Illustrations.

VOLUME VIII. 552 pages nith Illustrations.

VOLUME IX. 1060 pages, 66 plates and 210 other illustrations. Contributions to the Science of Medicine.

Dedicated by his Pupils to William Henry Welch, on the twenty-tlfth annivei-sai-y of his Doctorale. This volume contains 38 separate papei-s.

VOLUME X. 516 pages, 12 plates and 25 charts.

VOLUME XI. 555 pages, with 3S charts and illustrations.

VOLUME XII. 548 pages, 12 plates and other illustrations.

VOLUME XIII. 605 pages, with 6 plates, 201 figures, and 1 colored chart.

VOLUME XIV. 632 pages, with 97 figures.

VOLUME XV. 642 pages, with 87 Illustrations.

Twelve papers on pneumonia. By DBS. Chatahd Fabvan. E.merson. Makshall, McCrae, Steiner, Howard and Hanes.

A Study of Dianhcea in Children. J. H. JUsoN Knox, Jr., M. D., and Edwin H. Schorer, M. D.

Skin Transplantation. By John Staige Davis, M. D.

Epidemic Cerebrospinal Meningitis and Serum Therapy at The Johns Hopkins Hospital. By Frank J. Sladen, M. D.

VOLUME XVI. 070 pages with 161 figures.

Studies in the Experimental Production of Tuberculosis in the Genitourinary Organs. By George Walker, M. D.

The Effect on Breeding of the Removal of the Prostate Gland or of the Vesiculffi Semlnales, or of Both; together with Observations on the Condition of the Testes after such Operations on ^ hlte Rats. Bj George Walker, M. D.

Scalping Accidents. By John Staige Davis, M. D.

Obstruction of the Inferior Vena Cava with a Report of Eighteen Cases. By J. Hall Pleasants, M. D.

Physiological and Pharmacological Studies on Cardiac Tonicity in Mammals. By Percival Douglas Cameron, M. D.

VOLUME XVII. 686 pages with 21 plates nnd 136 flgurea.

Free Thrombi and Ball Thrombi in the Heart. By Joseph H. Hewitt,

Benzol' as'a Leucotoxln. By Lawrence Selling, M. D.

Primary Carcinoma of the Liver. By Milton C. Winternitz, M. D.

The Statistical Experience Data of The Johns Hopkins Hospital, Baltimore,

Md 1892-1911. By Frederick L. Hoffman, LL. D., F. S. S. The Origin and Development of the Lymphatic System. By 1'LORENCE K.

The Nuclei Tiiberis Laterales and the So-called Ganglion Optlcum Basale.

By Edward F. Malone, M. D. Venous Thrombosis During Myocardial InsufBciency. By Frank J. Sladen,

M D., and Milton C. Winternitz, M. D. Leuksemia of the Fowl : Spontaneous and Experimental. By Harri C.

SCH.MEISSER, M. D.

VOLUME XVIII. 446 pages with 124 figures. Fasciculus I.

A Study of a Toxic Substance of the Pancreas. By E. W. Goodpastdke

M " D., and George Clark, M. D. Old Age in Relation to Cell-overgrowth and Cancer. By E. W. Good

PASTURE, M. D., and G. B. W*islocki. M. D. The Effect of Removal of the Spleen Upon Metabolism in Dogs; Pre

liminarv Report. By J. H. King, M. D. „ t ,t

The Effect of Removal of the Spleen Upon Blood Transfusion. By J. H

King, M. D. B. M. Bernheim, M. D., and A. T. Jones, M. D. Studies on I'arathyroid Tetany. By D. \\'^nGHT Wilson, M. D., Thornton

Stearns, M D., J. H Jannev, Jr., M. D., and Madge DeG. Thurlow,

M. D. Some Observations on the Effect of Feeding Glands of Internal Secretion

to Chicks. By M. C. Winternitz, M. D. Spontaneous and Experimental Leukffimia in the Fowl. By H. C.

SCHMEISSER. M. D.

Studies on the Relation of Fowl Typhoid to Leuksemia of the Fowl. By

M. C. Winternitz, M. D.. and H. C. Schmeisser, M. D. Hyaline Degeneration of the Islands of Langerhans in Pancreatic Diabetes.

Bv M. C. Winternitz, M. D. Generalized Miliary Tuberculosis Resulting from Extension of a Tubercular

Pericarditis Into the Right Auricle. By M. C. Winternitz. M. D. Acute Suppurative Hypophysitls as a Complication of Purulent Sphenoidal

Sinusitis. By T. R. Boggs. M. D.. and M. C. Winternitz, M. D. A Case of Pulmonary Moniliasis In the United States. By T. R. Hoggs.

M. D., and M. C. Pincoffs. M. D. Gaucher's Disease (A Report of Two Cases in Infancy). By J. H. M.

Knox, M. D., H. R. Wahl, M. D., and H. C. Schmeisser, m. D. A Fatal Case of Multiple Primarv Carcinomata By E. D. Plass, M. D. Congenital Obliteration of the Bile-ducts. By James B. Holmes, M. D. Multiple Abscesses of the Brain in Infancy. By James B. Holmes, M. U. Gastric Carcinoma in a Woman of Twenty-six Years. By R. G. Hdsset,

M. D. Subdiaphragmatic Abscess with Rupture Into the Peritoneal Cavity Fol


rrhage. By R. G.


lowing Induced Pneumothorax for Pulmonary Ha

HussEY, M. D. Heart Block Caused by Gumma of the Septum. Bv E. W. Bridge.\ian,

M. D., and H. C. Sch.meissee, M. D. Analysis of Autopsy Records.

A. The Johns Hopkins Hospital. (Table Showing Percentage ot

Autopsies.)

B. The City Hospitals, Bay View. (Table Showing Percentage of

Autopsies.)

"The Monday Conferences."

Clinical Representatives on the Staff of the Department of Pathology.

Donation.

Fasciculus II,

The Role of the Autopsy in the Medicine of To-day. By M. C. Winternitz, M. D.

Experimental Nephropathy in the Dog. Lesions Produced by Injection of B. bronchisepticus into the Renal Artery. By M. C. Winternitz, M. D., and William C. Quinby, M. D.

Mesarteritis of the Pulmonary Artery. By M. C. Winternitz, M. U., and

H. C. SCH.MEISSER, M. D.

A Clinical and Pathological Study of Two Cases of Milary Tuberculosis of

the Choroid. By Robert L. Randolph, M. D., and H. C. Schmeisser,

M. D. The Blood-vessels of the Heart Valves. By Stanhope Bayne-Jones, M. U. Equilibria in Precipitin Reactions. By Stanhope Bayne-Jones, M. D. Carcinoma of the Pleura with Hypertrophic Osteoarthropathy. Report of

a Case with a Description of the Histology of the Bone Lesion. By

Stanhope Bayne-Jones, M. D, The Interrelation of the Surviving Heart and Pancreas of the Dog In

Sugar Metabolism. By Admont H. Clark, M. D. Congenital Atresia of the Esophagus with Tracheo-Esophageal Fistula

Associated with Fused Kidney. A Case Report and A Summary of the

Literature on Congenital Anomalies of the Esophagus. By K. D.

Plass, M. D. Ectopia Cordis, with a Report of a Case In a Fifteen-Month-Old Infant.

By James B. Holmes, M. D, Studies in the Mechanism of Absorption from the Colon. By Samuel

Goldschmidt, M. D., and A. B. Dayton, M. D. Report of Two Fatal Cases Following Percy's Low Heat Treatment of

Carcinoma of the Uterus. By V. N. Leonakd, M. D. and A. B. Dayton,

M. D. The Relationship in Typhoid Between Splenic Infarcts and Peritonitis

Unassociated with Intestinal Perforation. By A, B. Dayton, M. D. Left Duodenal Hernia. By A. B. Dayton, M. D.

Histological as Related to Physiological and Chemical Differences in Certain Muscles of the Cat. By H. Hays Bullard, M. D. A Method of Clearing Frozen Sections. By H. Hays Bullard. M. D. On the Occurrence and Significance of Fat in the Muscle Fibers of the

Atrlo-Ventricular System. By H. Hays Bullard, M. D. Studies on the Metabolism of Cells in vitro. 1. The Toxicity of a-Amino Acids for Embryonic Chicken Cells. By Montrose T. Burrows, M. D.,

and Clarence A. Neymann, Jl. D. The Significance of the Lunula of the Nail. By Montrose T. Burrows,

M. D. The Oxygen Pressure Necessary for Tissue Activity. By Montrose T.

Burrows, M. D. The Functional Relation of Intercellular Substances in the Body to Certain Structures in the Egg Cell and Unicellular (Organisms. By

Montrose T. Burrows, M. D. Studies on the Growth of Cells i'h vitro. The Cultivation of Bladder and

Prostrate Tumors Outside the Body. By Montrose T. Burrows,

M. D., .1. Edward Burns, M. D., and Yoskio Suzukl. M. D. The Study of a Small Outbreak of Poliomyelitis in an .\pai-tment House.

Occurring in the Course of an Epidemic in a Large City. By Montrose

T. Burrows, M. D.. and Edwards A. Park, M. D. Papilloma of the Larynx Report of a Case Treated with Radium with

Resultant Chronic Diffuse Thyroiditis. By William C. Duffy. M. D. Analysis of Autopsy Records. Autopsy Statistics.

(a) Bay View.

(b) Johns Hopkins Hospital. Report of the Photographic Department. General Improvements.

Donations.

VOLUME XIX. 358 pages with 29 plates.

The Structure of the .Normal Fibers ot I'urkinje in the Adult Human Heart and Their Pathological Alteration in Svphilitic Myocarditis. By 0. Van Der Stricht and T. Wixgate Todd. M. D.

The Operative Story of Goitre. Tie Author's Operation. By William S. Halsted, M. I).

Study of Arterio- Venous Fistula with an Analysis of 447 Cases. By CuRLE L. Callander. M, D.

VOLUME XX. 314 pages with SS plates.

The Pathology of the Pneumonia in the United States .\rmy Camps During the Winter of 1917-18. By William G. MacCallum. M. D.

Pathological Anatomy of Pneumonia Associated with Inlluenza. By William G. MacCallum, M. D.

Lymphosarcoma. Lymphatic Leukaemia. Leucosarcoma. Hodgkin's Disease. Leslie T". Webster, M. D.


CONTENTS

  • Interfacial Phenomena. With Especial Reference to Colloids and Enzymes. By W. M. B.WLiss
  • Factors Which Determine the Concentration of Calcium and of Inorganic Phosphorus in the Blood Serum of Rats. By Bexj. Kb.\mer and John Rowland.
  • A Study of the Results Obtained in Sixty-four Caesarean Sections Terminated by Supravaginal Hysterectomy. By JoHX W. Hauuis
  • Air Embolism Following Various Diagnostic or Therapeutic Procedures in Diseases of the Pleura and the Lung. By Karl Schlaepfer, M. D
  • The Effect of Saline Purgatives on the Absorption of Other Drugs. (Illustrated.) By D. I. Maciit. M. D. and E. M. Finesilver, M. A.
  • Localization of Cutaneous Nerves by Electrical Stimulation. Applied to Nerve-Block Anaesthesia. (Illustrated.) By Walter Hughsox 335
  • The Effect of Sodium Germanate Upon the Total Hemoglobin of the Albino Rat. By Joseph E. Nowrey, Jr 340
  • Giant Centrospheres in Xanthomatous Tumors. (Illustrated.) By David T. Smith 342
  • A Normal Pregnancy Following Insertion of the Outer Half of a Fallopian Tube Into the Uterine Cornu. By Thomas S. Clllen 344
  • An Operation for the Total Extirpation of Tumors in the Cerebello-Pontine Angle. A Preliminary Report. By Dr. Walter E. Dandy 344
  • Notes on New Books 345


INTERFACIAL PHENOMENA WITH ESPECIAL REFERENCE TO COLLOIDS AND ENZYMES

By W. M. Bayliss (University College, London)


Although much atteution is being given at the present time to the phenomena present in homogeneous solutions, and rightly so, it seems that there is need of more work on the mechanism of reactions in heterogeneous systems, and at the same time a clearer recognition of the fact that the majority of physiological processes take their course in such a system. Even when they appear at first sight to follow the relatively simple laws of mass action as applied to the conditions of homogeneous solutions, it must not be left out of sight that in the living organism they occur in the presence of various solid and liquid structures which may or may not have an important etlect on their course.

For this reason, I think that considerations of some such cases form the most useful purpose to which I can


devote the first of these lectures, which I am enabled to give by the generosity of the Herter Foundation, to whom I wish to express my deep sense of the honour conferred by the invitation and the pleasure it gives me to put my views before so distinguished an assembly. I wish then to call attention to some points in the mechanism of physiological processes which appear to require further and more critical examination. In doing so, I would at the outset make it plain that I have the highest appreciation of the value of the work which has been done and, where it is necessary to criticize it, the criticisms are made with the object of pointing out the


Lecture I of the Herter Series delivered before The Johns Hopkins University, March 7th, 1922.


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risk of supposing that certain phenomena are already adequately explained, when the statement applies only to some aspects of them. Conditions peculiar to special cases are apt to be extended to a whole group of phenomena. This is particularly so when the colloidal state is involved. For example, a certain group of investigators would explain all the properties of this state on a purely chemical, others on a purely physical basis. It is scarcely necessary to remark that we want to know what is the actual state of affairs, and if we find that the physical state as well as the chemical nature of our systems has to be reckoned with, we base our conclusions on foundations liable to crumble away if we omit either of these.

It is no simple matter to define satisfactorily what is to be looked upon as a heterogeneous system. Perhaps it may suffice for our present purpose to say that it is composed of parts which, although in contact, do not mix together. These parts were named "phases" by Willard Gibbs, to whom we owe so much of our knowledge of the factors controlling equilibrium in systems of the kind mentioned. It will be clear that there are interface.s of contact where the phases meet and that the molecules forming these surfaces are exposed to forces which are not identical with those in thie interior of the phases. For one thing, the molecules on the surface only are exposed to chemical action, so that the "active mass" is a function of the surface, not of the total mass present.

Some difficulty arises as to the dimensions required to claim the name of "phase." Single molecules, unless of very large size, cannot be possessed of the properties of surface, which imply the existence of a number of atoms joined together. The problem arises as to whether colloidal solutions obey Gibbs' "phase-rule," a point to wliidi we shall return later.

AVhat then are the most striking properties of surfaces as such? They are two, both conferring the possession of energj-.

1. Surface tension, when one of the phases is liquid, and the corresponding property when we have only to deal with solids or gases. This phenomenon is due to the mutual attraction of molecules, giving rise to the "internal pressure" of Young and Laplace and to the "cohesive" forces of Matthews.

2. Electrical charge, which may be given in three ways : —

(ff) By the deposition of ions by adsorption, as investigated by I'errin. For example, ferric hydroxide may have either a positive or a negative charge, according as it is suspended in acid or alkaline media.

(6) By electrolytic dissociation of the surface molecules. Thus, silica forms insoluble silicic anions and gives off hydrogen ions. The latter pass into the water, but only so far as electrostatic attraction to the opposite solid ion permits. The particles have thus a negative


charge, but are surrounded by a shell of positive ions in the liquid. Such systems have been called "electroIj'tic colloids" by Hardy; the large composite insoluble particle, consisting of unclianged matter with a layer of ions on the outside, is a "colloidal ion."

(el By electrolytic dissociation of whole molecules, with aggregation of one or other kind of ions resulting therefrom, the aggregated ion having a charge equal to the sum of those of its constituents. This happens in solutions of congo-red, as shown by my own work ; in soaps, according to McBain, who regards the colloidal aggregates as consisting of anions, undissociated soap and water in various proportions. To these McBain gives the name "ionic micelle." The work of Sorenseu and others shows that proteins behave similarly.

We may now proceed to discuss some special cases. Space forbids any great detail and I must be content with reference to points which seem to need further elucidation and to some in which my own work has played a part.

Turning first to the phenomena of adsorption, about which a variety of oi)inions still exists. While some have doubtless erred in attempting to reduce all the phenomena included under this head to changes in surface tension, on the other hand, there are facts which cannot be explained on a purely chemical basis. One of these latter was met with by myself. If we mix a dialyzed solution of the free acid of congo-red with colloidal aluminium hydroxide, free from alkali, a blue complex is precipitated which contains both the acid and the base, but not in chemical combination. They combine, however, slowly at room temperature, quickly if heated, to form the salt, which has the characteristic red colour of the congo-red dyes.

The problem concerns the nature of the forces which cause the attachment of one substance to the surface of another, without chemical combination being of necessity involved. The familiar formula of Gibbs, which gives the degree of adsorption in relation to surface tension, is based on thermodynamical theory. Like all such deductions, it can predict in what direction and to what extent a change of energy' will occur, but it does not pretend to explain how this happens. In other words, we need in addition the assistance of molecuhar dynamics, and I think that it is wisest to admit that at present we have no adequate general theory of adsorption. In all probability, tlie medianism is of different nature in different cases. AViiile diminution of ordinary mechanical surface tension plays a part in the adsorption of some organic compounds by cliarcoal, for example, in the case of the congo-red acid and aluminium hydroxide, above mentioned, where we have to deal with electrically charged surfaces of oi)posite sign, there is no doubt that electrical forces are chiefly responsible. It is beside the jtoint to urge the objection made by Matthews and to


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exclude electrical adsorption on the ground tliat the combination of sodium and chlorine ions to form sodium chloride would be such a case. The fundamental difference is that in adsorption the chemical nature of the substances concerned is unchanged, whereas when ions unite ill a true chemical way the properties are greatly altered. I may be allowed here to express a feeling that when chemists claim tlie numerous cases of association in which no change of properties occurs as being chemical in nature, they run some risk of obscuring the distinctive character of chemical ]>henomena.

Laiigmnir's view of adsoriition is sometimes stated to be a purely chemical one. Although, in the case of the taking up of ions by the surface of some crystals, it is intelligible that unsatisfied chemical allinities of atoms in the boundary of the space lattice may cause the deposition of other appropriate atoms, it is not so easy to see how a fatty acid is combined with the surface of charcoal or carborundum. Moreover, as Bancroft points out, if adsorbed chlorine is in combination with the surface atoms of charcoal, it is difficult to understand why it is not an easy matter to obtain compounds of chlorine and carbon by direct union. On the other hand, since the carboxyl groups of acids have a greater affinity for water than the hydrocarbon residue has, it is easy to see why, after deposition on a surface, they are arranged with the former groups projecting into the water phase. But why the acids are in greater concentration at the interface than in the body of the solution is not explained hereby. It may further be remarked that Langmuir's view of adsorption implies that an adsorbed layer can never be more than one molecule in thickness. This is doubtless often the case, but there is also evidence that in some instances the amount adsorbed is in excess of that possible in a layer of the dimensions given. It appears as if the attractive forces extended their action somewhat further outwards than to the depth of one molecule. But such cases need careful investigation and whatever the truth may be, it must not be overlooked, as is sometimes done, that surfaces become saturated, sometimes after taking up only a very small amount of material. The well-known formula of Freundlich, indeed, which takes no account of this fact, applies only to a limited region of concentration. The existence of saturation, therefore, is not evidence that any particular case is not one of adsorption. Although it seems to me that Langmuir's view does not account fully for the facts of surface condensation, there is no doubt that, after orientation on the surface, chemical combination with the material of this surface or between different kinds of adsorbed molecules may occur. This is an important point in relation to the action of enzymes. It will be seen that appropriate orientation may facilitate mutual combination, or oppose it. Cases of the latter kind have been described by Van Kruyt.


To return for a moment to the phenomena of electrical adsorption. Michaelis and Bona have recently stated that the staining of paper by .so-called "basic" dyes, that is, by salts which consist of a colourless acid in combination with a coloured base, is due entirely to chemical combination with salts contained in the paper. Although this statement is accepted by Prof. Matthews in his interesting article in Phi/siological Reviews, it is so directly in opposition to the work which I published some years ago in the Biocheinical Jourual and with experiments which are regularly made in my classes, that it is a matter of astonishment to me how skilful experimenters could have arrived at it. Since the actual facts are of interest in illustration of the phenomena of electrical adsorption, I may give a brief account of them. If filter paper is dyed with an acidic dj'e, such as cougo-red, the purer the l)aper, the less is the dye adsorbed. On the other hand, with a basic dye, the purer the paper, that is, the smaller the amount of inorganic salt present, the deeper is the colour. A small addition of a neutral salt, even sodium chloride, has a powerful effect in increasing the depth of stain with the acidic dye, of decreasing it with the basic dye. This effect of electrolytes increases greatly with the valency of the cation. The explanation of these results is simple. Paper immer.sed in water has a negative charge, as shown by Quincke. The acidic dyes, if colloidal, have also negative charges and in any case the coloured anion is negative. I'aper and dye are mutually repellent. Or, looked at from the point of view of energetics, if negative dye were deposited on negative paper, the magnitude of the charge would increase, which is contrary to the second law of thermodynamics. If the dye is positive, as the coloured cation of the basic dyes is, then mutual attraction between it and the negative paper occurs. Now suppose that a soluble inorganic cation, such as sodium, with a positive charge, is present. This will be deposited on the paper, partially neutralizing or abolishing its negative charge; the negative dye will be more easily adsorbed, the positive dye less so. There are various other phenomena which confirm this way of interpreting the facts, amongst others, the effect of inert liquids such as alcohol, which, by lowering the dielectric constant, lower the charge on the paper.

We may pass next to one or two problems concerning the large subject of colloids.

In all questions in which these substances play a part we must never forget that we have to deal with suspended particles of comparatively large size and therefore possessing interfaces of contact with the medium in which they are suspended, on which surface action of various kinds may occur.

The proteins are amongst the most important of the colloids. These, as composed of aminoacids, are "amphoteric" in nature, that is, they can combine with either strong acids or bases. At and near a certain small


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hydiogeu-iou concentration, called the "isoelectric point," they are free from capability of combination with either acid or base. Their physical properties, viscosity, osmotic pressure, electrical conductivity and so on, have minimal values in this region. So far as they owe their charge to electrolytic dissociation of their salts, they have none at the isoelectric point. A difficulty arises here if a charge can be conferred in any other way than by dissociation, say by adsorption of ions. In fact, as shown by Loeb, gelatin at the "isoelectric point" can be given a positive charge by cerium salts. What does the term "isoelectric point" mean here? It is a pure assumption to say that there is a "loose combination" between gelatin and cerium ions as between it and hydrogen-ions. In point of fact, although the term has a fairly definite meaning when used in relation to amphoteric compounds and hydrogen ions, there is a tendency to make it serve in cases where the state of affairs is altogether different. It was first introduced by Hardy in reference to the discharge and i)recipitation of arsenious sulphide by barium salts. Its application to the hydrogen ion alone is of later date. It must always be stated what ion is meant, since colloids are made isoelectric by various ions.

We have seen already that ions are adsorbed by surfaces and I'errin showed that the negative charge of cellulose could be nearly abolished by *V20 liydrochloric acid. Now in the case of proteins as amphoteric electrolytes, the hydrogen-ion concentration needed to make them isoelectric is a function of their relative strengths as acids and bases. If the isoelectric point of cellulose is to be interpreted in the same way, it would imply strongly acid properties. We must take into consideration the adsorption of hydrogen-ions, and the isoelectric point in this case means the concentration needed to abolish the negative charge which cellulose has in water, whatever may be its origin.

More objection nuist be taken to the use of the term in relation to the red blood corpuscles where the charge is due to the impermeability of the membrane for cations, while being permeable to anions. This will be considered in my next lecture. It is sufficient to point out here that this same property is responsible for the "Hamburger effect" of acids added to blood as increasing the apparent alkali reserve of the plasma.

It is a difficult matter in practice to find evidence of combination of the serum proteins with acid or alkali unless the hydrogen ion concentration is above 10 "' or below lO'** respectively. It was suggested by G-ustav Mann that uncombined proteins really exist in the form of internal ammonium salts, requiring a fairly high hydrogen or hydroxyl ion concentration to split the ring. But curves expressing the amount of combination with acid and base are very flat about the isoelectric region, so that it is not an easy matter to be certain on this matter. At all events, there is no appreciable combin


ation between the limits named. In a series of experiments which I have made recently, proteins did not appear to behave towards sodium bicarbonate as such strong acids as their dissociation constants would imply. But there is considerable uncertainty as to their actual molecular weight. In the case of gelatin, however, all the acidic groups appear to be much more equally dissociated than is the case with ordinary di- or tri-basic acids.

A property of importance in regard to colloids in general is that it is practically certain that no single molecule or ion is sufficiently large to satisfy the criterion of not diffusing through parchment paper. At the isoelectric point Sorenseu has shown that the Donnan eciuilibrium is unable to explain the low osmotic pressure of egg white and that there must be considerable aggregation. I have already referred to the cases of congo-red and soaps, in which the osmotic pressure in relation to the electrical conductivity is too low to be accounted for otherwise than on the assumption that the anions form aggregates. So that in any case either the undissociated colloid or its ions are aggregated. Now these aggregates must be large enough to possess surface properties, amongst others that of an electric charge of origin other than electrolytic dissociation of the molecules of the colloid themselves. In other words, a purely chemical theory cannot be the whole story, however satisfactorily an explanation of certain facts is afforded.

In many ways, the most interesting and important of colloidal substances met with in the animal organism is haemoglobin, and the various considerations which have been previously given have their application to it. The investigation is beset with many difficulties and the more work is devoted to it, the more seems to be necessary. 1 venture to think that at the present time too much attention is being given to the elaboration of details, intei'preted on a limited theory, while there is yet uncertainty about many of the fundamental facts. For example, the isoelectric point as regards hydrogen ions is stated to be on the acid side of the reaction of blood, so that the question arises as to how far the jireparations used were, wholly or in part, sodium salts. This possibility raises difficulties in deductions from the osmotic pressure measurements of Hiifner, which , more recent determinations suggest were high for free hiemoglobin. The question is of moment also in connection with the carriage of carbon dioxide bj' blood. Bohr stated that the preparations of haMuoglobin on which he determined the carbon dioxide dissociation curve were free from sodium. Again, according to the acidic dissociation constant of ha-moglobin, compared with that of carbonic acid, it would seem in any case that as much as 95 per cent of the available sodium is in combination with carbon dioxide. If this be so, theories of carbon dioxide carriage on the basis of interchange of sodium have little


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support. But the actual molecular concentration of the acidic groups is uncertain. Perhaps the most pressing need is a method of preparing uniform samples of htemoglobin, of known composition.

It will be remembered that the well-known equation of A. V. Hill was first put forward by him simply as a mathematical formula to express the experimental results obtained by Barcroft and his co-workers on the oxygen taken up by htemoglobin at different pressures. A definite interpretation was soon, however, put upon the two constants. While expressing the greatest appreciation of the valuable collection of facts, experimental and clinical, made under the inspiration of this view, I must confess to an uncomfortable feeling that it may turn out that little more than the facts themselves stand future investigation. For example, in expressing by Hill's formula the oxygen dissociation curve as it is in the blood, it is found that the exponent, n, has to be made of a value between 2 and 3. If therefore it expresses the number of molecules taking part in the reaction, or the order of the mass action equation of velocity, it must be that there are a number of reactions of different molecular order in certain relative proportions. Now I am told by a friend who has looked at the problem from the mathematical standpoint that it is not admissible to take the index by itself and find the relative proportions of the various reactions required to make it of the value found (about 2.-4). The equations themselves must be summated. This is a tiresome piece of algebra and seem to require the introduction of further constants to give determinate results. Moreover, it is remarkable that no direct evidence of the existence of such small aggregates in definite proportion has yet been obtained.

In respect to the numerous clinical estimations which have been interpreted on the lines of the Barcroft-Hill equation, the recent work of Lovatt Evans has shown that there is a rapid development of lactic acid in shed blood. This arises from the glucose and is prevented by the addition of 0.1 per cent of sodium fluoride. Since the pre.sence of this acid has a marked effect on the dissociation curve of oxy-ha»moglobin, and also on the alkali reserve, some doubt is thrown on results obtained more than fifteen minutes after removal, unless sodium fluoride had been added.

It is a common experience to find that if acidified hiemoglobin solutions are dialyzed, with the object of obtaining freedom from sodium, methii'moglobin is formed with some rapidity. If 1 per cent of boric acid be added, it was found by A. V. Hill that this change is prevented and the htemoglobin remains normal. Boric acid is a fairly efficient antiseptic against bacteria, but does not prevent the growth of moulds. The whole question of the relation of methiemoglobin to oxyhaemoglobin would repay further investigation, especially in connection with the manner of combination of oxygen.


Perhaps the former is the real oxygen compound, the latter something of another kind.

The validity of the phase rule as applied to colloids has its interest in the case of hsemoglobin. If it does apply, oxyhaemoglobin does not satisfy the requirements of a chemical compound. But it is to be remembered that Gibbs excluded from his treatment all factors except jiressure, volume and concentration. Since surface energj^ and electrical forces intervene in the case of colloids, the matter is as yet undecided. Experiments might be made with saturated solutions of ha?moglobiu in presence of a definite solid phase, say crystals, in which case the phase rule should hold. It is interesting to note that Cohn obtains evidence of a definite slight true solubility in the case of some proteins.

On the whole, it seems to me that the problem of ha?moglobin might be advanced if it were attacked from a quite independent point of view, taking account of possible effects of surface and without prejudice by previous theories. It is remarkable that the more careful the work with this substance, the more inexplicable it appears. As an illustration, I might refer to the recent researches of Adolph and Henderson on the heat of "combination" of oxygen and haemoglobin, in which values varying from 1500 to 15,000 were given by a method capable of an accuracy of 1 per cent.

A few remarks are next suggested with respect to enzymes. Here there is no doubt about the fact that their action is exerted at their surfaces. The phenomena are indeed particular cases of catalysis in heterogeneous systems. It is easy to show that many enzj^mes are active in media, such as alcohol of moderate strength, in which they are quite insoluble and can be filtered off. This is especially simple with urease; powdered soy beans can be extracted with 70 per cent alcohol, the filtrate is inactive, while the residue suspended in alcohol of the same strength hydrolyses urea with rapidity.

It is evident also that adsorption of the reacting substances takes place on the surface of the enzyme phase and that a rapid equilibrium, brought about by some agency or other, is reached. Although the view that there is temporary formation of unstable intermediate compounds between the enzyme and the substrate on which it acts is a favourite one, it must be pointed out that the existence of such compounds, other than mere adsorption, has not been demonstrated. Whether close approximation between the reacting molecules is sufficient, as .seems to have been the view of Faraday in the case of the combination between oxygen and hydrogen on the surface of platinum and other substances, or whether molecular forces brought into action raise the chemical potential, as suggested by Hardy; or again, whether the increased concentration raises the rate of reaction by mass action, remains as yet undecided. There is some difficulty in accepting the last view, if it be


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supposed that the reaction occurs after adsorption because the molecules are fixed in position and the effect of mass is of course due to the movement of molecules. It may be, however, that the increase in rate of reaction occurs in the layer of concentrated solution close to the surface immediately before adsorption. The view of Langmuir that the reacting groups are brought into favourable position, for reaction with one another, by appropriate orientation on the surface, has already been mentioned. It must not be overlooked that acceptance of this view excludes the application of mass action lormuliT' and the existence of intermediate chemical compounds with the enzyme as explaining the phenomena. But these facts by no means put it out of court. With regard to the former, it is sometimes found that reactions in systems known to be heterogeneous obey, for a part of their course, a simple unimolecular formula. But surely this throws very little, if any, light on the nature of the process; it does not prove that it is a simple chemical reaction, any more than the increase of money at coml)ound interest is. In many processes of the most varied kind, tlie rate of further change is projiortional to what is left unchanged.

If the activity of an enzyme is proportional to the amount of the substrate adsorbed on its surface, it follows that if other substances are present which are themselves also adsorbed, a part of the surface is unavailable for the substrate itself and the rate of reaction will bo slowed. I have investigated this in the cases of saponin and of amj'l alcohol in their retarding effect on the rate of reaction in urea.se. It is of interest to find that the effect on the enzyme is merely to slow the rate of action, which ultimately results in the same total change. That the explanation as a case of competing adsorption is correct is confirmed by the fact that the effect has a negative temperature coefficient ; that is, it is less at liigher temperatures, a characteristic of adsorption phenomena.

The regarding of enzymes as being in tlie colloidal state suggests a way of explaining many of the effects of the addition of foreign substances, of heat and so on. These may change the degree of dispersion. If the particles become larger by aggregation, the active surface of a given mass is decreased and vice versa. Enzymes are usually destroyed by boiling, but sometimes not. It ai)pears from the work of Chick and Martin on proteins tliat in the coagulation process the particles may sometimes merely stick loosely together and that they can then be redispersed by conferring an electric charge. In other cases, they fuse together to form a large single particle and redispersion is impossible.

Since the effect of an enzyme as catalyst is to accelerate the attainment of the equilibrium position when the reaction is a reversible one, it is clear that its effect may be either of a hvdrolytic or svnthetic nature according


to the conditions pre.sent. I have been unable to discover any evidence of the existence of an enzyme only able to effect a synthetic change, although statements of the kind have been made. The direction of the result produced by an enzyme in those cases where the action depends on the addition or removal of the elements of water, and these are the majority, is controlled by the concentration of water in the system. It is evident that in those frequent cases where the living cell exerts alternatively hydrolytic and synthetic action, say in the removal and storage of glycogen in the liver, there must be some mechanism by which the amount of free water can be changed as required. We naturally think of those colloids called emulsoids, whose particles contain different quantities of water according to the nature and concentration of the medium surrounding them. But there is also the possibility of adsoiqjtion of water on the surface. Probably both phenomena take place in the process of imbibition, the nature of which is not yet clear.

In connection with the equilibrium reached on the enzyme surface, there is another interesting problem awaiting investigation. If the constituents of a system are not adsorbed in the same proportion in which they exist in the natural equilibrium, it would be expected that the position of equilibrium under the action of surface catalysis would not be the same as the natural one or that produced by acid. In point of fact, certain cases have been reported where a difference of this kind exists, but whether it can be explained by differences of adsorption is unknown. I have been unable to find any change in the ethyl-acetate system on the addition of dried charcoal.

As a further case of adsorption, at all events as it appears to me, the cell-membrane or plasma-mendirane may be considered. This is not to be regai'ded as a fixed permanent structure, but as produced by deposition of cell-constituents which lower surface energy at the interface between protoplasm and surrounding medium. Thus, it changes with cell activity and is in equilibrium with the cell contents as they alter. Thus, there is no difficulty in the membrane becoming permeable in the active state of the cell to substances to which it is impermeable in rest. Moreover, when a fresh protoj)lasniic surface is ])roduced by mechanical action, a new membrane is naturally dejjosited on it. This is no doubt why large l)articles can be taken up in phagocytosis through a membrane which does not permit even sodium ddoride in solution to jtass. The particles actually break the membrane, which closes again behind them, in the same way as a needle can be passed through a soap film without bursting it, whereas a gas, such as hydrogen, nearly insoluble in tlie soap solution, only passes with extreme slowness.

It is not unlikely that a process akin to deposition of solid mav occur in the surface mend)rane, similar to


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that described by Eamsden with saponin, proteins and other substances. To account for its varying permeability, the structure suggested by the work of Clowes on phase reversal under the action of electrolytes seems to offer possibilities. A system of oil globules suspended in a watery protein solution can be converted into one in which drops of the watery solution are suspended in oil. It will be clear that a substance soluble in water but not in oil would be able to pass through the continuous watery phase of the former, but unable to pass in the latter case, since the only continuous phase is oil.

Finally, a word or two as to protoplasm itself. In its simplest form as in the clear pseudopodia of amoeboid cells, it is a liquid colloidal system or "sol." That it is colloidal is shown by the appearance of an immense number of particles under brilliant dark-ground illumination and that it is liquid is shown by the fact that


these particles are in Brownian movement. Under certain states of activity, it may become more jelly-like, its viscosity rises and the particles become fixed in a structure of some kind. I propose to refer to this matter again in the next lecture and for the present will merely call attention to the absence of any visible sign of structure whether of the nature of network, foam or otherwise. The production of such appearances by the action of fixing reagents was thoroughly investigated some time ago by Hardy, whose work has been rather unduly neglected by histologists. This action of fixing reagents is particularly striking in the case of the cellbody of the neurone, where Mott and Marinesco, independently, showed that there are merely granules or particles in Brownian movement in the living state. The Nissl bodies and neurofibrils are to be seen only after fixation, although of course the material out of which they are formed must be present in life.


FACTORS WHICH DETERMINE THE CONCENTRATION OF CALCIUM

AND OF INORGANIC PHOSPHORUS IN THE BLOOD

SERUM OF RATS


By Bexj. Kramer and John Howland


There have been a number of experiments upon animals, in the past, with diets deficient in calcium. Most of these have been made in an attempt to produce rickets. The chief investigators, in this period, have been Voit,' Dibbelt,- Aron and Scbauer,^ Stcelzner and Miwa,* and Mellanby. The bones of the animals have been examined. In many instances the diet has been carefully controlled so far as the amount of calcium was concerned but, of the authors mentioned above, only Mellanby has paid attention to the other factors, such as the fat, in the diet. Mellanby has reported no determinations upon the blood. Aron and Sebauer fed to a dog a diet containing only .07 per cent calcium which in the light of our studies, assuming that the dog reacts to a deficency of calcium in the diet as does the rat, should have produced a distinct lowering of the calcium in the blood. They found G.5 mg. Ca per 100 gnis. of whole blood, which is within normal limits. The diet, however, contained a large amount of beef fat (C per cent) which, in the light of present knowledge, may account for the noraial calcium concentration of the blood.

Experiments with diets deficient in phosphorus have also been maile, chiefly by Schmorl " and W. Heubner.' Neither reports analyses of the blood or serum. Within the past few years numerous attempts have been made to increase the calcium concentration of the blood by feeding or injecting calcium salts in large amounts. Among the investigators have been Heubner and Rona,' Denis and Minot," von Fenvvessv and Freund '" and


Clark. Analyses of the blood and serum for calcium have been made by various methods. The consensus of opinion is that while it is possible to increase the calcium concentration by the injection of large amounts of calcium, this increase is very temporary' and there is a strong tendency for the concentration to be maintained within nonnal limits. The normal calcium concentration cannot be exceeded even when large amounts of calcium are fed.

Injections of solutions of inorganic phosphate have been made and a temporary increase of serum phosphorus demonstrated (Greenwald," Iversen '=>), but there are no experiments, so far as we are aware, to show any permanent influence upon the inorganic phosphate of the blood or serum resulting from the injection or feeding of phosphorus in any form in excess.

We have attempted to study the factors that control the concentration of calcium and inorganic phosphorus in the serum of rats. The experiments of Dr. E. V. McColluni and of Miss Nina Simmonds, iu which the various dietary ingredients have been altered one at a time, have enabled us to do this in a satisfactory manner. We are greatly indebted to Dr. McColluni and Miss Sinimonds for the opportunity of making these studies and for the great assistance that they have aft'orded us, as well as for their entliusiastic cooperation.

Tlie animals were fed and housed in the Department of Chemical Hygiene and are the same animals whose bones and tissues have been studied in the collective


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I


investigation of rickets carried on by the Departments of Ciiemical Hygiene and Pediatrics.

There are two chief types of experiments. (1) Those in which animals were fed continnously upon the same diet, sometimes for a number of generations and (2) those in which the animals, after being on diets that were known to produce definite changes, were subjected to alterations in the shape of additions to the diet or to some physical influence.

In general the diets were made up so as to contain an excess of, or to be deficient in, some biologically important ingredient. To these diets were added various substances whose influence upon the concentration of Ca or P it was desired to study. By feeding several successive generations of animals upon the same diet it was possible to bring out, in some instances, deficiencies which were not readily apparent in the earlier generations. Some diets were so defective as to prevent reproduction. Others did not allow the animals to survive for even a reasonable length of time.

The ajjpearance and behavior of the animals, the morphological changes in their tissues, especially the bones, have been or will be referred to by others. Our interest has been focused chiefly upon the alterations in the concentrations of Ca and inorganic P of the serum of these animals.

In order to establish normal values for the concentrations of Oa and Inorganic P in the serum we have made determinations of these substances on a large number of rats fed upon diets which allow normal growth through at least four generations and maximum longevity. AYe believe the normal to be for Ca 9.5 and 10..5 and for Inorganic P 7-8.5 mg. per 100 cc. serum.

There were two chief basal diets. The one was composed of wheat 30, maize 20, rice 10, rolled oats 10, peas 10, Xavy beans 10, washed casein 10, the other of wheat 33, maize 33, gelatin 15, wheat gluten 15, NaCl 1, Ca Co 3 3.* The first diet was deficient in calcium, the second in phosphorus. Each had sufficient anti-xerophthalmic factor but there was not enough fat of good quality to support normal growth. The proteins of each diet were satisfactory. When the additions (usually small) were made to these diets the proportion of some of the ingredients was necessarily somewhat reduced but not enough to disturb essentially the character of the diet.

The additions were designed either to restore to the diet the deficiency of calcium or phosphorus or to test the effect of certain agents, themselves not containing calcium or phosphorus, upon the calcium and inorganic phosphorus concentration of the serum.

The animals were studied in groups. There were control animals for every experiment usually from the same litter. Our results chiefly represent averages, because the animals of one group were sacrificed at the same


These figures represent gms. per 100 gms. of diet.


time and the blood pooled. Occasionally, if the animals were large enough, estimations were made from the serum of one animal and compared with the pooled serum. The blood was obtained by cutting the carotid arteries and jugular veins under complete anesthesia. The blood after collection was centrifuged at once and the serum removed. The serum was not allowed to stand in contact with the clot.

The calcium was determined by the method of Kramer and Tisdall " and the inorganic phosphorus by that of Marriott and Hsessler.'^ The methods were frequently controlled by determinations on solutions containing known quantities of the respective element and bj' analyses of normal serum.


Serial


Di


et


Addition


Se^um




Ca


P


Ca


p



633


.64


.40


B.F. 5


10.0


8.4


3rd


630


.65


.45


C.L..O. 2


9.8


8.0


1st


694


.05


.45



5.6



1st


696


.05


.45



5.9



1st


761


.05


.45


C.L..O.


5.5


8.0


1st


639


.18


.45



7.5


8.0


2nd


618


1.22


.30



10.5 6.8


2.4 8.0


1st


683


.09


.45


C.L.O. 2


3rd


656


.09


.45


C.L.O. 2


8.8


7.0


2nd


684


.13


.45


C.L.O. 2


10.2


7.8


2nd


666


.17


.45


C.L.O. 2


9.0


8.8


2nd


636


.17


.45


C.L.O. 2


9.4


8.0


2nd


635


.21


.45


C.L.O. 2


9.3


8.0


3rd


657


.21


.45


C.L.O. 2


9.1


8.2


4 th


634


.25


.45


C.L.O. 2


9.7


8.2


3rd


658


.25


.45


C.L.O. 2


9.7


8.0


3rd


630


.65


.45


C.L.O. 2


9.8


8.0


1st


647


.65


.45


C.L.O. 4


10.5



2nd


652


.65


.45


C.L.O. 5


10.0


8.8


2nd


629


.65


.45


C.L.O. 5


9.8


8.2


2nd


651


.65


.45


C.L.O. 5


10.0


8.0


2nd


648


.65


.45


C.L.O. 7


10.2



2nd


639


.18


.45


C.L.O.


7.5


8.0


2nd


638


.18


.45


C.L.O. 1


9.6


8.0


2nd


707


.09


.42


S.L.O. 3


9.8


8.4


2nd


C.L.O.— Cod-liver oil.





S.L.O.— Shark-liver oil.





B.F.— Butter fat.





Figures for Ca and P in diet


xpressed as gms. pe


100 gm


. of d


Ca and P in serum expressed


as mgs. per 100 c.c.


of serun



In the second and third columns of the table are given the amounts of calcium and phosphorus in 100 gms. of the diet; in other words, the percentage composition as regards these ingredients. Column i states the nature and amounts of the substances added to the basal diet. In the fifth and sixth columns are given the concentrations of the calcium and inorganic phosphorus expressed in the terms of milligrams per 100 cc. of serum.

It will be seen that if the diet contains calcium about .6 per cent and phosphorus .4 - .5 per cent, and if the ani


September, 1922]


315


mal receives butter fat 5 per cent or cod-liver oil, 2 per cent, normal values for Ca and inorganic P are obtained in the serum even for several generations. If the phosphorus is maintained at the same level but the calcium reduced to .05 per cent, and if no fat is added, there is a marked decrease in the calcium of the serum, the phosphorus renmining normal. An increase in the calcium of the diet raises the calcium of the serum even with out the addition of any organic factor.

A most striking effect upon the calcium results from cod-liver oil administration. Even with so low a calcium intake as .0\)fc, cod-liver oil, 2 per cent, added to the diet, raises the calcium of the serum to 8.8 in the second and to G.8 in the third generation. Such a diet permits reproduction for at least two generations. As the calcium intake rises there is, in general, an increase in the calcium of the serum and when the diet contains only 0.21% calcium, the calcium of the serum is maintained at a nearly normal level for four generations. When the calcium of the diet reaches a certain concentration the addition of more calcium, with or without cod-liver oil, even in large amount does not further raise the calcium in the senim. This is shown in Table I and also in other tables. Determinations 039 and 638 in Table I illustrate several facts. A diet slightly deficient in calcium, containing no added fat but a normal phosphorus concentration, can bring about reproduction at least of one generation. That the diet is defective is indicated by its inability to maintain a normal calcium concentration. When to this diet so little as 1 per cent cod-liver oil is added a normal calcium concentration of the senim is found even in the second generation.

Determination 707 shows that shark's-liver oil is also very effective in nmintaining a normal calcium content in the serum despite a low calcium content of the food.

TABLE II.


Serial


Di


et


Addition


Ser


un.




c« 


P


Ol


P



761


.05


.45



5.5


8.0


1st


708


.05


.45


C.L.O. 2%

for 6 days


6.5


8.0


1st


762


.05


.45


C.L.O. 2 for 14 days


8.2


7.0


1st


Table II shows the influence of cod-liver oil upon the calcium concentration of the serum of animals receiving very small amounts of calcium. The animals were placed upon the diet without cod-liver oil when about twentyfive days old. They had been on the diet twenty-one days when the oil was added. No 761 represents the control animals of the same age. Cod-liver oil shows its influence in six days and in fourteen days the calcium of the serum has reached nearly normal limits.

A number of facts are illustrated in Table III.


Serial


Di


e.


Addition


Ser


um




Ca


P


Ca


P



649


.05

.05


.45 —45

C.L.O. 3

"c.LTors"


9.7



2nd


650


6.1



3rd


664


.13


.45


B.P. 8


4.4


8.0


3rd


624


.25


.45


B.F. 3


10.0


8.0


3rd


627


.21


.45


B.P. 8


9.4


8.0


4th


684


.13


.45


C.L.O. 2


10.2


7.8


2nd


656


.09


.45


C.L.O. 2


8.8


7.0


2nd


683


.09


.45


C.L.O. 2


6.8


8.0


3rd


635


.21


.45


C.L.O. 2


9.3


8.0


3rd


657


.21


.45


C.L.O. 2


9.1


8.2


4th


Determinations 649 and 650, 656 and 683 show the influence of generations. In both sets the phosphorus of the diet was adequate; the calcium was very low. Codliver oil was added to the diet. This was effective in maintaining the calcium of the serum at a normal or nearly normal level in the second generation but was unable to accomplish this in the third. Determinations 635 and 657 show tliat when the calcium of the diet reaches about .2 per cent and cod-liver oil is given, the calcium is maintained at a normal level even throughout the fourth generation. Determinations 661 and 627 show that a very slight increase of calcium has a profound influence on the calcium of the serum so that, when the calcium of the diet is increased only .08 per cent, the amount of butter in the diet remaining the same, the calcium concentration of the serum is maintained at a normal level even in the fourth generation, while without this increa.se the calcium concentration is less than half the normal even in the third generation. When the calcium of the diet is increased to .25 per cent, the calcium of the sei-um is maintained at a normal level in the third generation in spite of a reduction of butter fat to 3 per cent.

That cod-liver oil has a far greater effect upon the calcium of the serum than butter fat is shown by Nos. 650 and 661. In spite of the fact that one diet contains .08 per cent more calcium than the other and 8 per cent butter fat, the diet with 3 per cent cod-liver oil maintains the calcium of the serum at a much higher level than does the diet containing butter fat.

All the animals represented in Table IV were fed on a diet high in calcium, low in phosphorus and containing just enough anti-xerophthalmic substance to protect the eyes. The animals were placed on the diet at about forty days of age. At this time they usually weighed about 50 grams. They were fed on the diet for from twenty-five to forty days depending upon the condition of the animal. At the end of this time some of the control animals were sacrificed. To the diet of others, substances were added whose influence it was desired to investigate. A few animals were given water but no food for a period of three days and were then sacrificed.


U6


[No. 379


Serial


Di


e t


Addition


Scrum




Oi


P


Ca


p



618


1.22


.3



10.5


2.4


1st


660


1.22


.3



10.0


2.8


1st


749


1.22


.3



9.8


2.8


1st


675


1.22


.3




3.0


1st


822


1.22


.3



10.2


2.0


1st


668


1.22


.3


C.L.O. 2% 5 days


10.3


5.7


1st


736


1.22


.3


B.O. 2% 6 days


~


3.8


1st


745


1.22


.3


B.O. 2% 10 days


~


4.5


1st


768


1.22


.3


S.L.O. 2% 10 days


~


6.0


1st


769


1.22


.3


M.O. 20% 15 days


10.1


2.7


1st


770


1.22



O.O. 20% 15 days


9.9


2.4


1st


653


1.22


.3


B.F. 30% 10 days


10.2


4.4


1st


640


1.22


.3


B.F. 30% 14 days


~


7.0


1st


673


1.22


.3


B.F. 15% 12 days


~


4.8


1st


803


1.22


.27



9.4


2.0


1st


807


1.22


.32



10.0


2.0


1st


808


1.22


.30




2.6


1st


809


1.5


.3



10.0


1.8 1



674


1.22


.3


Starvation 3 days


"


8.6


~


B.O.— Burbot-liver oil. M.O.— MaiM oil. O.O.— Olive oil.


Upon this basal diet, with great uniformity, the calcium coucentratiou of serum is maintained at a normal level, while the inorganic phosphorus varies betweeu 2 and 3 milligrams. Fii-st generation animals alone are included in the table because reproduction upon this diet is impossible.

All the fish-liver oils employed and these include those from the cod, shark, and burbot (freshwater) raise the inorganic 1* concentration of the serum. This effect is the more pronounced the longer the period of admiuistratioii. The fact that normal figures were not reached was probably due to the short period of administration. The vegetable oils (maize and olive), even when administered in large amount, have no demonstrable effect. Butter fat occupies an intermediary position between the liver and vegetable oils. It has a distinct effect upon the phosphorus but it must be offered in larger amounts and for a considerable period of time.

When food is withheld for three days a rapid and remarkable increase of the inorganic phosphorus takes place. The inorganic phosphorus concentration of the serum rapidly reaches the normal level.


z


Diet


C 5


Serum


1


Radialioos from


Ca 1 P


Ca i P


u


749


1.22


.3



9.8


2.8


1st



663


1.22


.3



10.3


3.5


1st


U. V. Ferric chromium condenser spark.


646


1.22


.3




4.5


1st


U. V. Ferric chromium spark.


662


1.22


.3



10.2


5.4


1st


U. V. Cadmium spark.


669


1.22


.3



10.2


5.7


1st


U. V. Cadmium spark.


671


1.22


.3




2.7


1st


Cadmium spark filtered through glass.


716


1.22


.3



10.4


4.1 1st


U. V. Mercury vapor quartz lamp.


733 1.22|.3


9.6|3.2|lst


Ozone.


Table V sliows the effect of certain physical agents upon the inorganic phosphorus of the serum. The diet was the same as that given to the animals represented in Table IV, i.e., one which contained an excess of calcium and a deficit of P, so that while the calcium of the serum was at a normal level the inorganic phosphorus of the serum was invariably low. The animals were given the basal diet, beginning at about four weeks of age, for a period of twenty-three to thirty-eight days.

They were then exposed to radiations emanating from various sources. Spectroscopic analysis of the rays was made in each instance to determine the presence or absence of the ultra-violet. The mercury vapor quartz lamp yielded rays which gave, among others, a band iu the region of the second ultra-violet. A similar but more intense band was obtained with the cadmium spark. The inter-position of a plain glass filter blocked the second ultra-violet rays while it allowed the passage of a part of the first ultra-violet. The ferric chromium condenser spark gives rays which are rich in the first ultra-violet but poor in the second ultra-violet.

It will be seen from the table that following radiation by the mercury vapor quartz lamp, by the cadmium spark and by the ferric chromium condenser spark there is a definite increase in the inorganic i)hosphorus of the serum. The only exception is when the rays are filtered through glass. Following this there is no demonstrable effect. The rays from the ferric chromium condenser spark are the least, tho.se from the cadmium spark the most effective; those from the mercury vapor quartz lamp occupy an intermediate position. In all of these tests the time of exposure varied from fifteen to thirty minutes. The animals received ten treatments. The distance from the source of light was six inches. As there is a rapid formation of ozone by the mercury vapor quartz lamp a group of animals have been exposed to ozone for the same period and frequency as when radiations were employed. One of the animals had refused food for several days. The slight increa.se of inorganic phosphorus was probably the result of pooling the serui!i


September, 1922]


SV(


of this starving animal with that of the others. The increase over that from the basal diet in any event was not significant.

TABLE VI.


Serial


Diet



Ser


um




Ca


p



Ca


p



749


1.22


.3



9.8


2.8


1st


782


1.22


.47


Cas. 20%


10.4


4.4


1st





for 10 days





681


1.22


1.21


CaH PO,

4% for 10 days


5.0


7.2


1st


842


.05


.45



4.4


8.0


1st


846


.05


.56


NaHj PO.

.425%


4.5


7.2


1st


843


.05


.66


NaH, PO. .85%


4.5


"


1st


847


.05


.87


NaH, PO. .1.7%


~


8.0


1st


841


.05


.95


NaHj PO. 2.1.%


4.2


~


1st


844


.05


1.06


NaH, PO. 2.5%


4.1


8.6


1st


In Table VI are shown results obtained with animals fed upon both types of basal diets. The diet of some animals was high in calcium and low in phosphorus. The phosphorus was increased by the addition of casein or of secondary calcium phosphate. Both of these substances raised the inorganic phosphorus of the serum, but whereas the casein had no effect upon the calcium, the secondary calcium phosphate reduced this by fifty per cent, in spite of the large amount of calcium iu the diet.

The second tliet was very low in calcium and contained an adequate amount of phosphorus. The control animals on this diet have regularly from 4 to 5 mgms. of calcium with a normal phosphorus concentration in the .serum. The addition of acid sodium phosphate to this diet in increasing quantity produced practically no effect ui)on the concentration of calcium or phosphorus. There was a slight tendency for the calcium to diminish and the phosi)horus to increase in the .serum with the larger additions.

Summary

A study has been made of the influence of various factors ui)on the concentration of calcium and inorganic phosphorus in the sei'um of rats. The concentration of neither element has been distinctly increased beyond normal limits, either as the result of various additions to the diet or the use of physical agents. It seems fair to assume that this cannot be done by either of the.se methods. On the other hand, it is possible to reduce the concentration of these elements in the serum by feeding diets containing an insufticient quantity of the respective elements.


In order to bring this about there must be only enough of the organic factor represented by cod-liver oil or butter fat to prevent xerophthalmia and allow moderate growth. Even with a very small amount of calcium or phosphorus in the diet, compensation occurs and tlie concentration of these elements in the serum approaches tlie normal level if sufficient of the organic factor is included in the diet. A defect in the diet is accentuated in succeeding generations, so that a diet which gives a normal value for the serum calcium in the second generation may give a distinctly low figure in the third generation. Of tlie fats that have been employed in these experiments the fat from the livers of fish has been mucli more effective than butter fat. The vegetable oils (olive and maize) have been without influence.

When the concentration of either element in the serum is low as a result of a deficiency in the diet, it may be increased by increasing the amount of tlie respective element in the food. Up to a certain point even small additions to the diet have a pronounced effect upon the serum. Further additions have no effect whatever if a normal concentration has been reached.

"Wlieu the diet is defective in phosphorus and the phosphorus of the serum is therefore low, a marked increase of the serum phosphorus may be produced by starvation, by the addition of phosphorus to the diet in organic or inorganic form, by various fats, and by exposure to radiations from various sources which yield, among others, rays whose wave lengths are less than 3000 Aengstrom, units.

The relation of these findings to rickets and to calcification in general will be discussed in a subsequent communication.

BIBLIOGRAPHY

1. Voit, E.: Ztschr. f. Biologie, 1880, XVI, 55.

2. Dibbelt, W.: Verhandlungen d. d. path. Gesellsch., 1909, XIII, 33.

3. Aron, H. and Ssbauer, R.: Biochem. Ztschr., 1908, Vlll, 1.

4. Stoeltzner and Mlwa: Beitr. z. path. Anat. u. z. allg. Path., 1898, XXIV. 578.

5. Mellanby, E.: Experimental Rickets, 1921. Special Report, Series No. 61, Medical Research Council, London.

6. Schmorl, G.: Arch, t Exp. Path. u. Pharmacologie, 1913, LXXIII, 313.

7. Heubner, W.: Arch. f. Exp. Path. u. Pharm., 1915, LXXVIII, 24.

8. Heubner, W. and Rona, P.: Biochem. Ztschr., 1919, XCIII, 187.

9. Denis, W. and Minot, A. S.: J. Biol. Chera., 1920, XLI, 357.

10. von Fenyvessy, B. and Freund, J.: Ztschr. f. Immunitat., u. s. w., 1913, XVIII, 666.

11. Clark, G. W.: J. Biol. Chem., 1920. XLIII. 89.

12. Green wald, I.: J. Pharm. and Exp. Therap., 1918, XI, 281.

13. Iversen, P.: Biochem. Ztschr., 1920. CIX, 211.

14. Kramer, B. and Tisdall, F. F.: J. Biol. Chem., 1921, XLVII, 475.

15. Marriott, W. McKim and Haessler, F. H.: J. Biol. Chem., 1917, XXXII, 233.


318


[No. 379


A STUDY OF THE RESULTS OBTAINED IN SIXTY-FOUR CAESAREAN

SECTIONS TERMINATED BY SUPRAVAGINAL

HYSTERECTOMY

By John W. Harris

(From the Department of Obstetrics, The Johns Hopkins Hospital

and University)


Eai'dley Hollaud ' has recently published a statistical study of 4197 Cajsarean sections which were done in Great Britain during the decade from 1911 to 1920, and which in a way formed a continuation of a similar study by Armand Kouth - for the pre^'ious decade. He stated that in the series there were forty-six Ciesarean sections which had been terminated by supravaginal hysterectomy with eight deaths, a percentage of 17.1. As this is far in excess of the mortality in our service, it seemed that it might be interesting to study the results which we have obtained and to compare them with those reported by Holland.

From the early days of our service it was noted that the convalescence following supravaginal amputation Avas more satisfactory and much smoother thau that following most conservative sections, with the result that we gradually came to employ the former operation whenever it seemed justiliable, more particularly in two classes of cases; — First, as the method of choice for effecting sterilization after repeated Ciesarean section, and secondly, as a means of obtaining more ideal results upon patients who were already infected or who were admitted to the service after being long in labor.

In the earlier days of the service many conservative Caesarean sections were done in the second stage or late in the first stage, with the idea that the patient should be given a test of labor in order to ascertain whether the head would or would not engage. We gradually learned that this was an erroneous practice and resulted in a relatively high mortality. After this we limited the performance of conservative Caesarean section practically to such patients as could be operated upon at an appointed time before the onset of labor or as soon as possible after the appearjiuce of labor pains; while in patients who were seen late in labor or who already presented signs of infection, we either did not do a Ciesarean at all or removed the uterus after its performance. Some idea of the improvement of the results which followed may be gained from the fact that in the first fifty Ciesarean sections done in the service the mortality was twelve per cent, while in the following 173 operations done up to May 15, 1922 it became reduced to 2.88 per cent.

In 223 Ciesarean sections which have been done in the obstetrical service of the Johns Hopkins Hospital up to May 15, 1922 the uterus was removed by supravaginal hysterectomy in 64 instances. For purposes of study we have divided our 64 cases into two main groups — the first


including all patients from whom the uterus was removed at the first Ciesarean section, and the second those from whom it was removed at the second or third section, as shown in Tables 1 and 2. The indications, which were discussed in detail in Dr. Williams' paper "A Critical Analysis of Twenty-one Years' Experience with Ciesarean Section,"^ covered our material up to the end of 1921.

TABLE I. Supravaginal Hysterectomy at First Caesarean Section.


Late in Labor or Manifest Infection. .

Sterilization

Heart Disease

Atresia of Cervix

Neglected Transverse Presentation...

Myoma of Cervix

Hour-glass Contraction of Uterus. . .

Apoplexy of Uterus

Failure of Bag or Bougie

Uncontrollable Hsemorrhage

Dystocia following Ventral Fixation.


Total .


10 5 5 5

4 3 2 2 2 2 1


TABLE n. Supravaginal Hysterectomy at Repeated Caesarean Section.


Late in Labor or Manifest Infection .

Sterilization

Tearing of Uterine Incision

Blocking of Outlet by Condyloma. .


Total.


Our figures show that in the sixty-four patients there were three deaths, a gross mortality of 4.68 per cent, which is, roughly speaking, only one quarter as great as that reported by Holland. As will be seen below, none of the deaths were due to infection and only one of them was associated directly with the operation. The latter occurred at a second section upon a multiparous patient with a generally contracted pelvis, from whom the uterus was I'emoved for the purpose of effecting sterilization. Owing to the inexperience of the operator difficulty was experienced in ligating the left uterine artery, with the result that death from hiemorrhage occurred on the operating table.

The other two deaths were not connected with the operation and occurred under the following circumstances. The first was in a seventeen-year-old primipara.


September, 1922]


319


who came into the service on account of acute ulcerative endocarditis with broken compensation. As she was steadily growing worse, and as it was felt that the abdominal enlargement was aggravating her condition, it was decided to empty the uterus. It appeared that this could be accomplished most conservatively by Ciesarean section, and as it was furthermoi'e felt that future pregnancies should be avoided, the uterus was removed by supravaginal hysterectomy. Death occurred on the eighth day from the cardiac condition. At autopsy no sign of wound infection was present, but an acute thromboendocarditis with perforation of the aortic valves was found.

The second patient was a multipara who had been under treatment for several months for hypertension not associated with albuminuria. At the eighth lunar month she began to bleed as the result of premature sei)aration of the normally imi)lanted placenta. A ^'orhees bag was introduced which controlled the bleeding, but failed to dilate the cervix. Teu hours later the patient became alarniingl_v worse witli rapidly increasing stupor, marked increase in the blood pressure and the presence of fifteen grams of albumin per liter in the urine, from which it previously had been absent. Under the circumstances it was felt that the only chance for recovery lay in immediate delivery and that this could be accomplished most satisfactorily by ('a>sarean section. Upon incising the uterus we found the placenta completely separated and lying free in the uterine cavity, which contained several hundred cubic centimeters of blood. Haemorrhage had also occurred into the walls of tiie uterus and had led to such disassociation of its muscular fibers that contraction could not occur. As the organ remained soft and flabby in spite of the administration of pituitary' extract and manual irritation, supravaginal hysterectomy was done. The patient died in coma shortly after its completion. Although an autopsy could not be obtained, it was apparent that death had not occurred from hemorrhage, as the total amount of blood lost, including that retained in the uterine cavity, did not exceed six hundred cubic centinietei-s. In the circumstances, therefore, it seems justifiable to attribute the death to the pre existing renal condition, which had become accentuated by the ])remature separation of the placenta.

These three deaths represent a mortality of 4.G8 per cent, but, as has been indicated, only 1.5G per cent could in any way be attributed to the operation. Such results are far superior to those reported by Holland and it might be interesting, but in view of the absence of sufficient data it would be useless, to speculate as to the cause of the deaths in his series. In any event, our figures indicate that those of Holland give a distorted picture of the dangers following supravaginal hysterectomy, and indicate that the results obtained in our hands are essentially as satisfactory as in elective conservative sections done at an appointed time before the onset of


labor or within the few hours following the api)earance of uterine contractions. This is particularly emphasized when we consider that in a considerable portion of our cases the condition of the patient when first seen was such as to suggest that infection had already occurred.

In this connection it is interesting to ascertain whether we have unnecessarily sacrificed the uterus and thereby ended the reproductive career of our patients. This can be answered only by studying our cases in somewhat closer detail, and the verdict will depend in great part upon one's point of view. As is evident from Tables 1 and 2, the uterus was removed from eighteen patients solely for the purpose of eft'ecting sterilization. Five of such operations were done at the first and thirteen at a subsequent section.

AVhether the removal of the uterus in such cases was indicated depends altogether u])on one's point of view; first, concerning the justifiability of sterilization in general, and secondly, upon whether it can best be accomj)lished by removing the uterus. We have always taken the view that hopelessly deformed or mentally defective patients from the lower classes should be sterilized, aud, furthermore, we have made it a rule to sterilize all I)atients at the third section, considering that in such circumstances the woman had done her duty by society. Accepting such an indication for sterilization, it must be admitted that it can be effected in one of two ways, either by doubly ligating the tubes and burying the proximal ends between the folds of the broad ligaments, or by removing the uterus. If the patient is intelligent, this decision is, in great part, left to her and she is asked whether or not she desires to menstruate after the operation. If slie replies in the affirmative, the tubes are ligated, but if in the negative, the uterus is amputated as the easiest and most satisfactory method of attaining the desired end. In addition to these eighteen cases, the uterus was removed from five other women suffering with serious heart disease, as it was felt that in their cases sterilization should follow the first Caesarean.

Thus we have twenty-three patients in whom the prime or secondary object of the hysterectomy was sterilization, leaving forty-one on whom it was performed for other indications. Eighteen of these were done frankly on account of the patient coming into the service already infected or late in labor. In addition to these, the uterus was removed on account of atresia of the cervix in five instances, neglected transverse presentation in four instances, hour-glass contraction of the uterus in two instances, failure of the bag or bougie to bring about labor in two other instances and dystocia following ventral fixation of the uterus in one instance — a total of thirty-two cases. With the exception of the cases of atresia of the cervix, all of these thirty-two patients were oi)erated upon late in labor and after they had beea examined frequently by the vagina, and sometimes sub


320


[No. 379


jected to unsuccessful attempts at deliverj- by other methods. In several of the cases of atresia of the cervix the patient had a high temperature at the time of admission, and it was felt that a satisfactory result could be obtained only by the removal of the organ. Adding these cases to those in which sterilization was the prime indication, we find that they aggregate fifty-five in number, leaving nine others in which the operation was done for some other cause.

The justification for removing the uterus in the former group of cases lies in two considerations; first, the only death which occurred was an operative accident, as compared with the 9.4 and 26.5 per cent mortality following conservative Caesarean section performed late in labor or after attempts at delivery, as shown by Holland's statistics. Furthermore, as shown in Table 3, sixty of the uteri amputated were subjected to histological study and in twenty-three of them definite histological evidence of ascending infection was found in the shape of leucocytic infiltration or actual inflammatorj- reaction in the mucosa of the cervix and lower uterine segment. Moreover, in many specimens the presence of bacteria could be demonstrated hj suitable bacterial stains. On the other hand, in the twenty-.seven patients from whom the utems was amputated at an appointed time at the end of pregnancy or within six hours after the onset of labor, signs of infection were present in only one, whereas in the twentyeight patients who were operated ujwn late in the first stage or during the second stage of labor eighteen presented histological evidence of infection.

TABLE III. Histological Study of Amputated Uteri.


Duration of Labor


Total Specimens


InSammatoiy Changes


Percentage


Before or within 6 h. of Onset

6 to 18 hours


27 5

28


1

4 18


3.7 80.0



64.3




Total


60


23


38.3




While it is not our intention to contend that all patients with ascending infection would necessarily do badly, it does, however, seem to us that many of them would have done so, and, accordingly, we feel that we have adduced a satisfactory scientific justification for our course of procedure. Moreover, we feel confident that, had the conservative operation been done, or even one of the various types of extra-peritoneal, low cervical or flap operations employed, many more of our patients would have died and we should have had results comparable to those obtained in the first fifty cases of our series, as well as with the figures given by Keynolds,* Ronth,- and Holland ' in late operations.

This being the case, we do not feel that we have unnecessarily sacrificed the uterus, but, on the contrary, we are convinced that our procedure has resulted in saving the


lives of a considerable number of women who would otherwise have died.

It is generally believed that premature rupture of the membranes adds materially to the danger of operation, in that it favors ascending infection. In general this is doubtless true, but our material serves to show that ascending infection may be present without its occurrence, as on e.xamination of the history of the patients, in whose uteri histological evidence of ascending infection was present, it was found that the membranes were intact in nine. On the other hand, in the eleven patients from whom the uterus was removed after labor had been in progress for six hours or more and in which histological examination showed the ab.sence of ascending infection, it was found that the membranes had ruptured in seven and were intact in four instances. While such figures clearly indicate that premature rupture of the membranes does increase the danger of infection, they likewise indicate that the main factor concerned is the actual length of labor, and that whenever it has lasted for more than six hours the probability of ascending infection must always be considered and will increa.se with every additional hour the labor has lasted.

Furthermore, it is generally admitted that a temperature of 100.4° F. or more during labor raises the presumption of intrapartum infection. Upon studying our material with this in mind, we find that nine of the sixty-four patients presented elevations of the intrapartum temperature. The uterus from five of these presented signs of ascending infection, while in the other four it did not. To put it in another way, it might be stated that, in the twenty-three uteri which presented histological evidence of ascending infection, the condition was preceded by intrapartum fever in only five instances. In other words, the infection was preceded by elevation of temperature only in five out of twentythree instances. Accordingly, we may conclude that, while the occurrence of intrapartum fever raises the presumption of ascending infection, its absence by no means indicates that it has not already occurred. Here again we must draw a conclusion similar to that in connection with premature rupture of the membranes ; namely, that the great danger arises from the prolongation of labor rather than from any of the factors upon whose occurrence we have in the past laid such weight.

For these reasons we have come to regard the late CiBsarean as an extraordinarily dangerous operation and, in such cases, if we are not prepared to sacrifice the uterus, we must resort to pubiotomy or the destruction of the child if we wish to safeguard the patient, whereas, if we re.sort to radical measures, we may obtain results comparable to those following conservative Ca?sarean section done at an elective time either before or just after the onset of labor.


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REFERENCES

1. Holland: The Results ot a Collective Investigation into Caesarean Sections Performed in Great Britain and Ireland from the Year 1911 to 1920, Inclusive. J. Obst. and Gyn. Brit. Emp., 1921, XXVIII, Nos. 3 and 4.


2. Routh: On Cesarean Section in the United Kingdom. J. Obst. and Gyn. Brit. Emp., 1911, XIX, 1-233.

3. Williams: A Critical Analysis of Twenty-one Years' Experience wth Ccesarean Section. Johns Hopkins Hospital Bulletin, 1921, XXXII, 173-1S4.

4. Reynolds: The Superiority of Primary over Secondary Csesarean Section, etc. Tr. Am. Gyn. Soc, 1907.


AIR EMBOLISM FOLLOWING VARIOUS DIAGNOSTIC OR THERAPEUTIC PROCEDURES IN DISEASES OF THE PLEURA AND THE LUNG

By Karl Schlaepfer, M. 1).

(From the Surgical Clinic and the Surgical Hunterian Laboratory of The Johns Hopkins Hospital and University)


Probably it would not occur to any physician to suggest that air emboli following such common technical procedures as puncture to clear up the nature of an effusion in the thoracic cavity, or irrigation of a long-standing empyema cavity with an antiseptic solution such as bichloride of mercury^ liydrogeu peroxide, or Dakin's solution, or change of position of the tube draining such a cavity, may cause complications that could endanger the life of the patient. Roger,^ a French physician, in 1SC4 flrst called attention to these accidents in his observation on a child eight and a half years of age suffering from a chronic empyema cavity. While the customary irrigation of this cavity with an antiseptic solution, with the use, perhaps, of a flow stronger than during the other irrigations, was being performed, the child fell backward from her sitting position, unconscious. She showed clonic contractions of both upper and lower extremities, similar to those of epilepsy. This attack lasted one hour. The child lay drowsy for the rest of the day. By the next day all the symptoms were gone, and she felt normal. Basing their opinion on this incident and others similar to it, Roger, and later on other French physicians, Besnier,- Legroux,^ and Lepine,' concluded that these attacks were of nenous origin — an irritation of sensitive nerve fibres in the pleura, which transmitted this irritation to the central nervous system. They spoke of this reaction as a pleural eclampsia or pleural epilepsy.

These first clinical experiences were gained from accidents following irrigation of an old empyema cavity, as in Roger's case, or from diagnostic puncture of the chest wall for a supposed circumscribed abscess formation. For a long time irrigation was carried out without any complications. Then, in one case the patient fell unconscious ; in many instances there was an aura at the onset. Sometimes the patient complained of visual disturbances, and at the same time had a prickling pain in tlie discharging side. Headaclie and dizziness at times accompanied the onset. These initial signs were followed


by clonic or tonic contractions of one limb; or all the limbs were involved. The two forms sometimes alternated. Even the facial nerve was affected. These motor troubles were accompanied by eye symptoms, — dilated pupils on the affected side, on the opposite side, or on both sides. Respiration ceased or became of the CheyneStokes type. At the same time there was also abnormality of the heart action, the pulse becoming accelerated and very thready. At this stage the unconscious patient often had involuntary movements of the bowels. If a sudden change in the clinical picture did not take place, death terminated these so called pleural eclampsies. At autopsy there were very often no positive findings.

Ill cases where the patient recovered after several hours or even after some days, a paralysis very often persisted for a time, generally in the muscles which had been most affected by the motor disturbances. St. I'hilippe ^ observed a left-sided palsy in a case of leftsided empyema. No general rule can be made out about tlie relationship. In Walcher's " case the paralysis lasted for two days. The average duration was from a fraction of an hour to several hours. Aphasia of short duration was an exceptional symptom after these pleuro-pulmonary complications. Blindness was the most impressive sign noticed by the patient at the onset of the attack. It lasted for some hours or for days. During an attack the pupils were generally dilated, not reacting, and often unequal. Marble-like discoloration of circumscribed spots of the skin, as the forearm, or calf, was noticed in a few cases. The great variety in the clinical symptoms in these attacks is striking. The negative findings at autopsy strengthened Roger's opinion as to tlie nervous etiology of these complications.

Raynaud ' in 1875 also supported this belief in the origin of these pleural reflexes, as did Bouveret,' Brouardel,^ de C^renville,'" Desplats,^^ Dumontpallier/^ Gilbert,, and Letulle." Cordier ^^ and Gilbert tried to verify their o])inion by experiments on animals. In Da Costa's Modern Surgery '" Dr. Toland is quoted as hav

[No. 379


ing observed four cases of pleuritic epilejisy following irrigation with Dakin's fluid. Da Costa refers to Qu^nu as having seen intense dyspncea, great shock, disturbances of respiration and circulation, convulsions, and death follow injections of the blandest fluid (de C^renville: pleural epilepsy).

Similar accidents were observed after the performance of other therapeutic procedures, such as introduction of a probe or a rubber catheter into a narrow sinus of an old empyema cavitj'. In cases in which the canal had to be dilated by a laminaria stick, these reflexes also occurred. It is interesting to note that in all these cases with complications there was bleeding from the cavity, showing that a mechanical lesion of the fibrous wall of the canal or of its granulating surface had occurred.

Brandes ^' published the report of a case in which he tried to outline the extent of an empyema cavity by an injection of bismuth paste through the sinus. The rubber catheter apparently injured the wall of the cavity. Most of the bismuth escaped alongside of the catheter. The patient had a spasm, with deviation of the eyes, and died after 20 hours. At autopsy, bismuth was found in the smallest blood vessels of the cerebral cortex of both hemispheres. On examination of serial sections from tlie wall of the sinus, the bismuth could be traced up to the pulmonary capillaries and veins. The catheter had injured the granulations and opened some venules underneath. Bismuth was also found in other capillaries of the greater circulation.

I'uncture of the pleural cavity is quite a common procedure, familiar to every practitioner. For example, in the course of a pneumonia we suspect an exudate in the afifected pleural cavity, and perform a puncture to clear up the diagnosis. Or the clinical findings and the X-rays make it very likely that we have to do with an interlobar empyema or a lung abscess; the diagnosis is made certain by the puncture. A clinical picture similar to that given above may be encountered when carrying out any of these procedures. Two striking cases may illustrate the truth of this statement.

.lessen '^ was giving local ansesthesia to a patient thirty years of age with tuberculosis of the lung for a thoracoplasty over a tuberculous cavity, and was trying to make a special deposit of the fluid on the pleural side of the rib. At this moment the patient had an attack of coughing, and fell unconscious, with partial pallor, cyanosis, and clonic twitchings of the left arm. Bloodstained sputum was observed. Respiration and heart action were at first normal. Later on the reflexes were found to be absent. The pupils became narrow, and did not react. The patient died 16 hours after the onset of the attack. The autopsy showed a puncture wound in the indurated wall of the tuberculous cavity. The mucus


in the bronchi was stained with blood. IS'o air could be found in the vessels of the brain.

A second case, observed in Professor Clairmont's clinic (Zurich), was published by me in the Deutsche Zeitschrift fiir Chirurgie.-' A street car conductor, thirty four years old, had suffered with a recurrence of bronchitis which had developed into broncho-pneumonia. On the supposition of the existence of a circumscribed abscess, the right lung was punctured twice without result and without accident. Relying on the tj'pical clinical picture, the operator repeated the puncture in several places some days later with the same negative result. A week later a new attempt to help the patient was made. The X-rays showed in the right lung a circumscribed area filled with fluid. Resection of a .segment of the fourth rib was made under local auiBsthesia. Before going further a puncture was made and some bloody fluid was withdrawn into the syringe. For examination of this fluid the syringe was detached, the lumen of the needle being now in free communication with the atmosphere. At the moment the syringe was taken oft' the patient collapsed. He became unconscious, with fixed pupils, and clonic convulsions (twitchings) of all muscles. Some minutes later he became conscious again. Within two and a half hours he liad graduallj' recovered completely, except for loss of sight. The ophthalmoscopic examination revealed a normal fundus. The blindness lasted for three days. The patient had an increased muco-purulent expectoration. The signs of abscess cleared up without further operation. The patient improved every week, becoming finally normal after six months, when he resumed his occupation.

In these two cases the presence of air embolism seems very probable, but the diagnosis was not confirmed either by the clinical findings (ophthalmoscopic examination) or by autopsy.

Tlie first clinical observations of what proved to be experimental air emboli, unintentionally produced, were made in cases of accident in pneumothoraxtherapy for tuberculosis. The phthiseotherapists frequently employ artificial pneumothorax in cases of unilateral pulmonarjtuberculosis. It is e.specially to Forlanini -^ (1S82) that we owe the introduction of this procedure in medicine. He advised the so-called puncture method, in which the needle is introduced directly thi'ough the thoracic wall. Accidental puncturing of the lung may occur, and with this there may be trauma of some engorged veins in a tuberculous area underneath the surface of the lung, with sequelfe, which we shall consider later. This grave danger, with subsequent air emboli, led J. B. Murphy "' to suggest exposure by a preliminary incision of the parietal layer of the pleura, with introduction of the needle into the fissure of the pleural cavity. Brauer -- became a strong supporter of Murphy. He -was the first to recognize, in 1912, the danger of air embolism in thoracic work,


Septembkr, 1922]


323


aud rejected the opinion held by Roger and others as to these so called pleural reflexes. Wever's - experiments are convincing. Forlanini did not admit the possibility of the presence of air emboli. He maintained the old conception of the nervous origin of the convulsions.

Among the phthiseotherapists using pneumothoraxtherapy (Spengler,-* Saugmann,^" Giesemann,-" Suudberg,-" and v. ^'oornveld -*) air embolism as the cause of the fatal outcome during this treatment is generally accepted. tTessen believes that the frequency with which air embolism occurs is much greater than is recognized, and in support of his view is the statement of a colleague of Dr. von ^'oornveld, that he had given up this therapy because he had had three deaths in 12 cases. Many of the cases are registered as death from chronic myocarditis, cachexia or shock. This evidence shows that prominent physicians in Europe do not agree with the conception about jineumothoraxtherapy of Da Costa, who points out that there is little danger in the method, although possibly trouble may follow. That statement may perhaps hold good when the Murphy-Brauer method with the incision is employed. But in Europe, where the Forlanini-Saugmanu puncture technic is generally used, there is some danger that this method, even in the hands of an experienced physician, may cause air emboli, although the technic has been worked out to diminish to a great extent the possibility of danger. The first puncture presents the greatest risk, but in cases of a partial pneumothorax the danger is always present. When the operator is refilling or increasing the air content, the needle may either pass into a strand of adhesion, thereby opening a vein, or may pierce infiltrated lung tissue. The same complication occurs when the strand of adhesion breaks through near its attachment to the lung surface. It is on account of this possibility that the adhesions are cut through near their attachment to the pleura when Jacobaeus"^ thoracoscopy is employed. Even then the danger is not completely eliminated.

The following figures will give some idea of the frequency with which these complications may occur in the hands of even expert operators. Forlanini ^° had, in 10,000 punctures on 131 patients, 12 accidents, 5 with temporarily serious conditions, but all with final recoveries. In a second report he had in 1451 insufflations on 28 patients 4 complications without a death. Saugmann "^ records 970 operations on 51 patients without complications. In a second series '- he had in 215 first punctures and 5500 follow-up treatments 2 cases of embolus. Extracts from some histories may illustrate these accidents.

Saugmann ^' tried to introduce air into the chest of a man twentj'-two years of age, with unilateral pulmonary tuberculosis, which had existed for some five years. A first session was without result. A wide manometric excursion, corresponding to inspiration and expiration —


proof that the needle is in the pleural cavity — was not shown. At a second attempt the oscillations of the manometer were only minimal, and the procedure was given up. Later, a third effort was made with the same result. The patient was then told to take a deep breath. At this moment he turned pale, and complained of dizziness. The needle was taken out. The patient became unconscious, and vomited, and the breathing became very irregular. On the right side the pupil was large ; the eyes showed divergent strabismus. The pulse could not be felt. Although various stimulants were employed, the breathing stopped also. The autopsy revealed two prick lesions in caseous pneumonic lung tissue, with some suffusion of blood. No large blood vessel was implicated.

In a second case of Saugmann, ^^ a woman, aged thirtytwo, with a long-standing left-sided tuberculosis, the patient was very nervous. The inserted needle was connected with the manometer, but the latter did not show any wide excursions. An obstruction in the canal of the needle was supposed to be the cause and a stylet was passed. The patient took a deep breath. The stylet, on being withdrawn, showed some blood. At this moment the patient said that she felt giddy. The needle was suddenly withdrawn. The patient became pale. She lay on her back, unconscious, the eyes and the head turned to the left, with the pupils at first contracted, later dilated. Respiration and the heart's action became irregular; the puLse stopped and death occurred. At autopsy a mechanical lesion made by the needle in the lung tissue was found, with bloody suffusion of the whole area. On microscopic examination the damaged vein could be seen. In the vessels at the base of the brain, especially in the Sylvian artery, gas bubbles were found.

Speugler ^^ cites the case of a girl, eighteen years of age, with left pulmonary tuberculosis. I'neumothoraxtherapy was being carried on with good results. At one session, when the operator was trying to increase the air content of the cavity, the manometer showed by its wide oscillations that the needle was in the right place. Air to the amount of 250 c.c. was injected. The patient became agitated, made a quick movement with her left arm, and suddenly fell unconscious. The needle was taken out immediately. The pulse could not be felt. Pale, with dilated pupils, the patient was breathing quite superficially. A circum/^cribed marble-like area on her left forearm was noted. Clonic contractions of all the muscles were followed by tonic cramps in the rigiit upper arm. When she groaned, a palsy of the right facial nerve could be seen. For several hours she slept. Twitchings recurred several times, especially on the right side. She had an involuntary bowel movement. Death occurred after 3 days. Autopsy was refused.

Carpi,'*" when beginning the Forlanini technic on a patient who had been sick for 3 years, felt sure that the needle was in the pleural cavity. He started with 10 c.c.


324


[No. 379


of nitrogen. At this moment tlie patient complained of pains in the region of the punctnre. She had a sensation of pressure, became nnconscions, and had clonic convnlsions on the right side, which later implicated the left side also. Trismus and opisthotonus were caused by these contractions. The respirations became frequent and superficial, with a pulse of 140. For a short time the patient was cyanotic, and sweated profusely. The pupils reacted to light. This attack lasted only a few minutes. A.fter a short interval a second similar one followed. She made an uneventful recovery.

Before pa.ssing to the study of the etiology of these complications we shall consider the other procedures in which these serious accidents may occur.

Decortication (Delorme, Fowler) is advisable in cases of old empyema with a persistent sinus, to relieve the patient from the annoyance of a constant discharge of pus. Pelorme's ^" procedure tries to avoid the great disfigurement of the Schede thoracoplasty by including the dense fibrous thickened visceral layer of the pleura of the lung, so that the lung can expand to its primary volume. This thick membrane holds the lung down in its contracted state. Fowler,^** of Brooklyn, used the same technic at the same time without knowing of Delorme's procedure. A modification of this operation is Eansohoff"s '^ discission of the pulmonaiy pleura for cases in which it is impossible to liberate the lung from its encircling coat. Parallel incisions are made a quarter of an inch apart, crossed by a second series perpendicular to the fir.st. By this procedure expansion of the lung is probably rendered more gradual.

In these decortications many blood vessels, especially veins, in the superficial part of the lung are mechanically injured, the injuries- being analogous to those that may be caused when irrigating this wall with a strong flow, or when introducing a tube into this cavity, which ploughs a groove on the surface of the lung (Brandes' observation). Confronted in all these cases with similar pathological changes of the pleura and neighboring lung tissue, we encounter a similar danger of air emboli (Weill)." In pneumothoraxtherapy we have an analogous process when in a partial pneumothorax one tries to break up some adhesions. These adhesions may become loose, and leave a defect in the superficial layer of the lung. The lumen of an opened vein may be kept open by its position in fibrous, dense tissue, so that the air comes into direct contact with the venous circulation. To diminish this danger Jacobaeus,*^ in 1913, devised an endoscope for the thorax, through which he burns off the adhesive strands along the costal pleura (thoracoscopy).

Careful precautions have to be taken in mobilizing the costal pleura, especiallj' at the apex of the lung, when an attempt is made to insert a fat or a paraffin plug (Tuffier/- Baer*'M. The veins of these thickened pleurse communicate with the veins in the adjacent lobe of the


lung. The same danger is encountered in mobilizing the costal pleura for collapsing an empyema cavity, previously sterilized with Dakin irrigation.

In all the previously described operations the procedures were done near the chest wall. In pneumotoniy and pneumectomj' the operator comes nearer to the vessels at the hilus, with the larger veins under negative pressure, at least during inspiration. In these cases the possibility of complications from air embolism is always imminent, more so when sharp dissection is employed than when one comes up behind the necrotic wall with the Paquelin cautery or with a blunt instrument, such as the finger.

Passing through lung tissue "with the cautei'y in attempting to reach a bronchiectatic abscess in a patient thirty-one years old, Quincke ** was stopped by profuse bleeding. By means of gauze tampons the bleeding was controlled. The patient complained of a prickling sensation in both arms, and dizziness. He looked pale and collapsed with irregular, feeble pulse. The respirations became less frequent, and stopi)e(l. Fifteen minutes after the onset the patient was dead. At autopsy an open vein was found next to a bronchus which had been burned off by the cautery. The neighboring lung tissue was carnified.

Wever '^ cites a similar ca.se of a girl, nineteen years of age, with bronchiectasis. The intention was to open the cavities in several stages. During one of the procedures the patient coughed and at the same time profuse bleeding occurred in the wound, which was stopped by gauze packing. The patient vomited and had tonic cramps in both arms. She was unconscious, and turned her eyeballs upward, with widely dilated reacting pupils. Marble-like patches, with a distinct outline, on the body and the extremities indicated definite vascular disturbance. The speech was dysarthric. A left-sided palsy of the facial nerve was noticed. For 24 hours she remained about the same, after which she gradually recovered.

To clear up the etiology of these complications we must consider the conditions in the normal pleura so far as they concern its sensibility. From a series of experiments which I began in the surgical clinic in Zurich, Switzerland (Professor Clairmont). and which are being continued in the Surgical Hunterian Laboratory (Professor Halsted) of the Johns Hopkins University. I have come to the following conclusions :

From irritation of the normal pleura of different animals, mechanical, chemical, or by the electric current, we do not get a constant specific reaction by way of the central nervous system, as we should expect if the socalled pleural reflexes were the real source of the abovementioned complications in the different operations upon the chest. In the costal pleura we have sensory nerve fibres (intercostals), mechanical or chemical, irritation of which causes general protective movements, similar


September, 1922]


325


to those which we see iu other parts of the body (selfpi-otection) In smaller animals- (rats, guinea-pigs, rabbits) faradic stimulation of the parietal pleura causes clonic contractions of the upper extremity of the same side. In dogs I have obtained only local contraction of the thoracic musculature, with no movement in the adjoining extremity. Faradic stimulation of the pulmonary or mediastinal pleura produces no muscular contraction at all in any of the.se animals. Stimulation of the diaphragmatic pleura with the faradic current gives only contraction of the diaphrag-m. This fact seems to indicate that the diaphragm gets its sensory nerve supply from the phrenic nerve only and not from the neighboring intercostals also. In dogs, as was stated above, even by irritating an area of the diaphragm next to the thoracic wall one gets only a contraction of the diaphragmatic muscle. No contraction is seen in the neighboring intercostal musculature. With faradic stimulation of the parietal pleura adjacent to the diaphragm in smaller animals, contractions are observed, in addition to those in the chest muscles, in the upper and lower extremity of the same side. In dogs only local muscular contractions are seen.

The vagus (pneumogastric) fibres running with the bronchi and all the branches of the bronchial tree carry pain .sensation centripetally, causing protective movements in the upper extremity on the same side in small animals. After chemical irritation (with iodine) of the pleural cavity, the substance si)reads through the pulmonary pleura into the lung. Resection of the vagus nerve iu the neck inhibits these contractions. Stimulation or paralysis of the sympathetic nerve has no influence on these protective movements.

In all the cases of empyema in which these complications have been observed we have to deal with a longstanding condition. The pleura is thickened and covered with a granulating surface. The sensory nerve fibres in the area of the empyema cavity on the side of the thoracic wall are strangulated by mechanical pressure exerted by the encircling fibrous tissue. They are not functioning, and any reflex movements along these fibres are made impossible. Moreover, the lung tissue adjoining such a constantly discharging chronic infected cavity is altered and has become fibrous, as a conse(tuence of i-eaction against the infection. In the majoritjof the cases of empyema, perhaps in all (C. Beck),^" the primary focus of infection is iu the superficial layers of the lung; the pleural cavity becomes att'ected .secondarily. The fibrous tissue formation around an infected area represents a natural reaction of the body to combat the invading microorganisms. Around every lung abscess we liave tran.sformed tissue, jtartly infiltrated, partly fibrous, with the resultant changes around the blood vessels,


particularly the veins with their weaker walls. The tissues about a bronchiectasis show similar changes.

In an empyema cavity the only sensitive elements are the sympathetic nerves, running with the blood vessels into fibrinous deposits which outline the wall. In the lung we have the vagus fibres along the bronchioli.

These are the pathological conditions which are found in variable degree and extent in all cases in which we are threatened with an air embolus. In tuberculosis of the lung we find similar changes taking place. But in tluit disease, in cases suitable for pneumothoraxtherapy, we do not find such uniformity of arrangement of the indurated tissue. Places showing little change are close to fibrous areas. In the center of the pathologically indurated areas we may have a cavity, or at least a circumscribed necro.sis (caseous pneumonia). In these lungs we find all the different stages of the fight of nature against the invader. In all cases of air embolism the i)neumothorax needle alwaj's passes into an area showing a specific process of natural repair. The pleura in these cases may be still very sensitive, if only relatively snuill parts of the lung are involved by the tuberculous disease.

In the wall of an empyema cavity with its underlying, chronically inflamed lung tissue, in the neighborhood of every active infection in the lung, the blood vessels are involved in dense fibrous tissue formation. In an earlier stage, when the infection is still active, the tissue shows a certain amount of infiltration. The vessels are suspended between the fibrous strands. There is a constant pull on these vessel walls when these fibres contract, especially on the veins. When their walls are mechanically injured, the veins cannot contract to the full extent, being hampered by their attachments in this inelastic tissue. The musculature of the vein, beingweaker than that of the artery, is insufficient; the opening in the wall of the vein persists; from the hollow of the needle, or from the neighboring cavity (pleural cavity, bronchus) air or gas enters; and we have an air embolism. The result following any injury of the vessel wall shows the inability of these veins to contract to the same extent as they would do in normal tissue.

The blood in the lung gets its impulse from the right ventricle through the pulmonary arten*. The sucking effect of the left atrium and the larger veins of the lungs is not so efficacious. The combined force of these two factors, together with the act of respiration, nudies the current continuous. The sucking influence from the larger veins has a minor effect, in proportion as the vein is situated in the superficial parts of the lung, away from the hilus. A lesion of such a vein by an instrument allows the air, remaining under atmospheric pressure, (cavity, bronchus), or gas from a pneumothorax, to enter through the opening. The friction exerted by the air upon the vessel wall causes by reflex action a contraction of the


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[No. 379


muscles innervated by the sympathetic nerves. This contraction Is made ineffective by the attachment of the wall of the vein to the encircling flbrous tissue. At the same time this friction of air and blood along the wall stops the blood proximal to the air. This friction and the internal resistance of the air stops the movement in the blood column proximal to the lesion. No further movement is possible, unless the sucking action from a larger branch of a pulmonary vein comes to their assistance. The air embolism does not become clinically evident. This is the reason why this complication is not reported very often. In the different operations upon the chest wall a lesion of the veins happens quite frequently, but it does not make itself evident.

The force has to come from a larger vein under negative pressure in the neighborhood of the wounded vein in order to bring the air and the blood by aspiration into the left atrium. In several experiments on dogs the pressure in the pulmonary vein next to the hilus was tested with a manometer through an intercostal opening in the thoracic wall. The pressure was found to be — 0.3 to — 0.4 cm. water ; i.e., negative. From the left ventricle the air can be dispersed all over the body. The air embolism renders itself manifest especially by the trophic changes in the brain and in the eye-grounds (retina). lu the larger vessels the air causes a contraction of the wall; hence the smaller vessel may be closed at its junction with the larger vessel.

The distance between the point of the mechanical lesion of the vein wall and the junction of this vessel with a larger vein under negative pressure, whence the blood in the lacerated blood vessel, together with the air that has entered, can be aspirated, is the essential point in inaugurating the onset of a clinically manifest air endjolism.

In cases of unilateral tuberculosis of the lung treated by artificial pneumothorax the injured vein may become connected only with the air in the needle. Then we have the conditions described above. But when the accident happens when the gas is flowing out from the apparatus under pressure, the air embolism is much more dangerous. These are the cases ending in sudden death, in which at autopsy we find a great number of gas bubbles in all the smaller vessels of the brain.

The meclianism of air embolism in an empyema cavity, caused by a change of a tube, the use of a probe, dilatation witli a laminaria stick, irrigation, or injection, is well illustrated by Brandes' case, in which bismuth paste was found filling up the vein from the point of injury to a larger branch, with emboli in the capillaries all over the body, especially in the brain and in the intestine.

Mechanical injury of the blood vessel is facilitated in all abscess cavities by partial necrosis of the wall, caused by the continuous effect of the presence of pus. The blood vessels resist this process for a longer time, and


therefore become exposed. Under these conditions a strong current of fluid may open a vein. A proof of this statement is that in the history of these cases we find that the fluid was observed coming back mixed with blood.

The recorded cases of air emboli in thoracoplasty have the same physiological and pathological underlying causes as empyemata. The knife or the blunt dissecting finger is always the primary faulty agent. To diminish this danger the use of the cautery was introduced, particularly for operations in the deeper part of the lung (bronchiectasis, pneumotomy). A path to the abscess cavity is made through the lung tissue by burning which closes all vessels by a thick escharotic membrane. Near the hilus even the I'aqnelin cautery cannot close tin* pulmonary veins tight, as was shown by the cases referred to above. By using the finger as a gently advancing dissector (Lilienthal)*' this danger may be somewhat diminished. Moreover, we avoid the danger of i secondary hemorrhage when the eschars become loose in these infected wounds.

Complications showing a similar clinical picture may occur when we are |)erforming the operations on the chest cited above, in which another cause for the complications is present. Without a thorough knowledge of the clinical features of air embolism, the physician can never be sure of the real source of these accidents. After all, a number of cases on the border line will renuiin undetermined.

We are constantly having to deal with patients who have lost weight and strength during a long illness. Complications from various sources arise much moi-e easily in these patients, as they have no resistance. The quick relea.se of a large amount of fluid out of a cavitj- in the chest can produce a collapse, caused by a temporary anaemia of the brain (empyema, ecchinococcus). In conditions with chronic inflammation of the lung, such as empyema, some veins may be blocked by thrombi (v. Dusch).** If they are mobilized during a therapeutic procedure, these clots follow the same course as the air, and we have a very similar clinical picture. Vallin,^" in 1875, was the first to announce the opinion that these epileptiform attacks may be caused by capillary emboli from thrombi in pulmonaiy veins. Thrombi in the pelvic plexus or in the lower extremities may be mobilized during an operation. The pulmomny embolus gives a picture similar to that produced by a heavy air embolus. Autopsy will clear up the etiology. Chronic myocardial lesions may cause a collapse during an operation. The air embolus can be ruled out only by thorough examination. In a similar way insufticiency of the adrenals may become evident by a colla]ise with death. In only a few cases are the unimportance of the clinical signs and the short duration striking. The past histoiy of the.se patients reveals a neurotic constitution. No


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local signs of cerebral affection are seen. These conditions have a neuro-hysterical basis and are easily recognized.

The features of the clinical p'cture of air embolism following the different procedm-es mentioned above are .so varied that is is vei-y ditiicult to include them in a definite classification. Unfortunately, nuuiy of the histories of the reported cases do not mention several clinical features which we would expect in air emboli, and which would be of great importance in the differential diagnosis of these complications. More thorough examination of these conditions is necessai-y in order to furnish wider experience in such cases.

Pallor of the patient is often the first sign of imminent danger. Circumscribed patches of cyanosis on the skin present a marble-like appearance, a further proof of disturbances in the circulation. The patient complains of a sudden pain in the chest on the affected side. He cries out, saying that he feels giddy or ill and that he has black spots before the eyes. In many instances he becomes totally blind. Irritative pains in the injured side of the chest and severe headache aggravate the condition. The patient swoons, veiy often with no preceding aura. Unconsciousness comes on suddenly. The pulse is altered, perhaps cannot be felt any longer. Dyspnoea passes into stertor; the breathing becomes gasping; respiration stops. Involuntary evacuations precede the sudden death. In other cases pulse and respiration become very frequent. If the patient is still conscious, he complains of oppression in the chest. Later on the pulse becomes feeble. Respiration is of the CheyueStokes type. The pupils are generally widely dilated, and do not react. In a few reported instances they were different on the two sides, perhaps with strabismus of the convergent or divergent type. If death occurs shortly after the onset of the complication, an autopsy should be done as soon as possible after death, to eliminate any post-mortem changes in the brain, the organ of greatest importance for the diagnosis.

After having passed through the stage of these preliminary symptoms, the attack may come to an end, the patient recovering gradually from his comatose condition.

In the majority of all cases clonic or tonic convulsions give evidence of an excitation of the motor areas, beginning in the eye muscles or in the upper extremity, and very often becoming generalized all over the body. Spasm of the back muscles gives rise to opisthotonus. From the histories of the reported cases it is impossible to trace a definite relationship between the affected side of the chest and the location of the most violent convulsions. In the literature we find the greatest variation in this feature. These contractions are followed by a paralysis, which varies in location and duration. Usually it lasts only a short time, from some minutes to several


hours or several days. The paLsy is generally most marked in the limb that is most affected by the convulsions, and remains there longer than in the other regions, perhaps even for months. The eye-sight is altered in an analogous manner, its return to normal being governed by a similar law. The disturbances in the sensory-motor region are shown also by circumscribed areas of hyperajsthesia and anaesthesia, and by different forms of parsesthesia.

Examination of these cases of air embolism discloses the fact that the clinical findings are quite variable. From a study of the pathology and physiology of these lesions we must try to trace the effect of the temporary embolism in different organs, the presence of which was indicated by the clinical pictures just described.

The ophthalmoscopic examination during an attack and later on at periodic intervals is of the greatest importance. The findings in the reported cases are divergent. Raynaud found in his case a serous suffusion around the papilla and a venous hyperemia. Schnitzler ^ noticed in de C^renville's case a venous stasis combined with capillary hemorrhages. In the observation by Clairmont, which was published by me, no abnormality could be found immediately after the accident or later. In a case of air embolism following pueumotomy for gangrene of the lung, Becker ^^ saw the air passing through the arteries of the retina in the form of gas bubbles. Later on, when the air was passing thi-ough the capillaries, strands in the form of fine white silver glittering lines filled the fundus. Subsequentlj- the veins became enlarged. In the middle of the veins a white strand was seen (Stargardt).'*^ The air seemed to be carried in the center of the vessel. After a short time the fundus looked normal again. Even after weeks changes caused by temporary thrombosis of the smaller arteries and capillaries can be seen. In ca.ses with negative findings of the eye gi-ounds the source of embolism may lie in the occipital lobe.

The clonic and tonic convulsions with subsequent palsy are due to nutritive changes in the motor area of the cortex. Partial temporary thrombosis in the medulla oblongata is followed by sudden death due to blocking of the vital regulative centers of respiration. The microscopic examination of the brain in these cases will show the cause of death. Spielmeyer ^' found liquefaction of the cells of the cortex as a typical condition. He noticed also an incrustation around the nerve cells of the nerve plexus. The glia reaction was negative. In cases in which death had occurred after several days, Spielmeyer found marked proliferation of the glia cells in circumscribed areas.

Air blocking the smaller blood vessels in the intestine (stomach, duodenum) causes a typical ulcer formation in the mucous membrane. This was found by Keller '* in several cases at autopsy. When the patient has not partaken of meat before the operation which was com


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plicated by air embolism, a careful examination of the faeces during the following days will reveal traces of blood. Of course when the past history and the examination are suggestive of ulcer, or of tuberculosis of the intestine, this finding is of no use for the diagnosis of embolism. Albumin in the urine, and epithelial casts, — if they were not present before the accident — are quite significant of temporary' nutritive blocking of the renal system.

Besides these different clinical findings the evidence of injury of a blood vessel during the diagnostic or therapeutic procedure is of the greatest value. Blood on the stylet after puncture is very suggestive of a lesion. The patient may have at the same time an attack of coughing, and spit up mucus mixed with blood. Bleeding during irrigation of an empyema cavity or during the changing of a tube in such a cavity is a sign to be noted as one of imminent danger. In thoracoplasty and in pneumotomy sudden bleeding is also of great diagnostic value. In all accidents accompanied by the symptoms described above we must think first of an air embolus, unless we can rule it out by the lightness of the attack in a patient who has a nervous make-up (nervousness, neurasthenia, epilepsy) and is weak from a long illness.

The prognosis in any case of air embolism after an operation on the chest depends upon the amount of air entering the blood stream, and also upon the region in the brain which is most seriously involved. The extension of the condition, especially to vital centers, causes sudden death. The clinical signs during the first attack give a fairly practical indication of the extent of the lesion. If the patient survives the first attack, the outlook is not bad, and he will gradually within a short time recover from all the bad effects of the accident. The time necessary for convalescence is proportional to the seriousness of the clinical picture at the onset. In the great majority of cases all the symptoms are temporary; as time goes on, they disappear. The air passes through the capillarj^ system, causing a temporary blocking of certain areas in the brain, with concomitant nutritive changes. The heart action, which regulates the blood current, is very important in forcing the air through the capillaries, in this way diminishing the duration of the thrombosis in essential parts of the brain.

These points afford a sound basis for our therapeutic measures to combat the effects of these complications. The procedure that has been the source of the accident, must be stopped at once, thus removing the causative factor. The needle used in the puncture or injection or in the pneumothorax treatment is taken out. The irrigation in a case of chronic empyema is stopped. The tube or the probe causing the injury is removed. In pneumotomies where profuse bleeding shows that the vessel lesion is in the deeper part of the lung, packing the whole cavity with gauze will prevent the harmful air


embolus. For these cases more particularly the use of a positive-pressure apparatus, to diminish the negative pressure in the larger pulmonary veins, recommended by Tiegel,^^ has proved to be very satisfactory.

When confronted by an air embolus the physician concentrates his attention on strengthening the heart and increasing the blood current through the brain, in order to force the air from the capillaries as soon as possible and to drive it to the lung, whence it can be expelled from the circulation through the alveoli. Intravenous injection of heart stimulants helps to strengthen the action of the heart muscle. An intravenous injection of adrenalin is followed by a contraction of the vessels of the splanchnic area (intestines) and the skin. This is a suitable and helpful procedure to improve the blood supply of the brain. The head should be put low, to increase the effect of this procedure. Jessen " has reported good results from a venesection.

If the breathing stops, rhythmic traction of the tongue and faradisation of the phrenic nerve should be employed. Artificial respiration is contraindicated, because deep inspiration might enlarge the opening in the vein and give rise to a new embolus. Morphine should never be used because of its paralyzing effect on the respiratory center.

As a useful jiropliylactic procedure, when doing these operations on the chest, bend the head of the patient to one side so that it is not the highest point of the body. With the head in this jjosition, in case of air embolism, the air bubbles do not have the tendency to reach the capillaries of the brain. This was proved by experiments which I made on middle-sized dogs in the Surgical Hiinterian Laboratory of Professor W. S. Halsted. The dog was put on his right side, flat. Under ether ana"Sthesia an opening was made in the left chest wall, in the fifth intercostal space. An injection of 2 c.c. of air was made into the jiulmonary vein at the hilus. On ophthalmoscopic examination no air could be seen. A second injection of 2 c.c. of air was made. ^No air was seen passing through the blood vessels of the retina, but a gurgling sound was heard on squeezing the heart (left atrium I. As soon as the dog was put in a semi-erect position, air bubbles appeared in the eye-ground. The dog died without any convulsions. This experiment was repeated with the same findings.

Summary

1. Following various diagnostic and therapeutic procedures on the chest (puncture, irrigation of a cavity, changing tubes, introducing a probe or a rubber catlicter, bismuth paste filling, decortication, thoracoscopy, fat or paraffin plombage, pneuniotomy, pneumectomy), complications may ensue which have been proved experimentally and clinically to be caused by air emboli.


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2. The sudden release of a great amount of fluid in emaciated people with a neurotic constitution (neurasthenia, epilepsj') may cause a similar clinical picture. Because of its slightness and short duration, with lack of localized cerebi-al symptoms and serious sequelae, this condition is considered to be due to shock.

Chronic myocardial lesions or an insuflicieucy of the adrenals may cause sudden death during the performance of one of these operations on the chest wall.

A similar clinical picture is seen in embolism of the [)ulmoiiary artery. l>eath, accompanied by these signs, may occur also in a few cases of emboli in the brain following thrombosis of pulmonary veins. The autopsy will clear up the diagnosis.

3. It was demonstrated by experiments that we do not have a pleural reflex, even in the normal pleura, which would explain these complications. They are proved by the accidents in pneumothoraxtherapy to be due to air emboli.

i. The pathological condition of the lung and the pleural sheaths is the same in all these cases. The lung tissue shows, in a circumscribed area, a condition which is partly an infiltration, partly an induration of the tissue, where the blood vessels, especially the veins with their weaker walls, are fixed in a distended position. When mechanically injured, they cannot, with these rigid surroundings, collapse as usual ; the hole stays open. The distance of this wounded vein from a branch of a larger pulmonary vein under negative pressure, which can aspirate the air with the stagnating blood, decides whether the air embolism will become evident. Absence of this condition is the reason for the infrequency of air emboli.

5. The clinical pictures of these air emboli show a wide variety. They should be made more exact by further thorough study of these complications by means of ophthalmoscopic, stool, and urine examinations.

6. The best therapy is the preventive one. When an air embolus occurs, stop the operation immediately. Put the head of the patient low. Reinforce the heart action with stimulants. Adrenalin injections, administered intravenously, will increase the amount of blood passing through the brain by diminishing the blood supply in the splanchnic areas (intestines, skin).

REFERENCES.

' Bull, et Mem. de la Soc. med. des hop. de Paris, 1865, i, 132138. Also: Union med., Paris, 1864, 2.s., XXIII, 69-73.

"Note sur un cas de mort subite par syncope survenue pendant roperation de la thoracent6se. Mem. de la Soc. med. de Paris, 1875.

' Note sur un cas de mort snbite. Mem. de la Soc. m§d. des hop. de Paris, 1874, 72.

' Note sur un etat paretique developpe dans les membres du cote correspondant a un empyfeme. Mem. de la Soc. med. des hop. de Paris, 1875.


"a Epilepsie hfimiplegique pendant le lavage de la plevre. Jour, de med. de Bordeaux, 1904, XXXIV, 127.

=b Notes et reflexions sur un cas de pleuresie purulente d'emblee. Explosion d'une crise d'epilepsie hemiplegique; gu6rison. M6m. et Bull. Soc. de m^d. et chir. de Bordeaux, 1886, 292 (discussion, 328). Also: Jour, de med. de Bordeaux, 1885-6, XV, 509-514.

"Gaz. m6d. de Strassbourg, 1876, 3 s., V, 1-5. Also: M6m. Soc. de Med. de Strassb., 1876-7, XIII, 20-34.

' Des morts inopinees pendant ou apres la thoracocentese et des convulsions epileptiques a la suite des injections pleurales. Bull. et Mem. de la Soc. des hop. de Paris. 1875.

" Traite de I'empyeme. Paris, 1888.

'Communication a Soc. med. des hop. de Paris, 12 Nov., 1875 (Arehavski).

" Des manifestations encephaliques de la pleuresie purulente. Rev. m^d. de la Suisse romande, 1888, VIII, 5; 65.

"Etude sur les accidents dits reflexes survenus apres I'opgration de I'empyfeme. Jour, de Soc. med. de Lille, 1883, V, 761; 801. Also: Semaine med., Paris, 1888, VIII, 277.

'- Des accidents reflexes survenant apres I'operation de I'empyeme. . Paris m6d., 1883, VIII. 229-233. Also: Compt. rend. Soc. de biologie, Paris, 1881, 7 s., II, 293.

" Gilbert, A. et H. Roger. Etude exp6r. sur le pneumothor. et sur les reflexes d'origine pleurale. Rev. de med., 1891, 977.

"Letulle. Semaine m^dicale, 1890, (quoted from Cordier).

"^ Des accidents nerveux au cours de la thoracocentese et de I'empyeme. Lyon et Paris. 1910.

"Modern Surgery. Sth edition. Philadelphia, 1920. P. 1018.

" Biu Todesfall durch Embolie nach Injektion von Wismutpaste in eine Etapyemfistel. Miinch. med. Wochenschr., 1912, Nr. 44, S. 2392.

" Arterielle Luftembolie und die Technik des klinstlichen Pneumothorax. Deutsche med. Wchnschr., 1913, Nr. 26, S. 1245.

"Ein Fall dreitagiger Erblindung nach Probepunktion der Lunge. Deut. Zeit. f. Chir., 1920, CLIX, 132.

"'A contribuzione della terapia chirurgica della tisi. Gazetta degli Ospedali, agosto-nov., 1882.

=' Surgery of the lung. Jour. Amer. Med. Assn., 1898, XXXI, 151.

^ Die Behandlung der einseitigen Lungenphthisis mit ktinstlichem Pneumothorax. MUnch. med. Wchnschr., 1906, Nr. 7, S. 338.

^'Zerebrale Luftembolie. Diss., Kiel, 1914. Beitr. z. Klin. d. Tuberkul., 1913, XXXI, 159.

"In: Brauer & Spengler: Erfahrungen u. Ueberlegungen zur Lungenkollapstheorie. Beitr. z. Klinik d. Tuberkulose, 1909,

XIV, 419.

^' Klin. Erfahrungen iib. d. Behandlg. d. Lungentuberk. to. kiinstl. Pneumothorax-bildg. Beitr. z. Klin. Tuberk, 1915, Bd. —

XV, H. 3, S. 305.

^ Operationszwischenfalle und Komplikationen beim Anlegen und Nachfiillen des kiinstlichen Pneumothorax. Brauers Beitrage, 1915, XXXVIII, 21.5.

-' Drei Todesfalle mit Obduktion nach Behandlung mit kiinstlichen Pneumothorax. Brauere Beitrage, 1913, XXVI, 30.

°* Ueber Emboliebildung bei der Behandlung mit kiinstlichen Pneumothorax. Beitr. z. Klin. d. Tuberkul., 1915, XXXIV, 305.

^ Eine neue Methode der Beseitigung v. Verwachsungen bei Pneumothoraxbehandlung d. Lungentuberkulose. Hygeia, 1914, H. 15, S. 230.

™ Die Behandlung d. Lungenschwindsucht m.d. kiinstl. Pneumothorax. Ergebn. d. inn. Med. & Kinderheilk., 1912, Bd. IX, S. 621.

" Klin. Erfahrungen iib. d. Behandlg. d. Lungentuberk. m. kiinstl. Pneumothorax-bildg. Beitr. z. Klin. Tuberk., 1915, Bd. XV, H. 3, S. 305.


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'=Zur Technik des kunstl. Pneumothorax. Beitr. z. Klin. d. Tuberk., 1914, Bd. XXXI, H. 3, S. 571-644.

Idem. Loc. cit.

^ Idem. Loc. cit.

/re; Brauer & Spengler: Klin. Beobachtungen b. kiinstl. Pneumothorax. Beitr. z. Klin. d. Tuberkulose, 1911, Bd. XIX, H. 1, S. 1-335.

'"Die Grundsatze und die Durchfiihrung der Pneumothoraxtherapie in der Behandlung der Tuberkulose. Korrespondenzbl. f. Schweiz. Aerzte, 1914, XLIV, 545-566.

" Du traitement des empy^nies chroniques par la decortication du poumon. Gaz. des Hop. de Paris, 1896, 69, 1445.

A treatise on surgery. Philadelphia. 1906.

=' Discission of the pleura in the treatment of chronic empyema. Ann. Surg., 1906, XLIII, 502.

" De la mort subite dans la pleur^sie. Revue de medicine, Paris. 1887, VII, 33-63.

" Endopleurale Operationen unter Leitung des Thoracoskops. Beitr. z. Klin. d. Tuberkul., 1915, Bd. XXXV, H. 1. S. 1-35.

•"Etat actuel de la chirurgie intrathoracique. Paris, 1914.

•" Beitrage zur Klinik des kiinstlichen Pneumothorax. Ztschr. f. Tuberkul., 1913, Bd. XXIX, H. 3, S. 129.

" Ueber Pneumotomien. Mitteil. a.d. Grenzgeb. d. Med. & Chir., 1901, I, 1.


'•Zerebrale Luftembolie. Diss., Kiel, 1914. Beitr. z. Klin. d. Tuberkul., 1913, Bd. XXXI, S. 159.

"Personal communication.

" Personal communication.

" Ueber gefahrdrohende Zufalle und plotzlichem Tod nach Thorakotomie. Berl. klin, Wchnschr., 1879, Nr. 35, S. 521-526.

"Convulsions eclamptiques S. la suite de la thoracocentgse. Bull, et M6m. de la Soc. med. d. hop. de Paris, 1870, VI, 261-267.

'-" l7j : Cerenville, Des manifestations encephaliques de la pleuresie purulente. Revue med. de la Suisse rom., 1888, VIII, 19.

"/>!.• Wever, Cerebrale Luftembolie. Diss., Wiirzburg, 1914, S. 63.

'•= Luftembolie im Auge. Beitr. z. Klin. d. Tuberkul., 1913, XXVIII, 479.

" Ueber die anatomischen Folgen der Luftembolie im Gehirn. Verb. d. deutsch. Kongr. f. inn. Med., Wiesb., 1913, XXX, 359-365.

'*Brfahrungen iiber kiinstlichen Pneumothorax. Beitr. z. Klin, d. Tuberkul., 1912, XXII, 165.

'" Experimentelle Studien iib. Lungen- und Pleurachirurgie. Mitt. a.d. Grenzgeb. d. Med. & Chir., 1907, Bd. Ill, Suppl.-bd., S. 789.

" Arterielle Luftembolie und die Technik d. kiinstl. Pneumothorax. Deutsche med. Wchnschr., 1913, Nr. 26, S. 1245.


THE EFFECT OF SALINE PURGATIVES ON THE ABSORPTION OF

OTHER DRUGS*

By D. I. Macht, M. D. and E. M. Finesilver, M. A.

(From, the Pharmacological Laboratory. Johns Hopkins University)


Introductory

The present investigation was begun as the result of a personal experience. While suffering from an attack of the grippe one of the writers took for the relief of a severe headache a full dose of aspirin which had generally proved efficient for the purpose on other occasions. In the present instance no relief was experienced. The patient then swallowed a tablet of phenacetin and salol, also without relief. This was followed by five grains of pyramidon and this also did not prove any more efficient in the relief of the headache and pains than the other antipyretics. On pondering over the cause of the ineffectiveness of the above medication it was recalled that, before having recourse to the antipyretics, the patient had taken a saline purgative. It therefor occurred to him that possibly the saline purgative might have played a role in preventing or retarding the absorption of the drugs which were taken subsequently to it. It is well known that the pharmacodynamics of saline purgatives consists chiefly in the poor absorbability on the part of the intestinal walls of certain ions such as those of magnesium sulphate, phosphate, etc., and the accumulation of fluid in the intestines through the osmotic action


Read before the Association of American Physicians, May 4, 1922.


of the unabsorbed salt which, instead of being absorbed, actually draws more fluid into the intestinal lumen. This peculiar property of the saline piirgatives considered in connection with the above described clinical experience led the authors to inquire into the effect, if any, that such saline purgatives might have upon the absorption and excretion of other drugs given by mouth simultaneously with or subsequently to the taking of the laxative.

Phenolsulphonphthaleix

Administration by stomach Tube. — One of the most convenient drugs for beginning such a study was thought to be the dye, phenolsulphonphthalein. This drug, as is well known, is rapidly and completely excreted by the kidneys irrespective of the method by which it may be administered. The rate of excretion can be easily determined quantitatively by the colorimetric method; hence its widespread use as a kidney function test.

Accordingly, this drug was administered to a number of dogs with a given quantity of water through the stomach tube. At the end of two and one half hours the dogs were cathcterized and the amount of phenolsul])honphthalein excreted by the kidneys in the two and one half hours was determined colorinietrically. A few days later the same animals were given exactly the same amount of phenolsulphonphthalein in exactly the same amount of water as in the previous experiments, but on


September, 1922]


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this occasion the water coutained 5 pei' ceut of sodium sulphate in solution. The fluid together with the dye and sodium sulphate was administered through a stomach tube as before, and the total output of phenolsulphouphthalein was again determined at the end of two and onehalf hours. It was found that the amount of dye in the urine this time was less than one-half the amount excreted in the previous or control experiments. Similar experiments with phenolsulphouphthalein with and without sodium sulphate were performed on rabbits, with similar results; that is, it was found that the sodium sulphate solution markedly retarded the absorption and consequent excretion of the dye.

The same procedure was repeated on other occasions on both dogs and rabbits, but, instead of sodium sulphate, solutions of magnesium sulphate were used. It was found that this saline cathartic acted very much like sodium sulphate, in so far as it markedly retarded the absorption and excretion of phenolsulphouphthalein.

Experiments on Intcstin<il Loops. — In order to analyze the above phenomena more in detail, the following experiment was performed. A cat was anesthetized with ether, a laparatomy was made and the small intestines were exposed. Two loops of the small intestine, each 25 cm. long, were tied otf, leaving the vascular supply intact. Into one of the loops there was injected 10 c.c. of a solu tion of sodium sulphate (10 per cent) plus 1 c.c. of phenol.sulphonphthalein. Into the other loop was injected 1 c.c. of phenolsulphouphthalein in 10 c.c. of plain water. The abdominal wound was closed and the animal put in a cage. At the end of one hour the cat was killed with ether, the abdomen was opened again and the two loops of the intestine were examined. It was evident at once that one of the loops was greatly distended, like a sausage, while the other was collapsed. The two loops were excised and the fluid contents of each were measured. In the control loop, into which 10 c.c. of water had been injected, there were found only 1 or 2 c.c. of intestinal contents, whereas in the other loop, the one with the saline solution, the volume of the fluid had increased to over 30 c.c. Each loop of the intestine was washed out with water, the washings were combined with the fluid contents already obtained, and the amount of dye present in the intestinal loops was determined. It was found that whereas in the control over 50 per cent of the plienolsulphonphthalein had been absorbed by the intestine at the end of one hour, in the second loop there was jiresent over 90 per cent of the dye. It was thus evident that the sodium sulphate had retarded markedly the absorption of the phenolsulphouphthalein from the intestinal lumen. This difference in absorption between the two loops was further strikingly corroborated by immersing each loop separately in a weak alkali. On treatment with the alkali, the mucosa of the normal loop became intensely red in color, showing that the dye had


penetrated inside the cells. On the other hand, the second loop showed very little pink discoloration, thus indicating that very little of the dye had been absorbed. On extracting each intestinal segment with alcohol, the phenolsulphouphthalein in the tissues themselves was extracted, and a much greater amount of dye was obtained from the control loop. Several such experiments were performed with similar results.

Intraniuscukir Injections. — It was then thought possible, though by no means probable, that perhaps the administration of saline purgatives by the stomach might exert some effect on the excretion of phenolsulphonphthalein even if the dye were given by injection. Accordingly, to settle this point experiments were made first on .some rabbits. The usual kidney function test was performed on two rabbits by injecting each of them with 1 c.c. of phenolsulphouphthalein intramuscularly. At the same time each animal received 50 c.c. of water through the stomach tube. The urine was collected at the end of the first hour and at the end of the second hour and the amount of dye excreted was determined by the usual procedure. A few days later each of the same animals was again injected with 1 c.c. of pheuolsulphonphthalein intramuscularly, and simultaneously with the injection there was introduced through the stomach tube about 50 c.c. of water containing 5 per cent of magnesium sulphate. The output of the dye was again determined at the end of the first and second hours, and it was found that the excretion of phenolsulphouphthalein was markedly delayed in the experiments in which the salines had been administered by stomach. Precisely similar results were obtained with sodium sulphate.

Following these studies on rabbits, similar experiments were performed on three dogs. Phenolsulphouphthalein was first injected intramuscularly and the kidney function of the dogs was determined by measuring the dye output at the end of the first and second hours. A few days later the same dogs were given the same amount of dye by injection, but before the injection each dog received by stomach tube a quantity of sodium sulphate. It was found in the latter series of experiments that there was a distinct diminution in the phenolsulphonphthalein output. Following these experiments some days later another normal kidney function test was performed on the same animals, and two days later the same animals were injected with the dye and magnesium sulphate was administered by stomach tube. In this case also the phenolsulphonphthalein output was diminished or retarded by the administration of the salines. From the above experiments it was evident that the administration of saline purgatives by stomach, tended to retard the excretion of phenolsulphonphthalein, not only when given by mouth but also after injection of the drug.

Observations on Man. — Inasmuch as the phenolsulphonphthalein test is a perfectly harmless one, and inasmuch


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as saline purgatives can be taken with impunity by almost every one, a series of tests were made to discover whether the above experimental findings hold good for man. Accordingly, a number of observations were made on the authors themselves, their colleagues and friends. The results obtained indicated that the administration of saline purgatives, definitely and in most cases markedly, affected the kidney output of phenolsulphonphthalein. More detailed results of these experiments appear elsewhere.' In this place it is sufficient to state that the conclusions drawn from a study of phenolsulphonphthalein are of immediate clinical importance, as may be illustrated by the following case described to the authors by a genitourinary surgeon.

Protocol. — A middle-aged man came to a physician for treatment of the bladder. In connection with a routine physical e-\amination, a phenolsulphonphthalein test for kidney function was made and to the doctor's surprise and consternation it was found that the phthalein output for the first two hours was very low, about 16%, thus pointing to a derangement of kidney function. The patient in question was in the habit of drinking every morning a dose of a well known bitter water. During the course of treatment he became tired of taking the above bilter saline purgative and changed about to a daily dose of compound licorice powder. It happened that the physician soon after made a second kidney function test and to his surprise he found that the kidney output had improved and was 38%.

The above history is not an unusual one. In the present paper, however, the research undertaken deals not so much with the influence of saline purgatives on the absorption and excretion of drugs administered by injection as with experiments concerning the effect of salines on the absorption and excretion of various drugs introduced into the stomach and intestines. A large number of drugs were studied in this connection and the results obtained are summarized briefly in the following pages.

Absorption of Potassium Cyanide

In order to put the above experimental data as obtained with phenolsulphonphthalein to a crucial test experiments were made with solutions of the powerful and rapidly acting poison potassium cijanUlc. In these experiments cats and dogs were kept under anesthesia, laparotomy was performed, and a loop of the small intestine was tied oft' without interference with the mesenteric circulation. A blood pressure tracing and a tracing of the respiratory movements of the chest were recorded on a kymograph. After preparation of the animals, a given dose of potassium cyanide solution in a definite amount of water (10 c.c. of 57o solution of KCX) was injected into the intestinal loop and the blood pressure and respiration curves were studied from the beginning of the injection until stoppage of the respiration and the heart of the animal. The time of death was then noted. In another series of animals the same procedure was carried out with the exception that the potassium cyanide was adminis


tered in a solution of sodium sulphate (10 c.c. of a 5% solution KCN in 10% XajSOu). Blood pressure and respiratory tracings were taken again and the time of death was noted. It was found that, when potassium cyanide was given together with the sodium sulphate, its ab.sorption and the subsequent pictui'e of cyanide poisoning were greatly retarded, thus indicating that sodium sulphate tended to delay the absorption of the potassium cyanide by the intestinal mucosa. The following tracing illustrates one of the experiments. In this experiment potassium cyanide was injected into a loop of intestine together with sodium sulphate. It will be noted that the heart did not cease beating until about half an hour (36 minutes) after the injection of the poison. In control experiments, the same dose of cyanide injected in plain water solution produced death in about 7 minutes (Fig. 1).

In another experiment, after the injection of a 4% solution of potassium cyanide in 20 c.c. of sodium sulphate 10%, the animal lived 70 minutes, whereas the control dog, receiving the same quantity of cyanide in aqueous solution, died in seven minutes.

Absorption of Chloretone.

Chloretone was selected as representative of the hypnotic group of drugs. This drug has been used extensively in this laboratory as an anesthetic for cats and dogs. The eft'ect of saline purgatives on the absorption of chloretone is most strikingly illustrated by the following experiments.

Exp. A. — A cat weighing 3 kilos was anesthetized with ether, laparotomy was performed and a loop of the small intestine, 20 cm. long, was tied off. Into this was introduced 100 mgms. of chloretone, per kilo weight of cat, dissolved in 20 c.c. of water. The abdomen was closed and the cat put in a cage. The effects of the ether anesthesia soon passed away but the anesthetic properties of chloretone quickly began to manifest themselves, so that before the end of an hour from the beginning of the experiment the animal was completely anesthetized by it.

Exp. B. — A cat weighing 3 kilos was anesthetized with ether as above, a loop of the intestines, 25 cm. long, was tied off. and into this was introduced a chloretone solution, 100 mgms. per kilo weight of cat, dissolved in 20 c.c. of a 5% solution of sodium sulphate. The cat was put in a cage. IDffiects of the ether soon passed away, but the anesthetic action of the chloretone did not manifest itself, and at the end of an hour the cat was sitting up quite contentedly. It was killed afterwards with ether.

Absorption and Excretion of Some Opiates

Several opium derivatives were studied in this connection.

Apomorphiii. — It is well known that apomorphin produces vomiting when introduced by mouth or injected into an animal, through being absorbed into the circulation and carried to the vomiting center in the medulla. A number of dogs were first given small amounts of apomorphin hydrochloride (30 mgms.) dissolved in 50 c.c. of water. The on.set of vomiting after the administration of the drug was noted. This varied for individual dogs,


THE JOHNS HOPKINS HOSPITAL BULLETIN. SEPTEMBER. 1922


PLATE XLI


3 2-^'



Fic. 1. — Experiment, June 7, 1921. Dog, 10 kilos. Ether anesthesia. Injected into intestinal loop 25 cm. long. 10 cc. of KCN 59f in a lO'/r solution of Na ^ SOj. Death occurred at 4.30 P. M. Beginning of injection was at 3.54 P. M.


September, 1922]


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but was pretty constant for any one animal. The same animals were given the same doses of apomorphin in the same volume of fluid on another occasion. In the second series of experiments, however, instead of water, a solution of sodium sulphate (5%) was used as a vehicle. It was found that in the latter series of experiments vomiting came on much later and some times not at all.

MMphhic. — To study the effect of salines on the absorption of morphine the bimeconate salt was found to be very snitable. A cat was anesthetized with ether and two loops of the small intestine of about the same length were tied off. Into one of the loops were injected 10 c.c. of water in which was dissolved 50 mgms. of morphine bimeconate. Into the other loop the same amount of morphine bimeconate was injected but dissolved in 10 c.c. of a afc solution of sodium sulphate. At the end of one liour the cat was killed, each loop of intestine was carefully cut out, and the intestinal contents of each were measured. The control loop at the end of one hour contained less than 1 c.c. of fluid. The loop containing the saline purgative at the end of one hour was found to contain about 35 c.c. of fluid. The contents of each loop plus the washings of the mucosa were then examined for the content of morphine bimeconate. After first coagulating the proteids and filtering, a colorimetric determination of mecouic acid with ferric chloride was made. It was found by this method that the specimen from the loop containing sodium sulphate gave a much deeper color reaction for meconic acid than the control loop, thus indicating tliat much less of the bimeconate had been absorbed in the first case than in the control.

Pantopon. — The ett'ects of pantopon on the respiration were studied. A rabbit was trained to lie quietly on a board without anesthesia and its rate and depth of respiration were registered by means of a stetliograph. This rabbit was then given through a stomach tube 20 mgms. of pantopon in 20 c.c. of water. The results obtained are illustrated by the following protocol.

Rabbit— 2000 grams. 11:30 A.M. — Respirations — 58 per minute 11:45 A.M.— " —56 "

11:50 A.M.— " —56 "

11:55 A.M.— •• —56 "

12 M. — •• —56 "

Pantopon, 20 mgms. in 20 c.c. of water is introduced by stomach tube.

12:04 P.M. — Respirations — 52 per minute 12: OS P.M.— " —50 "

12:10 P.M.— " — 4S '•

12:16 P.M.— " —46 "

12:28 P.M.— •• — 44 " " and very shallow

12:35 P.M.— • —40

12:45 P.M.— " —40

12:55 P.M.— •• —40 "

Exp. 2. — The same animal was used the following week and in this case the same amount of pantopon was administered by stomach tube, but mixed with 20 c.c. of a 5% solution of sodium sulphate.


Rabbit— 2000 grams. 11:15 — Respirations — 52 per minute 11:30— " —50 "

11:45— " —50 "

11:48 — " — introduced 20 mgms. of pantopon in 20 c.c.

of sodium sulphate solution, 5%.

12 M. — Respirations — 50 per minute

12:15— '• —50 "

12:30- " —50 "

12:40— " —54 "

12:45— " —54 "

12:55— " —50 "

1 — " —50 " 1:30— " —50 "

2 — " —50 "

2:30— " —46 " " first effect of narcotic

is noticeable.

3 — " —44 "

The above protocols illustrate strikingly the difference in the narcotic action on the respiration due to the dift'erence between the absorption of aqueous and saline solutions of pantopon.

Other Alkaloids

In addition to the opium derivatives a number of other alkaloids were tested. Among these were cocain, strychnin, atropin and quinidin.

Cocain. — The effect of saline laxatives on the absorption of cocain is illustrated by the following experiments.

Exp. Nov. 10, 1921. — Dog. 7.15 kilos. Ether anesthesia. Laparotomy was performed and a loop of the small intestine, 30 cm. long, was tied off. Into this loop were injected 10 c.c. of a 2% solution of cocain hydrochloride plus 20 c.c. of water. A blood pressure and respiration tracing was made and the first appearance of convulsions was noted. It was found that the cocain in this case very rapidly produced toxic symptoms, as indicated by failure iu respiration, fall in blood pressure, and onset of tremors. Death occurred in 35 minutes after the injection of the alkaloid into the intestine.

Exp. Nov. 15, 1921. — Dog: 6 kilos. Ether anesthesia; laparotomy. A loop of small intestine, 50 cm. long, was tied off and into this were injected 20 c.c. of cocain-hydrochloride solution (2%) and 15 c.c. of a 5% solution of sodium sulphate. Blood pressure and respiration tracings were made and tremors and convulsions were looked for. There was no appreciable effect on the blood pressure or respiration or on the nervous system noted in the course of one hour from the time of injection. The dog was sacrificed later for another purpose.

From the above experiments it is evident that while in the control animal the loop was shorter in length and consequently the absorbing surface smaller and the amount of cocain injected was much less than in the saline experiment, nevertheless, toxic symptoms developed much sooner, thus verj- strikingly .showing the inhibiting efl'ect of sodium sulphate on the absorption of cocain hydrochloride.

Strychnin. — Experiments with strychnin were made on rabbits and dogs. The procedure was to tie off loops of intestine, inject a given dose of strychnin and note the first appearance of convulsions. It is hardly necessary to state that the experiments were performed under gener


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al anesthesia. It was found that when sti-ychnin was administered in a solution of sodium sulphate the convulsions came on a little later than normally. Thus for instance, in one rabbit 4 mgms. of strychnin nitrate were injected into a loop of the small intestine, 15 cm. long, under paraldehyde anesthesia. The injection was followed by convulsions in 28 minutes. In another rabbit of the same weight the same dose of strychnin in a 5% solution of sodium sulphate was injected, and the convulsions did not set in until 35 minutes after injection. In dogs a similar difference in the time of onset of convulsions was noted. In all these experiments pains were taken to have the same degree of anesthesia in both the control and experimental animals, in order to eliminate differences in nervous response due to that source.

Atropin. — The effect of salines on the absorption of belladonna alkaloids is well illustrated by the following experiments.

Exp. May 11, 10.i2. — A cat was anesthetized with isopiopyl alcohol by the method described by one of the authors elsewhere.^ A loop of intestine, 20 cm. long, was tied off. Into this was injected a mixture of 4 c.c. of tincture of belladonna root plus 10 c.c. of water. The response of the heart to vagus stimulation with an induction coil was noted and the size of the pupils was also observed. It was found that the injection of belladonna produced a definite mydriasis in less than 5 minutes and paralyzed the vagus control of the heart within 10 minutes, after injection.

Exp. May 11, 1922. — A cat was anesthetized with isopropyl alcohol, as above. A loop of intestine, 25 cm. long, was tied off. Into this loop was injected the same amount of tincture of belladonna as above plus 10 c.c. of a 5% solution of sodium sulphate. The first appearance of mydriasis in this cat was noted after 15 minutes. The failure of vagus inhibition of the heart did not occur until after 20 minutes.

Quinidin. — Inasmuch as quinidin is employed extensively at present in the treatment of certain cardiac conditions, some experiments were made with this alkaloid. Equivalent amounts of quinidin sulphate were injected into loops of intestine, dissolved in water in some cases and in sodium sulphate solution in others. It was found by chemical tests that the absorption of this alkaloid was delayed bj' sodium sulphate. Very accurate quantitative determinations in this case, however, could not be made owing to the fact, emphasized by Lipkin ^ and others, that quinin and quinidin are rapidly decomposed in the intestinal canal. Ample evidence was nevertheless obtained to show a difference in absorption between the aqueous and saline solutions of the drug. Heart Drugs

Digitalis. — Some 15 experiments were made with digitalis on cats. A sample of digitalis tincture was first assayed by the cat method and its minimal lethal dose was found to be 0.86 c.c. per kilo. This tincture was u.sed in some experiments as such, while in others it was evaporated to drive off the alcohol and diluted with saline to the original volume. Experiments were made first by administering a large dose of digitalis through


the stomach tube and determining the time of death with and without the addition of sodium suli)hate. This method, however, was not found to be the most suitable on account of vomiting and other disturbances, and so resort was made to loop experments. In these experiments long loops of the intestine were tied off and a dose of digitalis sufticient to kill the animal was injected. Observations were then made on the rapidity of exitus with and without saline purgatives. The experiments were made under ether or isopropanol anesthesia.- The following protocols will illustrate the findings.

Exp. A. — Cat, 2.2 kilos. Ether was given and then isopropyl alcohol. After laparotomy a loop of intestine, 55 cm. long, was tied off without interfering with the circulation. Into this 10 c.c. of the evaporated tincture of digitalis were injected together with 20 c.c. of water. The injection was made at 2:35 P. M. The cat was dead at 4 : 30 P. M. Tlie volume of the contents of the loop after death was found to be 20 c.c.

Exp. B. — Cat weighing 1.8 kilos. Anesthetized as above. After laparotomy, a loop of intestine, GO cm. long, was tied off. Into this was injected 10 c.c. of evaporated tincture of digitalis plus 10 c.c. of water plus 10 c.c. of a solution of sodium sulphate (3%). The time of Injection was 2:50 P.M. The cat lived until 8:45 P.M. The contents of the loop after death was found to be 50 c.c.

Exp. C. — Cat, 2.0 kilos. Anesthetized as above. Loop of intestine, about 50 c.c. long, was tied off. Into this was injected 10 c.c. of evaporated tincture mixed with 15 c.c. of castor oil. Injection was made at 10:25 A. M. The cat died at 11:55 A.M.

The above experiments as well as others clearly brought out the fact that when digitalis was administered together with saline laxatives its absorption ami consequent toxic symptoms took place much more slowly than when the drug was administered with water.

ConiHilhiria. — Several experiments were made on cats and dogs with fluid extract of convallaria. This drug was found to be much more toxic than digitalis, but the relative absorption with and without sodium sulphate was the same as in the case of digitalis.

Antipyretics

Of this group of drugs quite a number were studied and numerous experiments were made both on animals and men because one of them had been the starting point of the whole research. The following antipyretics were examined ; sodium salicylate, salol, aspirin, antipyrin, acetphenetidin and lactophenin.

Sodium t<ulic!/hitc. — The test for this drug being very simple, experiments with it were made both on rabbits and man. The drug was administered to rabbits in some cases by stonuu-h tube, in other cases by intraperitoneal injections, and the time of the first appearance of salicyl in the urine was noted. In man sodium salicylate was administered by mouth and the time of the first appearance in the urine was determined in tlie same waj'. The test for salicyl was made by the addition of ferric chloride and the characteristic violet color was obtained. In doubtful cases the urine was acidified and shaken with ether and the test with ferric chloride was


September, 1922]


335


repeated. It was found, both in the case of rabbits and in the clinical cases, that the excretion of salicylate in urine was markedly delayed by the pi'evious or even simultaneous administration of various saline purgatives, such as magnesium sulphate, sodium sulphate and sodium and potassium tartrate. It was not delayed however, by the administration of other laxatives of the non-saline variety, such as castor oil and cascara sagrada. The following experiments will illustrate some of the findings.

Exp. A.- — Rabbit weighing 1800 grams was catheterized and the urine tested with ferric chloride with a negative result. At 11 A. M., 5 c.c. of a 2% solution of sodium salicylate plus 20 c.c. of water was introduced into the stomach through a sound. The urine gave a positive salicyl test at 11:45 A. M.

Exp. B. — Rabbit weighing ISOO grams, with negative urine test, was given at 11 A.M. 5 c.c. of sodium salicylate (2%) plus 20 c.c. of a 1.5% solution of sodium sulphate. The first positive test for salicyl in the urine did not appear until 2:40 P. M,

Exp. C— Rabbit weighing 2000 grams. At 10:15 A. M. introduced into stomach 20 c.c. of a 3% solution of sodium sulphate. 10:45 A.M. introduced into stomach 2 c.c. of sodium salicylate (2%). 11:50 A. M. salicyl test — urine negative. 12:45 P. M. salicyl test — urine negative. 2:30 P. M. salicyl test — urine negative. 3:45 P. M. — urine negative. No positive test was obtained that afternoon.

Exp. D. — Rabbit weighing 1500 grams, injected with 2 c.c. of a 2% solution of sodium salicylate intraperitoneally, at 3:05 P. M. Positive test for salicyl in the urine was obtained at 4:15 P. M.

Exp. E. — Rabbit weighing 1800 grams injected with 2 c.c. of sodium salicylate (2%) intraperitoneally at 3:15 P. M. Urine gave ixisitive test for salicyl at 4:20 P. M.

Exp. F. — Rabbit weighing 2000 grams given 5 c.c. of cascara sagrada tincture in water by stomach tube, and this was followed by an intraperitoneal injection of 2 c.c. of a 2% solution of sodium salicylate, at 2:45 P. M. Urine gave a positive test for salicyl at 4:10 P. M.

Exp. G. — Rabbit weighing 1800 grams was given 10 c.c. of castor oil by stomach tube. 15 minutes later injected with 2 c.c. of a 2% solution of sodium salicylate intraperitoneally. Urine gave a positive test in one and a half hours.

Exp. H. — Rabbit weighing 1500 grams. At 1 P.M. given 15 c.c. of a 5% solution of sodium sulphate by stomach tube and simultaneously given an injection of 2 c.c. of sodium salicylate (2%) intraperitoneally. First indication of salicyl in the urine did not appear until 4 P. M.

Salol. — Experiments with phenyl salicylate, or salol, were made on man. As is well liuowu, when this drug is administered by mouth, a positive test for salicyl is normally given in about an hour, to an hour and a half. It was found that the absorption and consequent excretion of salol was delayed by the administration of saline laxatives. Thus one of the authors was found to excrete the drug normally in a little over an hour. The same subject when taking salol after a previous dose of magnesium sulphate (V2 ounce) did not give a positive urine test for nearly three hours.

Aspirin. — Experiments with aspirin or acetylsalicylic acid were made on animals and on man. It was found that the absorption of the drug was markedly delayed by the previous and even simultaneous administration of


saline purgatives. In order to make sure of the first appearance of the salicyl after this drug, the tests for it were made with special care. In all cases whether dealing with urine or with other fluids the specimens were first hydrolyzed by the addition of a little sodium hydroxide and boiling, in order to make sure of bi-eaking up all the aspirin into salicylic acid. Solutions were then carefully titrated and made very slightly acid and then definite quantities of ferric chloride were added.

The effects of salines on the absorption of aspirin are most strikingly illustrated by loop experiments. Cats were u.sed in these experiments. Under anesthesia the abdomen was opened and two loops of the small intestine of approximately the same length were tied off. Into one of these 0.3 gi'am of aspirin suspended in water was introduced through a funnel and the loop was tied otf. Into the other loop the same quantity of aspirin was introduced with the same amount of fluid but for this second loop instead of water a solution of sodium sulphate (2.5%) was used. The abdomen was clo.sed and the cat was killed an hour later. The contents of each loop were carefully collected and measured. It was found in every case in which the aspirin was introduced in aqueous solution, that most of the fluid at the end of one hour had been absorbed. In the loops into which aspirin was introduced with sodium sulphate, the volume of fluid at the end of one hour instead of being decreased was actually increased to two or three times the original volume. The contents of each loop plus the washings of each loop were examineit chemically for the amount of aspirin remaining in each and the quantity of the drug in each loop was compared colorimetrically. It was found that at the end of one hour about three times as much aspirin was absorbed from the loop with water than from the loop into which the aspirin was introduced with sodium sulphate. In other experiments, similar differences in absorption were found even after two hours.

Experiments with aspirin on man yielded very similar results. These experiments were made on a large number of students and also on the authors themselves. Aspirin was administered alone with some water, and some days later the same subjects took the same amounts of the drug with saline purgatives, such as magnesium sulphate, sodium sulphate, a Seidlitz Powder, or sodium phosphate. It was found that in most cases normally the first appearance of the salicyl in the urine occurred within one hour after administration of tlie drug. When, however, the aspirin was taken after a saline purgative, no positive test could be obtained from the urine within anywhere from three to six hours or longer. In fact, when the experiment was performed in the afternoon, no positive test could be obtained on that day. No such effect was noted after purgatives of the non-saline type.

Antipyrin. — A few experiments were made with antipyrin on man and on other animals. The drug was


33G


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detected by means of the ferric chloride reaction, which gives a red color, and the isonitroso test made by the addition of sodium nitrite, acidifying with sulphuric acid and shaking out with chloroform. Experiments on intestinal loops were performed with and without sodium sulphate and clearly indicated that the absorption of the drug was markedly delayed by the administration of sodium sulphate. The same phenomenon was noted after the administration of antipyrin by mouth to man.

Lactophenin. — The following experiment with lactophenin will serve to illustrate the effect of saline laxatives on the absorption of antipyretics belonging to the phenetidin group. Two loops of intestine were made in a cat and into each of these 50 mgms. of lactophenin was introduced, into one together with water, into the other together with a solution of sodium sulphate. After one hour the contents of each loop were tested for indophenol, by boiling with concentrated hydrochloric acid and treating with chromic acid. It was found that the sodium sulphate markedly delayed the absorption of the drug.

Potassium Iodide and Hexamethylenamine

These two drugs, it is well known, are among those which are most rapidly excreted by the animal organism. Not only are they very rapidly absorbed but they also very soon appear in most of the secretions of the body. In the urine hexamethylenamine can be detected within 20 minutes, while iodides appear in the urine in less than 10 minutes after ingestion. It was therefore with special interest that experiments were^niiide with both of these di'ugs on man.

Hcjcainethylcnaminc (urotropin) was administered to a large number of normal subjects in doses of 0.5 gram by mouth and the time of the first appearance was ascertained. The same subjects on another day were given the same drug together with or following a dose of saline laxative and the time of the first appearance in the urine was compared with the normal. The test used was the so-called Hehner's test which detects both hexamethylenamine and free formaldehyde. It is performed by taking 0.5 c.c. of the urine and mixing it with an equal quantity of fresh milk and then stratifying it with 1 c.c. of 50% sulphuric acid containing a trace of ferric chloride. A violet ring very soon appears. It was found that, whereas normally after the ingestion of 0.5 gram of urotropin its excretion could be detected in the urine in about 15 minutes, the same subjects when taking the drug witii a saline purgative showed a delay in the excretion. Thi.s delay was in some cases not very remarkable but in other subjects it was very striking, as may be illustrated by the following ])rotocols.

Exj). D. M. — Given 0.5 gram urotropin together with a Seidlitz Powder. The first appearance of a positive test in the urine was 214 hours after the administration of the drug. The normal time of appearance of the drug in the same subject was 20 minutes.

Exp. N. W. — Normal excretion of urotropin was 15 to 20 min


utes. The subject was given half an ounce of magnesium sulphate and this was followed by 0.5 gram of urotropin. A faintly positive test was obtained 2 hours after administration.

Exp. O.K. — Normal excretion of urotropin about 20 minutes. EJxcretion after half an ounce of magnesium sulphate was lii> hours later.

The experiences with potassium iodide were not so striking as those with urotropin. Twenty minims of the saturated solution were given to various subjects with and without saline purgatives. The normal excretion of iodide could be detected within 15 minutes. After the previous administration of magnesium sulphate a slight delaj- was ob.served. Thus, in one subject the first positive indication of iodide in the urine was one hour after ingestion. In another it was 15 minutes. In still others the drug could be detected in half an hour after taking by mouth. The test employed was by the addition of 0.5 c.c. of a 2% solution of sodium nitrite, acidifying with 0.5 c.c. of dilute sulphuric acid and then shaking out with chloroform. A positive test was indicated by a pink or violet coloration of the chloroform.

Antiseptics

Phenol. — Several years ago one of the authors in studying lavage in acute carbolic acid poisoning showed that the most suitable solution for lavage in acute carbolic acid i)oisoning was a saturated solution of sodium sulphate.* In the light of the present investigation the results obtained in this toxicological study of carbolic acid poisoning were not surprising. The sodium sulphate undoubtedly served two purposes : it not only served as a solvent for the washing out of the carbolic acid, but it also acted as a purgative and, above all, as an agent which tended to prevent the further absorption of the poison. This experiment with sodium sulphate solution in the treatment of carbolic acid poisoning prompted the authors in the present investigation to make some observations on the effect of saline purgatives on the absorption of mercuric chloride.

Bichloride of Mercury. — Two sets of experiments were made. In one series rabbits were used. To some of these an aqueous solution of mercuric chloride was administered by stomach tube. To others the same quantity of bichloride, proportionate to weight, was administered together with or following a previous administration of sodium sulphate. It was found, when a concentrated solution of saline was employed, that the animals succumbed more quickly in the first series of experiments than in the second. In other words, the sodium sulphate seemed to retard the absorption of the mercury and promote its elimination by purgation. The following two protocols may serve as illustrations.

Exp. Dec. 21, 1921. — Rabbit A, weighing about 2000 grams, was given by stomach tube 125 mgms. of mercuric chloride In 20 c.c. of water. The animal died in 12 hours.


Septemuer, 1922]


337


Kxp. Dec. 21, in.il. — Rabbit B, weighing about 200 grams, was given by stomach tube 125 mgnis. of mercuric chloride in 20 c.c. of 7.59'c sodium sulphate solution. The animal was alive on the following day, Dec. 22nd. when a similar dose of bichloride in sodium sulphate was administered. The rabbit died a day later.

In some of the experiuieuts on rabbits the bladder was catheterized and an attempt was made to detect the presence of mercury in the urine. The method employed was similar to those described by Vogel and Lee'^ and later by Elliott." A spiral of copper wire was placed in the urine and concentrated hydrochloric acid, 10 c.c. in 200 c.c. of urine, was added. The flask was thoroughly shaken and allowed to stand over night. The copper wire spiral was then heated in a small hard glass tube and any mercury that might be adherent to it was volatilized and allowed to deposit on gold foil. By this method the experiments .seemed to indicate that more mercury was absorbed and consequently excreted in the urine of the control animals than in the case of tho.se which had had a previous dose of sodium sulphate. Inasmuch as these e.vperinients, however, were not very satisfactory, a quantitative study of the absorption and excretion of bichloride was made on cats by means of intestinal loops. Three such experiments were performed and in each case the figures obtained clearly indicated that the sodium sul[)hate distinctly retarded the absorption of the bichloride. Tlie following protocols illustrate well these findings.

Exp. Dec. 2(1, tt)2l. — A. — A cat weighing about 3 kilos was anesthetized with ether. The small intestine was tied off just below the pylorus and again above the iliocoecal valve. Into this intestine was injected 50 c.c. of water containing 450 mgms. of mercuric chloride. The abdomen was closed and the animal was kept narcotized until its death which occurred in about three hours. The intestine was then cut out, its contents collected and the surface of the intestine thoroughly washed. The washings were mixed with the intestinal contents. The total volume of fluid was examined quantitatively for mercury.

Exp. Dec. 2(1, 1021. — B. — Another cat weighing about 3 kilos was treated in the same way as above. The whole small intestine was tied off above and below and 450 mgms. of bichloride were injected in 50 c.c. of 5% solution of sodium sulphate. This experiment was begun about 11 A. M. and the animal was still living narcotized quite late in the afternoon. The exact time of death was not noted, but the cat was found dead the following morning. The whole small intestine in this case was also cut out and the contents and washings preserved and worked up for mercury.

The macroscopical appearance of the intestines in lOxperiments A and B were quite different. The mucosa in cat A showed violent irritation. It was extremely congested and hemorrhagic and contained a bloody mucus. In cat B the intestinal mucosa appeared more normal. It was mucii paler than in the previous experiment and did not show so much irritation or hemorrhagic exudate.

Chemical tests for mercury were made by first treating tiie intestinal contents of each cat with concentrated hydrochloric acid and a little potassium chlorate. The solutions were boiled for several hours until all traces of


chlorine had been driven off. They were then diluted in each case to the same volume. The solutions were then divided into two parts and examined for mercury by two independent workers. The mercury was precipitated with hydrogen suljihide and filtered tlirough Gootch filters. The sulphide was then weighed. The quantity of mercuric sulphide obtained in Experiment A was 10.5.6 mgms. an amount equivalent to 123.21: mgms. of mercuric chloride. The mercuric sulphide obtained from the intestinal contents of cat B was 141.2 mgms., an amount equivalent to 1C1.78 mgms. of mercuric chloride. The difference Iietween the above figures indicates that less mercury was absorbed from the intestine containing the sodium sulphate than from the one in which the bichloride had been introduced in aqueous solution. Two other sets of experiments on cats yielded similar quantitative differences.

Discussion The experiments described above are quite sulficient to establish beyond doubt that saline purgatives can and do affect markedly the absorption and consequent excretion of other drugs. Bearing in mind the pharmacodynamics of saline purgatives such a result is not altogether surprising when both the saline and other drugs are administered by mouth or introduced into the intestinal lumen. More remarkable, however, are the findings in the case of injections such as were made with phenolsulphonphtiialein in man and in other animals and with sodium salicylate in rabbits. It is evident that the absorption and excretion of drugs are influenced to some extent by saline laxatives even when such drugs are given by injection after the purgatives have been previously administered by stomach tube. The explanation of this phenomenon is not altogether clear. Undoubtedly, however, it must have something to do with the concentration of tlie blood and the distribution of fluids in the body. It lias been known for some time that saline purgatives will concentrate the blood, as is indicated by the blood count, and this has been recently emphasized by the work of Underbill.^ This probably plays a role in the pheuo niena studied in this paper. The findings obtained are of an immediate practical interest. It is evident that it is not immaterial whether drugs are administered with or without certain cathartics. The salines will markedly retard the absorption of many drugs, such as antipyretics, digitalis, etc., and the therapeutic results expected in clinical cases may consequently not be attained. The authors liave studied tlie relation of other laxatives such as calomel, cascara sagrada, castor oil, etc., in this con nection but have not found any important influence exerted by these drugs on the absorption and excretion of other medicaments. The exact duration of the inhibitory effect of saline purgatives on tlie intestinal mucosa has not been made a subject of exhaustive study by the authors. It will, of course, vary with the drugs and the


338


[No. 379


purgatives used and also with the individual subject. This inhibitory or retarding influence on the absorption of medicaments should, however, be born in mind by the careful clinician who may wish to obtain a prompt response on the part of the patient to the effects of any given medication.

Another important bearing which the above findings serve to reveal is the usefulness of saline purgatives and lavage with saline purgatives in cases of poisoning. Such a procedure is certainly an innocuous one and in the light of the present investigation would tend to minimize the absorption and promote the expulsion of any toxic substances remaining in the intestines. Summary

1. The absorption of a large number of drugs from the gastrointestinal tract was studied by chemical and physiological methods iu animals and in man.

2. It was found that all the drugs examined were markedlj' delayed in their absorption from the stomach and intestines when they were administered subsequently to or even simultaneously with saline laxatives.

3. The absorption and subsequent secretion of phenolsulphon])hthaleiu and sodium salicylate were markedly


affected by saline purgatives, even when the former drugs were administered by intramuscular or intraperitoneal injection.

4. Laxatives other than those of the "saline" type exerted no important effect on the absorption of the drugs from the stomach and intestines.

5. The above findings have an important practical bearing on the therapeutic administration of medicaments.

6. The above experiments, especially those with phenol and mercuric chloride, furthermore, speak in favor of a more extensive emploj'ment of saline laxatives in the treatment of toxicological cases.

REFERB5NCES

1. Macht, D. I.: Jour, of Urolog}', 1922, VII, 271.

2. Macht. D. I.: Proc. Soc. Exp. Biol, and Med., 1921, XIX, 85.

3. Lipkin, I. J.: Annals of Tropical Med. and Parasit., 1919-20, XIII, 149.

4. Macht, D. I.: Bull. Johns Hopkins Hosp., 1915, XXVI, 98.

5. Vogel, K. M., and Lee, O. I.: Jour. A. M. A., 1914, LXII, 532.

6. Elliott, J. A.: Jour. A. M. A., 1917. LXVIII, 1693.

7. Underhill, J.: Jour. Pharm. & Exper. Therap., 1922, XIX, 2, 135.


LOCALIZATION OF CUTANEOUS NERVES BY ELECTRICAL STIMULATION, APPLIED TO NERVE-BLOCK ANAESTHESIA


By Walter Hughson

(From the Anatomical Laboratory of The Johns Hopkins University)


In a report published in the Anatomical Record,' the author described a new method of teaching the distribution of cutaneous nerves to students in Gross Anatomy. This procedure, suggested by the work of Trotter and Davies - on cutaneous sensibility, consists of stimulation of the cutaneous nerves through the skin with a unipolar electrode. By this means, the point of emergence of the cutaneous nerve from the deep fascia and the subsequent course can be easily identified ; the area of distribution of the nerve is clearly outlined by the tingling sensation.

With the rapidly increasing use of local anaesthesia in surgical procedures, of all kinds, methods liave become pretty well standardized. Any suggestions, therefore, can now be expected only to add some refinement to a technic which in expert hands is already nearing perfection. The intelligent use of local antesthesia in its wide field of application requires a highly specialized knowledge of the distribution of all cutaneous nerves. Though any region may be aft'ected by the use of paravertebral nerve-block, this method is not always justifieil by the scope of^ the surgical procedure. Simple regional anesthesia has many advantages over the more generally used infiltration-method which need no exposition here, and has been widely u.sed since the publication of Cush


ing's paper in 1900.^ It is to the former type of anaesthesia that this method of accurate localization of nerve trunks can be applied.

The suggested method in na way absolves the operator from the necessity of being familiar with the general position of the nerve trunks. No amount of knowledge, however, can prepaj-e one for the individual variations which are constantly met with in the position, point of emergence from the fascia, and above all the ultimate distribution of superficial cutaneous nerves. These variations may occur even on opposite sides of the same individual and undoubtedly are responsible for nmny of the unsatisfactory results obtained in block anaesthesia.

To meet these diflficulties such a localization of cutaneous nerve trunks furnishes a method of almost absolute accuracy. A single dry cell, a small induction coil,* and an indifferent and a stimulating electrode constitute the entire apparatus needed. A faradic current too weak to cause muscular contraction will give the tingling sensation over the entire distribution of the nerve when the stigmatic electrode is applied immediately over the nerve trunk. When the anatomical position of the nerve trunk

Harvard Instrument Company.


THE JOHNS HOPKINS HOSPITAL BULLETIN. SEPTEMBER. 1922


PLATE XLII


Fig. 1. — Localization of Cutaneous Nerves by Electrical Stimulation, Applied to Nerve Block Anissthesia.



I M M M M U

-— S^ C^ ^ ITS



Fio. 2. — Localization of Cutaneous Nerves by Electrical Stimulation Applied to Nerve Block Ansesthesia.


•33 6


September, 1922]


339


is abnormal or when there is some uncertainty as to its exact location, careful search with the stigmatic electrode over the expected area invariably reveals the nerve. Sensory nerves over practically the entire body can be located in this way.

Having determined the size of the area to be anaesthetized and located the nerve trunks supplying the region, the injection of novocain, or any other appropriate drug, at the points determined will give the required anaesthesia over the entire area of the cutaneous nerve.

With this accurate localization the point may be marked on the skin f at any time previous to the oi)eration, as in the ward by the interne, and the surgeon is required simply to inject the anaesthetic at the points indicated. Smaller amounts of the anaesthetic are sufficient when injection is thus made either directly into the nerve sheath or into its immediate vicinity. The illustration (Fig. 1) is made from a case in which 0.5 c.c. of novocain was injected at each of the points marked by a star. The resultant cutaneous an;esthesia involved the whole anterior surface of the thigh, as shown by the black outline, and lasted for a period somewhat over two hours.

Even greater accuracy may be obtained when injecting the anaesthetic by using the hypodemiic needle as the stimulating electrode, a suggestion made by Dr. H. H. Young. This can readily be done by simply soldering a fine copper wire to the needle or by the use of a specially prepared hypodermic syringe as shown in Figure 2. To the outlet of the syringe there has been attached a small set screw which readily permits making the necessai-y connection with the induction coil, and in addition does away with the somewhat awkward needle and wire; the latter however has the advantage of being very easily made. It is not necessary to insulate the needle when used in this way. After insertion of the needle through the skin at the jire-determined points, a weak current is passed through the needle as stigmatic electrode and the nerve trunk located with remarkable accuracy. Deviation of the needle-point less than a millimetre to either side of the nerve trunk will cause a loss of the stimulating


t We have used an excellent indelible skin-inl? for this purpose, described by N. S. Finzi, Brit. M. J. 1, 52, Jan. 12, 1918.


effect. When the nerve has thus been positively identified, the aniesthetic may be injected. Prompt dulling and then complete loss of sensation results. The procedure is in no way painful. Actual stimulation of the nerve trunk with this mild current causes no more intense sensation than stimulation through the skin. This method is also applicable to nerve trunks lying deep in the muscle tissue before they have emerged from the fascia.

Of course, no new principle is involved in the method suggested ; it is simply a refinement of one branch of the general technic of producing local aniesthesia by the injection of a drug in the region of the nerve trunk. This method has not been used for actual clinical purposes but sufficient direct observations have been made to indicate that it can have definite practical use in a great variety of surgical procedures. This is particularly true as regards the extremities and the neck. A certain amount of intelligent cooperation on the patients' part is necessary but this can readily be obtained by first demonstrating the sensation to be expected by stimulating some obvious trunk such as the ulnar or radial.

Conclusions Localization of sensoiy nerve trunks by unipolar stimulation through the skin furnishes a method whereby nerve-block anaosthesia can be produced with the greatest accuracy and certainty. Difficulties due to anatomical variations are done away with and the surgeon is enabled to deal with each case individually. The apparatus required is easily prepared and the procedure is in no way painful or unpleasant to the patient.

FIGURE LEGENDS

Fig. 1. — Crosses indicate position of nerve trunks and points of injections of anaesthetic. Black line outlines area of ansesthesia obtained.

Fifi. 2. — Record syringe fitted with set screw for attachment of wire.

BIBLIOGRAPHY

1. Hughson, W. : Electrical Stimulation of Cutaneous Nerves. A Teaching Method. Anat. Record, 1922, XXllI.

2. Trotter, W. and H. M. Davies: EJxperimental Studies in the Innervation of the Skin. Jour. Physiol., 1909, XXXVIII, 134.

3. Gushing, H.: The Employment of Local Anaesthesia in the Radical Cure of Certain Cases of Hernia, with a Note upon the Nervous Anatomy of the Inguinal Region. Annals of Surgery, 1900, XXXI, 1.


THE JOHNS HOPKINS HOSPITAL BULLETIN


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(Foreign postage, 50 cents.) Price of cloth-bound volumes, $5.00 each.


340


[Xo.:}79


THE EFFECT OF SODIUM GERMANATE UPON THE TOTAL HEMOGLOBIN OF THE ALBINO RAT

By Joseph E. Nowrey, Jr.

(From the Department of Medicine. The Johns Hopkins University, Baltimore, Maryland)


In a previous pubUcatiou it has been sliown tliat gernianiuni dioxide in alkaline solvition is nontoxic and noncorrosive for the tissues of the albino rat when administered by subcutaneous injection.^ It has also been pointed out that such a solution will cause a marked increase in the number of erythrocytes in the peripheral blood when administered in the same manner.- A further study has demonstrated that this rise in the red blood corpuscles is accompanied by an increase in the number of nucleated red cells in the bone -marrow of the.se animals, indicating that stimulation of the erythrocytogenic tissue has taken place as the result of the action of this compound.^

The physiological action of germanium compounds is, at present, almost wholly unknown. In spite of the evidences of increased bone-marrow activity obtained from the examination of histological preparations, it was thought possible to settle the question of a real or an apparent increase in the number of the erythrocytes in the blood. If there were a real increase in the number of erythrocytes, the total hemoglobin should be higher in treated animals than in normals, providing the hemoglobin parallels the red cell count.

The purpose of the present study was to determine what change, if any, takes place in the total amount of hemoglobin in the blood of the albino rat after the administration of germanium dioxide in alkaline solution.

Experimental

One gram of chemically pure germanium dioxide was sifted into 200 milliliters of distilled water. Sufficient normal sodium hydroxide was then added to dissolve the oxide, and make the solution distinctly alkaline. The excess of alkali was neutralized by the addition of enough normal hydrochloric acid to bring the solution back to a 1)H of 7.0-7.2. The solution was then sterilized by boiling, and after cooling the volume was made up to 2.")0 milliliters by adding sterile distilled water. The addition of the alkali converted the dioxide into sodium germanate according to the equation:

(JeO, + 2NaOH = Na.GeOj + H.O. As will be shown in a future paper, it is in this form that germanium is most efficient as an erythi-opoietic agent. The addition of an excess of alkali is therefore imperative.

Two series of mature rats were selected, one series of seven animals being used as tests, and the other series of six reserved as controls. Bed blood cell counts and hemo


globin estimations were made on the rats of both series, and immediately after these were completeu, the test rats were injected with a dose of 17 milligrams of the sodium germanate solution per kilo of body weight. This is equivalent to a dose of 10 milligrams of the dioxide per kilo.

For a period of three weeks preceding, and during the course of the experiment, the rats received the same varied diet, in order to safeguard against any variations in the action of the germanate solution that could arise from dietary influences. The work of Hooper, Kolls, and Wright * shows that fasting and various diets will strongly influence the action of arsenic compounds; and it is quite possible that the same factors have an influence on the action of other substances.

One week after the initial counts and hemoglobin determinations were made, the red cell counts were repeated and the hemoglobin redetermined on the rats of both series. Immediately after this was done, total hemoglobin determinations were made on all the animals, employing the following technique which is a modification of the method described by Jaquet and Suter.'

The rat was placed under ether amiesthesia and tied to a small animal board by its four extremities. The board was fastened upright over a large beaker in such a manner that the blood and perfusing fluid would drip directly and without loss into the vessel. The ventral surface of the rat was then washed with a five per cent solution of sodium citrate in order to prevent the blood from clotting on the animal's fur. The abdominal and thoracic cavities were then opened by a large inverted T-shaped incision. These cavities were immediately flooded with the sodium citrate solution in order to lessen the tendency toward clot-formation.

While the heart was yet beating, the left ventricle was opened, and a cannula was inserted through the ventricle and into the aorta, and held firmly in place by a ligature. Precaution was taken not to allow the tip of the cannula to pass into the subclavian or carotid arteries, as this would cau.se an incomplete perfusion. A clamp held the cannula in position and kept its weight from dragging the aorta out of ])lace. The hepatic vein was then cut at its site of emergence from the liver, and the perfusing fluid, physiological saline, was allowed to pass tiirough the animal's vascular system. As soon as the fluid had begun to circulate, the rat was suspended by means of a cord tied around its upi)er incisors, and the ligatures were removed from its extremities, in order to facilitate the return flow of blood from the feet. The reservoir con


September, 1922]


341


taining the perfusing fluid was placed at a height of two meters above the aniuial's heart.

The perfusion was allowed to continue until the fluid leaving the animal was colorless. By this time the liver and kidnej's were very pale in color and practically bloodless. An interval of from two and one-half to three and one-half hours was necessary to accomplish this, and from 1000 to 1100 milliliters of the perfusing fluid were required to complete each perfusion.

The blood volume of the rat was then estimated bj' means of Hatai's forniulfe ° which are based on the work of Chisolm.' These formula; are as follows:

1. For rats under 150 grams bodj' weiglit, (Both sexes)

Blood volume, (in c.c.) = (Body weight ia grams )'X 0.099.

2. For male rats 150 grams and over,

Blood volume, (in c.c.) = (Body weight in grams )»-»X0.09G08.

3. For female rats 150 grams and over.

Blood volume, (in c.c.|^(Body weight in grams )°-°X 0.10194.

On the basis of this estimation sufticient distilled water and hydrochloric acid were added to the perfusate to obtain a final dilution of one part of blood in 250 parts of tenthnormal hydrochloric acid. The action of the hydrochloric acid converted the hemoglobin into acid hematin. The estimation of the total hemoglobin was made directly from this <lilution, using the permanent glass standard described by Newcomer.* The jjreliminary hemoglobin determinations were made by the same method.

In this experiment the red cell counts were determined from samples of blood drawn from the tip of the animal's tail. Tiiis method is, in the writer's opinion, the method of choice for the purposes of the present study, and preferable to the method of obtaining the sample of blood directly from the heart of tlie rat. The danger of puncturing the pleura is very great in such a small animal, and as Biirker, Ederle, and Kircher have shown," the production of a unilateral artificial pneumothorax causes a true polycytha^mia, and such an accident would nullify the results of his experiment.

Resi'lts

The results are summarized in the tables.

Following the injection of tlie germanate .solution there was a marked rise in the number of erytlirocytes in the peripheral blood of the test rats.

It will be seen from the tables that in the control animals, the hemoglobin readings, as determined by using a measured quantity of blood, and those obtained from the pei'fusate diluted on the basis of Hatai's formula?, are consistent. This would indicate that for the nonnal rat, the blood volume may be estimated within a reasonable degree of accuracy by the use of these formulje. In the


test rats, however, on comparing the hemoglobin readings obtained by the two methods, considerable discrepancy was noticed, as the estimated concentration of hemoglobin obtained from the total hemoglobin determination was always higher than that obtained from a measured sample of blood. It was apparent, therefore, that Hatai's formuliT? could not be used to estimate the blood volume of the rats treated with the germanate solution. The most plausible explanation of the discrepancies between the partial and the total hemoglobin determinations in these animals is the assumption that an increase in the blood volume has occurred.

This experiment, then, furnishes an additional evidence that the increase in erythrocytes following the injection of sodium germanate is due to a true erythropoiesis and is not a relative effect due to a simple concentratiou of the blood plasma, such as Bertelli, Falta, and Schweeger,'" and Lamson " have shown to occur after the administration of epinephrin. The results of the present study show clearly that no concentration of the blood plasma has occurred, for if such were the case, the concentration of hemoglobin based on a normal blood volume would be lower in the total hemoglobin determinations. Since these, on the contrary, show a distinct tendency to run higher than the corresponding readings obtained from a measured amount of blood, there is furnished a strong indication that the blood volume is increased following the injection of sodium germanate solution.

Summary and Conclusion

Two series of albino rats were taken. One lot of seven animals was used as tests, and the other series was reserved as controls. Eed cell counts and hemoglobin estimations were made on all, and the test rats were then injected with 17 milligrams of sodium gernuinate per kilo of body weight. One week later red cell counts and hemoglobin readings were done again on all. The rats of both series were then perfused and total hemoglobin estimations were made from the diluted perfusates.

A study of the results shows that in the controls there was no significant change in the red cell counts, in the hemoglobin concentration, or in the blood volume. In the test animals there was a marked increase in the number of erj-throcytes per cubic millimeter of blood, a moderate increase in the concentration of the hemoglobin, and a variable increase in the blood volume. There was no evidence of a concentration of the blood plasma in any of the animals.

It is therefore concluded from this and the pi'eceding papers that the increase in the red cells of the blood of the rat following the injection of sodium germanate is in all probability due to a stimulation of the bone-marrow and not an efl'ect which follows a concentration of the plasma.


342


[No. 379




TABLE


I.— CONTROLS.





First


Dctermi


a,io„.



Se


cond Determination.



E


s


.sS


1


s



i "


1-0


e




d~


St

9t


•2


OS



«|


11<^


If





e i


.S


n E


E i


a « 


o S 2


o "



&


B.5


iS^


^


(B.H


£^


fSS.


5 gx


5s


1.


167.


9.00


10.63


177.


9.25


9.94


10.09


9.62


9.72


2.


188.


8.10


12.01


195.


8.28


11.73


11.83


11.06


11.16


3.


179.


7.97


11.73


181.


8.00


11.73


12.10


10.34


10.66


4.


241.


8.43


11.87


245.


8.56


11.73


11.59


13.58


13.42


5.


137.


8.52


11.04


137.


8.73


11.45


11.65


8.29


8.43


6.


232.


8.50


10.35


240.


8.64


9.73


10.74


13.32


13.82


Mean.


190.6


8.42


11.27


195.8


8.54


11.05


11.33


11.03


11.20


TABLE IL — TESTS.


First Determinati(


256.

240. 219. 192. 200. 113. 148.


n E


8.30 7.84 7.64 S.06 9.28 7.27 7.74


Second Determination.


10.35 10.76 10.90 11.73 12.14 10.63 10.21


10.96


260.


9.93


253.


9.12


228.


9. IS


190.


9.24


215.


10.07


124.


9.62


161.


9.58


204.4


9.52


11.04 11.73 12.01 12.42 12.42 11.32 11.04


11.71


16.32 12.70 12.77 13.81 13.62 12.67 13.44


13.62


14.29 15.17 13.90 10.80 11.79 7.58 9.30


11.83


21.13 16.42 14.96 12.01 12.928.48 11.32


13.89


The two determinations were made one week apart. The sodium germanate was injected into the test animals immediately after the first determination.

I wish to thank Dr. A. O. Kolls for many helpful suggestions.

REFEREHS'CES

1. Hammett, F. S., Muller, J. H., and Nowrey, .T. E., .Tr.: ,T. Pharmacol, and Exp. Therap., 1922, XIX, 337.

2. Hammett, F. S., Nowrey, J. E., Jr., and Muller, J. H.: J. E.\p. Med., 1922, XXXV, 173.

3. Hammett, F. S., and Nowrey, J. E., Jr.: J. Exp. Med., 1922, XXXV, 507.

4. Hooper, C. W., Kolls, A. C, and Wright, D.: J. Pharmacol, and Exp. Therap., 1921, XVIII, 133.

5. Jaquet, A., and Suter, F. : Corresbl. f. Schweiz. Aerzt., 1898, XXVIII, 104.

6. Donaldson, H. H.: Memoirs of the Wistar Institute, 1915, VI, 165.

7. Chisolm, R. A.: Quart. J. Exper. Physiol., 1911, IV, 207. S. Newcomer, H. S.: J. Biol. Cheni., 1919, XXXVII, 465.

9. Biirker, K., Ederle, R., and Kircher, F.: Zentralbl. f. Physiol., 1913, XXVII, 623.

10. Bertelli, G., Falta, W., and Schweeger, C: Zeitschr. f. klin. Med.. 1910, LXXI, 23.

11. Lamson, P. D.: J. Pharmacol, and Exp. Therap., 1915, VII, 169.


GIANT CENTROSPHERES IN XANTHOMATOUS TUMORS

By David T. Smith

(From the Department of Embryology, Carnegie Institution of

Washington, and the fiurgical Pathological Laboratory,

Johns Hopkins Hospital, Baltimore)


When normal cells begin to degenerate in tissne-cultnres a series of very characteristic but complicated changes takes place. The mitochondria break up into fine granules, form vesicles, or disappear entirely (Lewis and Lewis, 1915) ;^ in some cells there is a great accumulation of granules and vacuoles which stain with neutral red (W. H. Lewis, 1919),= while in others the most striking feature is the formation of a giant centrosphere (W. H. Lewis, 1920).' It is with the evidence of degeneration afforded by the latter that we are concerned in this communication. The centriole in tissue-culture cells that have been fixed and stained seems always to be in the form of a granule, single or double, usually the latter. The peculiar differentiated region around the centriole is called the centrosphere and corresponds to the central body, the aster, or astrosphere of the dividing egg and cell (W. H. Lewis, 1920).^ This structure is at one side or one end of the nucleus and generally in direct contact with it. Dr. Lewis found that the mitochondria and


degeneration granules have a marked tendency to arrange themselves around the centrosphere and to move in radiating paths to and from this structure. The pigment in true pigmented cells, as well as pigment granules that have been taken in as foreign bodies, shows the same peculiar arrangement about the centriole and centro.sphere. When the cell begins to degenerate under certain conditions, the centrosphere increases enormously in size, becoming as large as or even larger than the nucleus. In this giant centrosphere Dr. Lewis distinguishes a centriole, single or double, immediately surrounded by a clear medullary zone, and beyond this a larger cortical zone. The granules and vacuoles are arranged about this outer cortical zone.

In studying a group of xanthomatous tumors which contained many giant cells, much blood pigment from other cells, and numerous foam cells, the writer noted that the granules in the pigment-bearing cells were arranged about a clear area, often the size of the nucleus


Septemp.eu, 1922]


343


or a little larger, which otcui)ieil the center of the cell and displaced the nucleus to one side (Fig. 1). By careful focusing it could be determined that this clear area was a spherical body, one portion of which was in direct contact with the nucleus (Fig. 2), and that around this sphere was an outer shell of granules, complete except where it came against the nucleus. Often the cells were so filled with granules as to obscure all other cellular structures (Fig. 1). In the sections from which the figures here shown were taken (Surg. I'ath. No. 3S'J0), nearly all the pigmented cells contained these clear central areas. A few cells showed an accumulation of granules to one side of the nucleus without the formation of this area (Fig. 3).

In several types of cells in tissue-cultures the neutral red granules which accumulate in degenerating cells are at first scattered; later they begin to collect near the nucleus, probably about the centriole. Later still, as the mass of granules increases, a clear centrosphei'e forms in the center of it, the granules surrounding its periphery. In xanthomata there are indications of a similar process, of which Figure 3 probably represents an intermediate stage before the appearance of the clear central area. Earlier stages were seen, but most of the cells were in the later stages and contained centrospheres of various sizes. Owing to the fact that the cells are orientated at various angles in the plane of the section, some cells, especially those in which the nucleus and centrosphere are parallel to the plane, show these relationships much better than the others. Although no definite structures can be made out in these spherical bodies, such as have been described by Dr. Lewis in tissue-culture cells, it is evident that we are dealing with giant centrospheres in these xanthomatous tumors. The details of the structure in the giant centrosphere cannot always be seen, as was shown by Lewis and Webster (1021) * in their study of giant cells in cultures from human lymph-nodes.

Seventeen xanthomatous tumors were studied with special reference to the presence or absence of these giant centrospheres. The latter were present in nine, or a little over 50% of the cases. Of these, seven also contained foam cells, which have been regarded as evidence of degeneration of tissue (C. Smith, 1912). '^ One specimen (Surg. I'ath. No. 5151) contained foam cells but no giant centrospheres. The cells containing the pigment appeared very degenerate and it is possible that they had reached that late stage in the process in which the giant centrosphere is lost, as has been described by Dr. Lewis (1920) in tissue cultures. There were eight tumors in this series which had neither giant centrospheres nor foam cells. In the cells of these tumors the nuclei stained well, the growth seemed active and there was no evidence of degeneration.


It is well known that pigment occurs in several types of tumors and it was therefore thought advisable to examine some of these to ascertain if the giant centrosphere is present in all, or if it is a special characteristic of the xanthoma group. The study included 25 benign pigmented moles, 50 malignant pigmented tumors, 20 giant-cell tumors of bone containing blood pigment, and 10 bone sarcomata containing blood pigment.

In some of the cells of the moles it could be seen that the granules were grouped at one side or one end of the nucleus, about what was probably the centriole, but there was no evidence of a giant centrosphere.

In the pigmented tumors many of the tumor cells were undergoing degeneration, but although tlie pigment frequently appeared to be collected about the centriole, no giant centrospheres could be found. Occasionally, however, in cells that had taken in pigment liberated by the death of the tumor cells, these structures could be seen.

In the giant-cell tumors no giant centrospheres or foam cells could be seen. In every one of these cases the growtli api)eared to be active and healthy and resembled very clo.sely that seen in the eight xanthomata which did not contain giant centrospheres or foam cells. The types of cells containing the pigment in the giant-cell tumors of bone appear to be identical with those that contain the pigment in the xanthomatous tumors, and one would therefore expect to find giant centrospheres in this class of tumors also, if the growth of the tumor became arrested and degeneration began. No evidence of either giant centrospheres or foam cells was found in the bone sarcomata.

Dr. W. H. Lewis, in his study of the giant centrosphere in tissue cultures, has shown that the so-called Plimmer body (cancer-cell inclusions, bird's-eye inclusions, etc.) and the giant centrosphere are identical. He points out that the presence of the giant centrosphere is evidence of disturbance in the metabolism of the cell and suggests that these bodies might be found in pathological conditions other than cancer. This prediction has been fulfilled in part by the discovery of the giant centrosphere in xanthomatous tumors.

It should be remembered that this discovery was purely an accident. If the.se cells had not contained pig mcnt granules forming a corona about the giant centrosphere, this body would never have been observed in sections prepared according to our routine pathological technique. This suggests that, with more refined cytological methods, definite alterations in the cells might be found in a number of abnormal conditions which are now considered as functional because they show no definite pathological changes with the usual technique.

Summary

Giant centrospheres were present in the pigmentcontaining cells of nine out of seventeen xanthomatous


3U


[No. 379


tumors examined. They are probably to be regarded as evidence of degeneration.

BIBLIOGRAPHY

1. Lewis and Lewis, 1915: Mitochondria and other cytoplasmic structures in tissue culture. Amer. J. Anat., vol. 17, p. 339.

2. Lewis, W. H., 1919: Degeneration granules and vacuoles in the fibroblasts of chick embryos cultivated in vitro. Johns Hopkins Hosp. Bull., vol. 30, p. SI.

3. Lewis, W. H., 1920: Giant centrospheres in degenerating mesenchyme cells of tissue cultures. J. Exper. Med., vol. 31, p. 275.

4. Lewis, W. H., and L. T. Webster, 1921: Giant cells in cultures from human lymph-nodes. J. Exper. Med., vol. 33, p. 349.

5. Smith, C, 1912: The histology and nature of the so-called foam-cell tumors. Surg., Gyn., and Obst., vol. 14, p. 551.

EXPLANATION OF FIGURES Fig. 1.— Photomicrograph of a xanthoma (Surg. Path. No. 3890) which shows that a large number of the cells are filled with


pigment granules. Some of the cells appear as circumscribed black splotches in which no details can be distinguished. In some of the others can be seen the clear central area which is the giant centrosphere. One cell, which is seen to the right and a little below the center, shows both nucleus and giant centrosphere, the latter surrounded by a corona of granules. (X iSO.)

Fig. 2. — Camera-lucida drawing of a cell containing pigment granules and a few adjacent nuclei from the same section as Fig. 1. The nucleus appears somewhat swollen and the outlines of the cytoplasm are indicated merely by the extent of the granules. The giant centrosphere occupies the center of the cell and displaces the nucleus to one side. The granules are collected about the centrosphere except where it comes in direct contact with the nucleus. (X2600.)

Fig. 3. — Another cell, showing a younger stage in the accumulation of granules from the same section. The nucleus is swollen and the pigment granules are massed at one end of it. probably around a centriole. There is no evidence of a centrosphere. (X2600.)


A NORMAL PREGNANCY FOLLOWING INSERTION OF THE OUTER HALF OF A FALLOPIAN TUBE INTO THE UTERINE CORNU


Bv Thoias S. Cullen


In the Johns Hopkins Hospital Bulletin for September, r,)21, Dr. Henry N. Shaw reported the case of a patient upon whom I had operated on Oct. G, 1919. Her right tube and ovary had been removed by another surgeon several years before.

She entered the Johns Hopkins Hospital with signs of a left tubal pregnancy. On opening the abdomen I found a left coruual preguancy. This was removed but the outer half of the left tube was saved, and its inner end was sutured into the uterine coruu.

She subsequently became pregnant but lost her baby on March 11, 1921, at the end of the seventh month. There was a placenta praevia.


On June 26, 1922 I received the following letter from Andrews, S. C. "Mrs. J. E. L. asked me to write you that she gave birth to a seven and a half pound baby girl this morning at eight-thirty. Dr. I). S. Porter was with her, labor was perfectly normal, both mother and child are doing finely so far."

Those who use eveiy effort to save the tubes and ovaries or part of them will from time to time be disappointed and may be forced to do a second operation. The results in this case, on the other hand, clearly demonstrate what may occasionally be accomplished hy judicious conservatism.


AN OPERATION FOR THE TOTAL EXTIRPATION OF TUMORS IN THE CEREBELLO-PONTINE ANGLE. A PRELIMINARY REPORT

By Dr. Walter E. Dandy


The most frequent tumor in the cerebellopontine angle is an encapsulated endothelioma arising from the leptomeninges. Rather loosely embedded in the lateral wall of the brain-stem, it is potentially a benign tumor by virtue of its encapsulation. Its complete removal offers a permanent cui'e to the afflicted individual but its extirpation has been attended by a mortality so high as to render such attempts inadvisable. In fact, the complete removal of such tumors with recovery has been regarded as impossible. As a result, a partial intracapsular enucleation has been the operation which has seemed to offer most to the patient, but it is obvious that such treatment of a potentially benign lesion is most unsatisfactoi-y


to the patient, for the tumor must inevitably recur.

Five years ago, 1 completely removed such a growth from a patient who has since remained well. The growth was extirpated in tofo by careful dissection around the tumor. Subsequently, two other tumors of the same type were similarly removed, but the results of such a method were too capricious and the mortality was too high.

Gradually a procedure has been evolved by which I believe these neoplasms can be successfully removed and with relative safety; the mortality should be little higher than from a subtotal removal of the contents of the tumor. The last two patients with cerebello pontine tumors have been treated by this procedure and are well. The last


THE JOHNS HOPKINS HOSPITAL BULLETIN. SEPTEMBER, 1922


PLATE XLIII


^M-H


^^m^i


Fig. 3.


ii


September, 1922]


345


patient was a particularly bad risk because of a partial hemiplegia and hemianiT?sthesia and inability to swallow. She quickly recovered from the operation. In one patient, the operation Avas performed in two stages; in the second in one stage. The latter metliod is far preferable because in the interim between stages the capsule becomes friable and more difficult to handle.

The purpose of this preliminary report is to present the salient features of the operative procedure. A bilateral suboccipital exposure of the cerebellum is performed with as much exposure of the affected angle as possible. The


interior of the growth is removed with a curette. Following this, the capsule is picked up with forceps and beginning at the upper and lower poles, carefully drawn away from the medulla, pons and mid-brain. The traction brings into view the several small veins and arteries crossing from the brain-stem to the tumor. These vessels are ligated individually with silver clips or tine silk ligatures and divided. Gradually, in this painstaking way, the whole tumor is delivered from its bed without bleeding, and without trauma to the brain-stem. The cranial nerves stretched by the tumor are automatically liberated as the capsule falls away from them.


NOTES ON NEW BOOKS

Human Embryology and Morphology. By Arthur Keith. 4 th Edition. Cloth, $10.50. (New York, Longmans, Green d- Co.: London: Edward Arnold, 10.il.)

In the fourth edition of his Human Embryology and Morphology, Sir Arthur Keith has emphasized the importance of studying the development of the human fetus in relation to the general facts of comparative anatomy and evolutionary history. This unique approach is essentially characteristic of the broad conceptions of the author and is most important because morphological embryology of today must assist in unravelling the problems of philogeny and growth, or remain relatively sterile. The presentation is clear and excellent throughout and furnishes a particularly appropriate introduction to the study of both human and comparative anatomy. The numerous views which have been advanced regarding many phases of embryology have evidently been searchingly analysed and no criticism can be made of those that the author accepts. But these views have been presented as final and in this the book fails to meet one of the most important requirements of a modern text-book. The student should be given every opportunity of deciding for himself in regard to opposing theories and should not be taught, too finally, any individual theory as established truth. With the addition of a few pages devoted to the presentation of both sides of certain disputed points, this book would have stood alone as embodying the two most important requirements of a text-book: a fundamental and inspiring viewpoint, and a presentation sufficiently broad to bring the student face to face with the problems of a growing science.

R. S. C.

Arterial Sclerosis. By Louis F.iugekes Bishop. {London: Henry Frowde; Hodder d Stoughton, 1021.)

It is greatly to be deplored that, in the making of books of which there is no end, a more critical judgment should not be exercised in determining not only the value of the content but the length of many of the volumes that are offered to an ever credulous public with the stamp of approval conveyed by the publisher's imprint.

It is difficult to understand why there should be, apparently, so great a popular demand for a work of the type of this volume, as to necessitate a third printing, that adds nothing of constructive value to our knowledge of arteriosclerosis. The work is an extraordinary mixture of the personal experiences and opinions of the author together with many excerpts from the writings of others, the whole being combined in such a way as to lead often to much confusion in the interpretation of the text. In his preface the author states that "clinical medicine either in the hands of reputable or disreputable practitioners is always anticipating


the advance of knowledge," and further "the needs of physicians who have been trained in other specialties have been kept in mind and simplicity has been chosen rather than complexity."

In discussing the relationship of arteriosclerosis to blood pressure it is difficult to follow the author's apparent meaning. We disagree with the statement that "low blood pressure in arteriosclerosis may be regarded as an exception that always demands explanation." The following sentence quoted in full will illustrate our difficulty in interpreting the text— "The term blood pressure is often used where high blood pressure is meant. With light thrown on the meaning of the context this does not lead to confusion, though, of course, during life there is always pressure of blood in the vessels." (Chapter VI, p. 65, line 6 from top.)

We confess that we need light on "the context" and admit being led to confusion. We must, however, leave to his readers the ultimate decision as to the author's success in respect to the simplicity of his text.

The causes of arteriosclerosis discussed in a chapter of 16 pages contains not a single reference to the vast and fundamentally important literature upon the subject. The chapter entitled "The Natural History of Arteriosclerosis" following directly that devoted to the etiology, should be properly considered as a part of the former. Here again we have no reference to the literature and a delightful preliminary allusion to the lite history of the oyster is followed by a most confused and inconsequential discussion of the subject. That the causes of arteriosclerosis are chemical and physical is a patent truism, but that the evolution of the disease process would in any large number of cases fall in with any such standard type as outlined on page 41, we seriously question. Great stress is laid upon high blood pressure apparently as evidenced by the systolic level, with but infrequent reference to the significance of the diastolic figures.

The chapter devoted to diet in arteriosclerosis and that upon the chemistry of the proteins should be read to be appreciated. The latter has at least the great merit of representing the statements of workers in this field from whose writings this chapter is largely constructed.

The essential purpose of the volume is apparently an exposition of the author's theories of the causal relationship between protein food intoxication and pathological cardiovascular changes, as well as his methods of therapeutic procedure in arteriosclerosis.

E. P. C.

Olinical Electrocardiography. By Frederick A. Willius. Cloth, fo.OO. {Philadelphia and London, W. B. Saunders Company 1022.) This volume covers in an elementary way the fundamental principles of the physiology of the galvanometer in its adaptation to clinical use, together with a short chapter on the mathematics in


volved, and brief statements of the mechanism concerned with the various cardiac arrhythmias.

During the past two years a number of similar worlis have appeared each dealing with the subject in a way peculiar to the individual author. It is, however, very doubtful that any genuinely new knowledge has been contributed through these sources or even that the debatable points involved have been in any sense cleared up.

In a work intended primarily as an introduction to the subject, one would wish that dogma might be less emphatic and that a more conservative judgment as to the conclusions arrived at from the interpretation of the galvanometric records might obtain.

Although it is quite correct to say that a negative "P" wave may indicate a shift in the pace-maker, such a statement unqualified may easily lead to confusion and convey a false impression. In Chapter X dealing with the abnormalities of the "P" wave, this earlier impression is in a measure corrected, though it is greatly to be desired that the text devoted to this phase of the subject be revised.

Paroxysmal tachycardia is dealt with in a little over a page and there is no adequate discussion of sinus tachycardia in relation to what the author calls paroxysmal sinus tachycardia and paroxysmal auricular tachycardia. Are we to accept a paroxysmal tachycardia of sinus origin as proven, and is the term paroxysmal auricular tachycardia to be used to connote the presence of an ectopic and heterogenetic rhythm with negative "P" waves in all three derivations? Unless we consent to this view it is, of course, incorrect to say that the "P" wave is always inverted in paroxysmal tachycardia auricular in origin (p. 79). In the references to the literature following the chapter devoted to the auricular tachycardias there is no reference to Bouveret's original article which even to-day should be read by every student interested in this subject.

In the discussion of auricular fibrillation, although the present interpretation of its mechanism based upon the work of Lewis and his co-workers is referred to, no allusion is made to the development of localised areas of block, which is the underlying fault leading to the condition of fibrillation and really distinguishing it from flutter, and no reference in this connection is made to the fundamental work of Meyer, Mines and Garrey.

In the light of our knowledge of the pathological physiology of flutter it is not correct to imply that the "P" wave is always inverted. While in some instances the "P" wave appears as a distinctly negative wave, in the vast majority of flutter records it can be read only as a diphasic curve (See Figs. 52 to 58), a fact which gives rise to the striking similarity seen from case to case, the only clinical condition of which this can be said to be true. The character of the "P" wave in the presence of a circus movement within the auricle must depend upon the relationship of the direction of the spread of the wave to the plane of the given derivation.

In discussing the subject of notched QRS complexes (p. 109) no allusion is made to the variation in these deformities which may occur under change of position and forced respiration seen "so conspicuously in many instances, and depending upon the relationship of the plane of the derivation concerned to the direction of the axial spread of the excitation wave. To base sweeping conclusions upon notching of this complex in any statistical relationship to the clinical diagnosis without first determining their variability, seems to us wholly unwarranted. *


The chapter devoted to the abnormalities of the "T" wave is based upon a purely theoretical conception of the author without any adequate proof and cannot fail to confuse and mislead the reader who has no fundamental knowledge of the subject. Such an attempt to elucidate the mystery of the "T" wave can serve no genuinely useful purpose and the effort to classify clinical-pathological states on the basis oi the negativity of the "T" wave in any combination of derivations is too absurd to merit discussion.

The history of the development of our knowledge following the introduction of the galvanometer as an instrument for the study of clinical cases is the story of a brilliant and extraordinary insight into more than one obscure corner of medical science; its use, however, as suggested by the figures from pages 162-182 of this volume can only give the uninitiated an utterly false conception of the usefulness of the instrument in clinical procedures.

E. P. C.

Textbook of Physiology. By Russell Burton-Opitz. (Philadelphia and London: W. B. Saunders Co., 1920.)

The author's aim, as he states it, is to supply the medical student with a text-book in the subject of human physiologj-. As such, as far as it goes, the book accomplishes its end in a satisfactory way. Physiology covers a vast field and the making of a book of this kind is no light task. The number of subjects are too many to be fully understood by any one man. Hence, that the product of the author's labor should contain errors was inevitable. Some of these will gradually get corrected in the newer editions. But the fact that older authors do not succeed altogether in this leads us to be not too sanguine. To illustrate my point I need only refer to the figure on p. 147 of the present book, showing the distribution of electrotonus along a nerve. The figure is Pfluger's down to the last detail of symbol and character of type, and the explanatory note below the figure is a free translation of Pfluger's explanatory note buried in the text of his monograph. For these facts the reader need only to turn to Figure IS. Plate V and p. 465 of the original. Yet not only does Burton-Opitz ascribe this diagram to American authorship but that master of text-books in Physiology, Professor Howell, chronically persists in doing likewise (see his Sth Edition). The error is clearly one of misapprehension or of oversight, and will persist, I presume, to the stroke of doom. In Burton-Opitz's book there is another figure — an original one — which clearly shows that here the author is describing something he does not well understand. The text describing the figure bears this out. I refer to the description of the principle, and application in physiology, of a thermopile, as given on page 97. The thing is a failure. Wlien the author touches topics nearer his own fields of research one at once perceives the note of authority, and it is in these parts on the blood and circulation that the book has especial merit. Now this unevenness in text-book writing suggests that it is high time that physiology as a text for the student should no longer be written by one man. It has been suggested that, to avoid the difficulty of getting a dozen specialists to co-operate on one book, the subject could be satisfactorily covered by a dozen small treatises or special chapters separately printed. These could be revised and reprinted independently of each other and even by new authors as the older ones dropped the task. The convenience of handling and carrying the smaller volume need not be dwelt upon. Let us hope that the day of the cumbersome text-book is nearly ended. C. D. S.

CONTENTS

  • The Excitatory State. By Dk. W. M. B.wliss, M. A., D. Sc, F. R. S.
  • A Study of Frozen Sections Througli a Cadaver Showing The Anatomical Relations of a Large Uterine Myoma. (Illustrated.) By Dr. .1. WniTRiuiiE Williams
  • The Changes in the Para-Ocular Glands Which Follow the Administration of Diets Low in Fat-Soluble A: With Notes of the Effect of the Same Diets on the Salivary Glands and the Mucosa of the Larynx and Trachea. (IlluBtrated.) By SiiiXNosi'KE Mori, M. D
  • Note on a Modification of the Chromaffin Reaction, With Observations on the Occurrence of Abdominal Chromaffin Bodies in Mammals. (Illustrated.) By G. B. WisLocKi
  • On the Cardiac Complications of Gonorrhoea. (Illustrated.) By W. S. TiiAVER. M. D
  • Post-Encephalitic Behavior Disorders in Children. By Le.«lie B. Hoiima.n
  • Tolerance and Acquired Tolerance of the Mesenchyme Cells in Tissue Cultures for Copper Sulphate and Sodium Arsenite. By JiLiis La.ne Wilson
  • Note on the Abdominal Chromaffin Body in Dogs. By George B. Wislocki and S. J. Crowe
  • Notes on New Books


THE EXCITATORY STATE

By W. M. Baylis,^?, M. A., I). Sc, P. R. S.


In thi.s lecture I wish to tall atteiitiou to certain phemonena associated with the excitatory state or state or activity. At the end of the previous lecture 1 mentioned the reversible change in protoplasm produced by ail electrical shock, in which the liquid sol changes to a jelly with more or less solid properties. The use of the term "gel" has become somewhat indefinite and is sometime.s used to mean any kind of precipitation of a colloid. The term "jelly" is sutliciently clear as typified by gelatin when cooleil.

Similar changes have been described in cell-division, fertilization and so on, and the method of centrifuging the cell has proved very useful in work of this nature.

A point of interest is that if changes in consistency are indicative of activity, the protrusion of pseudopodia


Second lecture of the Herter Series. Hopkins University on March 8th, 1922.


Delivered at Johns


is not active but passive, since the Brownian movenient.s persist. Thi.s conclusion does not exclude active contraction elsewhere on the surface, however, and it seems dilticult to account for the spherical form of leucocytes when stimulated otherwise tlian by a contraction of some kind over the whole organism. At the same time, all that the fact really implies is tliat the surface tension is equal all over.

The chief practical interest of the contractility of protoplasm is in its relation to the properties of the capillary blood vessels, which are essentially protoplasmic. This point will come up again in my next lecture.

Nerve and muscle (inclnding the heart) are commonly regarded as the excitable tissues pnr excellcuce. But this is due to the fact that tlieir response to stimulus is rapid and obvious. All living tissues are clearly excitable in so far as they respond by change of some

kind to external influences. It is owing to the rapid character of the excitation proce.ss in the former tissues that so much investigation has been devoted to it in these cases. There are two aspects of muscular contraction to which I would call attention in connection with the problems of interfaces to which my first lecture was devoted. The first of the.se concerns tlie mode of production of tlie state of tension which the work of A. V. Hill has shown to be the primarj' fact. His researche.'^ liavc taught us the manner in which the magnitude of a contraction depends on the length of the fibres when the contraction begins. This means that the controlling factor is one of surface, not of volume; it is a question of surface energy, not of osmotic energj- or of imbibition. The conclusion is confirmed by the results of experiments made to determine the temperature coefficient of the contractile stress. The most accurate and careful work has shown that this is negative; that is, the more powerful contraction is at the lower temperature. As previously mentioned, a surface tension phenomenon is implied thereby. The known production of lactic acid naturally suggests that the deposition of this acid or of its hydrogen ions on the muscle fibrils is the cause of the rai.sed surface tension, as originally suggested by Haber and Klemcnsiewicz in their classical paper on "Interfacial forces." The presence of lactic acid as the cause of the contractile stress leads to a few remarks on the puzzling facts of "tonus" or "posture" in muscle. It has long been known that smooth muscle is able to enter into a state of shortening in which, as it appears, there is little or no expenditure of energy. This shortening is more or less reduced by rise of temperature; hence again, a surface tension effect is suggested. Since it nnist be supposed that in the usual form of contraction return to the normal length is as.sociated with removal in some way of the lactic acid which causes the shortening, I have ventured to put forward elsewhere the view that the touic state might be due to a prolonged remaining of the acid and that some influence may be required to set in action the process which results in its removal. This would be something of the nature of an inhibitory process. Roaf has recently described experiments with a numganese dioxide electrode and finds that in a veratrinized muscle the acidity remains with the shortening, and that when decerebrate rigidity is reflexly inhibited, the acidity deci-eases. The latter fact shows that the muscle remains acid during the tonic contraction. But the behavior of the manganese electrode is not completely understood. The work of Sherrington on plastic tonus" has directed attention to the presence in voluntary muscle of phenomena similar to those of smooth muscle. Various researches have shown the existence of certain features in common. I may mention my own work in which the heat production was found to be very small, and that of Roaf which indicates minimal pro


duction of carbon dioxide. If the theory of the nonremoval of lactic acid, as suggested above, be applied to voluntarj- muscle, it seems that it would be necessary to assume the existence in voluntary muscle of a distinct innervation process to set in action the relaxation, as in fact is the case with the inhibitory nerves of smooth muscle. This is no doubt a difficulty, but the theory of Bottazzi, according to which tonus is due to sarcoplasmic contraction, does not seem to me to offer a satisfactory solution. It is tempting to connect the .sympathetic innervation of voluntan' muscle with the tonic state, but it is remarkable how little direct evidence of this has been obtained. The sympathetic innervation renuiins a mystery.

The .second aspect of the excitation process to which I would refer is the increased permeability of the cell membrane which is found to accompany it. The imper meability of the cell membrane in ordinary conditions to most of the ions with which it is in contact is, of cour.se. the rea.son why living cells have so high an electrical resistance. When the membrane is made permeable, the resistance falls to a notable degree, as in Osterhout's experiments with sodium ions and with ana'sthetics. These facts are the basis of Stewart's well known method of determining the proportion of plasnui to cor{)uscles in blood.

I have already pointed out how impermeability to salts may be due to impermeability to one of the ions, the opposite ion being allowed free course, as far as permitted by electrostatic attraction. The result of such a state would be the pre.seiue on the outer surface of the cell of au electric charge of the sign of the permeating ion. Suppo.se that we place electrodes on two distant places of a muscle cell or a nerve fibre, and connect them to some instniment which indicates any potential difference between them, such as the string galvanometer. It has been known for many years that, provided that no injury has occurred, the two points are equipotential. Let us now stimulate one of these place-s. Our galvanometer shows by its deflection that the two electrodes are no longer equipotential, and further, that the stimulated region is electrically negative to that at rest. Now we know that we have changed the cell membrane at the stimulated spot into a permeable one, so that the polarization rendered possible by the inipenneability to one ion is abolished and the potential dift'ereuce between the two sides of the membrane at the electrode on the point at rest is no longer opi)osed, while the direction of the current shows that the outside of the membrane is electropositive to the inside. We have, in other words, by making the excited spot jiermeable, made the interior of the cell as it were accessible to the leading off electrode. The ions which were arranged in two oppositely chargeil layers have been enabled to mix together and neutralize their effect. It is not, therefore, that excitation produces

a negative state, but the permeability brought about by it enables the polarized charge at the resting electrode to become evident. This is, briefly, the theory associated with the name of Bernstein, who pointed out that the nature of electrical conduction in solutions necessitated the view that the electrical changes observed in active tissues must be ionic in origin. In support of this conclusion, Bernstein measured the temperature coefficient of the electromotive force of some bioelectrical phenomena and found it to be of the very low value of that of the electrical conductivity of solutions of electrolytes. Some earlier views had associated it with a kind of chemical reaction, which would have had the \isual high temperature coefficient of such phenomena. If follows naturally, from what has been said, that if a muscle is in a state of tonic contraction, an inhibition of this state under one electrode would make that spot appear to be positive to that still in contraction. It is of some interest to note that the complex electrical changes described by Orbeli as occurring in the ureter can be interpreted fairly satisfactorily as being due to waves of inhibition followed by waves of contraction similar to those in the intestine preceding and following a mass of food material and i)ropelling it onward (the "law of the intestine" or "myenteric reflex").

My object in calling attention to the electrical phenomena in muscle and nerve is partly also to refer to some points connected with such phenomena as they occur in the heart and in the secreting glands.

But "before passing to these, I would mention the work of Starling and his collaborators on the work of the heart muscle. At the beginning of this lecture it was pointed out how A. V. Hill had shown that the magnitude of the contractile tension developed in a muscle flbre depends on the length of the fibre at the initiation of the contraction. Now Starling has demonstrated that the same law holds for the heart muscle and that all the phenomena of the output of the heart in relation to arterial pressure, venous inflow and so on, are to be explained on the basis of the various degrees of filling of the ventricles at the moment of systole. The facts as applied to this organ are embodied in the "law of the heart."

The electrocardiogram is of course a complex case of the electrical change in a muscle flbre as described above, made complex by the fact of the excitation progressing as a wave along an elaborate system of conducting channels. I merely wish to express a doubt as to whether conclusions drawn from changes in the form of the curve have any great significance in relation to the actual working of the heart muscle. We must bear in mind that, owing to the potential difference led oft' being due to slight differences in the time of arrival of the excitation process at difterent spots, any state which alters even very little either the rate of the wave in one part


or its duration at one spot may exercise a profound influence on the actual form of the "ventricular complex." In the work which was done by Starling and myself on the dog's heart, we were able, by varying the temperature of the air used for artificial respiration, actually to invert the electrical change, without, so far as could be detected, producing any effect on the mechanical beat. The work of Mines and other.s showed further that, under certain conditions, the mechanical beat could be abolished, while the electrical change remained normal. This merely shows, however, that it is the excitation process that is responsible for the electrical change. Although, according to Einthoven, there may still be a very small contraction, detectable by sensitive methods, the independence in degree of magnitude of the electrical and the contractile change is clear. Again, the form of the "ventricular complex" is sometimes brought into relation with the rapid rate of conduction along the Purkinje tissue as compared with that in the contractile muscle. It has recently been shown that the heart of the chick embryo of 50 hours gives a similar electrocardiogram to that of the adult, although histological examination reveals a structure uniform throughout. Of course, these considerations do not apply to the use of the electrocardiogram in the investigation of block, extra-systole, auricular fibrillation and so on, but it appears to me that great caution is necessary in interpreting varieties of form of the ventricular complex itself.

In conclusion, I wish to refer briefly to the electrical phenomena of secretion, especially in the mammal. It is somewhat remarkable that practically no further work has been done in this region since that of Bradford and myself many years ago. It is now time for a renewed investigation in the light of modern electrical theory. But in the meantime it is worth while to examine how far the facts made out by Bradford and myself are to be explained on the lines of present views of the secretory process. In the first place, it appears that during the period of rest following activity of a secreting gland there is a steady accumulation in the cells of material which is to serve as the precursor of the contents of the secretion. This process is associated with the consumption of oxygen and comes to an end when a certain quantity has been produced. It seems to be controlled by mass action and to proceed automatically, without the intervention of nervous impulses. It is on the whole very similar to the restitution phase of muscular activity. Moreover, like the actual contractile phase of muscle, the act of secretion, when excited by nerve stimulation or by drugs, seems to be independent of oxygen. In the gland, what probably occurs is something like this : the material present in the cell, or some part of it, is split up into smaller molecules, so that the osmotic pressure in the cell rises. This causes attraction of water and momentary distension of the cell. But at the same time that

end of the cell iu relation with the lumen of the alveolus and thus with the duct loses its semi-permeability, so that water, carrying with it solutes from the cell, escapes to the duct. Such a current flowing through the cell from the lymph space to the duct continues as long as osmotically active matter together with the permeable state of the one end of the cell remains. The process is similar to the current of water flowing through a tube of sugar solution if the one end is open, the other end closed by a semipermeable membrane and immersed in water. It was found by Bradford and myself that the electrical change produced in the submaxillary gland of the dog on stimulation of the chorda tympani nerve was definitely associated with passage of water, although the state of knowledge at the time did not permit the more detailed explanation. Now, if the cell membrane of the secreting cell


has properties similar to that of the i"ed blood corpuscles, namely, permeability to anions, impermeabOity to cations, the sign of the electrical difference between the fundus and the duct end of the cell in the excited state corresponds to what would be the case on the hypothesis outlined above. A similar change has recently been found by Anrep and Daly in the pancreas excited by secretin. When the sympathetic supply to the salivai-y gland is stimulated, there is a small electrical effect in the opposite direction to that of the chorda. The meaning of this requires elucidation. It is important that future work should be done, at all events in the first instance, on dogs, since it is only in these that the effects of the chorda tympani and of the sympathetic supply are clearly differentiated.


A STUDY OF FROZEN SECTIONS THROUGH A CADAVER SHOWING THE ANATOMICAL RELATIONS OF A LARGE UTERINE MYOMA

By J. Whitridge Williams


In May 1921, Professor Weed informed me that he had received from the Anatomy Boai'd a female cadaver, which he thought might be pregnant. His records showed that the body had come from the morgue, and that the death certificate of the coroner stated that the woman was 26 years old and had died from natural causes.

Upon examination I found the partially frozen body of an apparentlj' middle-aged colored woman, which presented no abnormalities except an abdominal tumor which reached to within three fingers of the xiphoid cartilage, but I could not determine whether it was a pregnant uterus or a tumor of some other kind. It was determined to freeze the cadaver thoroughly and then to section it in various planes in the hope that we might secure information concerning the relations of the pregnant uterus to the rest of the body and particularly to the pelvic floor.

After the body had been thoroughly frozen, the head and the legs were removed, and the trunk was bisected by a sagittal mesial section, which was so accurately made that it involved the pubic joint and the urethral canal. It was then found that instead of a pregnant uterus we had to deal with a lobular myoma, whose largest lobe distended the abdominal cavity, while a smaller lobe completely filled the pelvic cavity, with its lower pole protruding 2 cm. below the line joining the lower margin of the .symphysis and the tip of the last sacral vertebra. In other words, the smaller lobe filled out the pelvic cavity and distended the pelvic floor just

Lantern demonstration before the American Gynecological Society, May 19 22.


as a child's head in the second stage of labor before it reaches the vulva.

Two other sagittal sections were made through the right half of the body — one being 3 cm. to the right of the mesial section, and the other 3 cm. to the right of the second. The left half of the bodj' was divided by four oblique sections, which roughly corresponded to the oblique planes which Hodge employed so advantageously in studying the anatomy of the pelvis. The first of these extended through the promontory of the sacrum and the top of the symphysis, while the othere were parallel to it and at varying distances below the plane of the superior strait.

Mr. Max Bra*del was kind enough to make tracings of the sagittal sections, from which he prepared the accompanying pen and ink drawings (Figures 1-3). The oblique sections were allowed to thaw in a solution of carbolic acid and formalin and were then photographed. After careful dissection the viirious landmarks were identified and have been indicated by Mr. Broedel in the retouched photographs. I take this opportunity to express my sincere thanks for his artistic work, without which this communication would be of but little value, but I am especially indebted to him for his interest and advice during tlie study and dissection of the specimens.

The object of this communication is to demonstrate the topographical relations of a large myoma and to study the changes which it has produced iu the pelvic structures, as well as to show the dift'erences between the distention of the pelvic cavity by a myoma and that resulting from the head of a child at the time of labor. Witli this in view, I shall briefly describe the conditions existing in sagittal sections 1 and 2, then I sliall consider the degree of distension of the abdominal and pelvic cavities resulting from the presence of the tumor, as well as the displacement and compression of the abdominal contents. This will be followed by a description of the tumor itself, and the changes produced by it iu the ovaries and the uterine ligaments. I shall then take up the consideration of the oblique sections through the left half of the body which illustrate the anatomical distortion produced by the pi-esence of the tumor in the pelvic cavity, and finally I shall study the changes in the vascular supply of the pelvic viscera, as shown in figure 10.

Beginning with the study of the sagittal sections, figure 1, which represents the right side of the vertical mesial .section tlirough the trunli, shows that we have to deal with a large myoma whose upper extremity reaches to the level of the twelfth dorsal vertebra and which has compressed and displaced the abdominal and jwlvic contents to an extraordinary degree. It is seen that the tumor consists essentially of two main portions — the larger one arising by a comparatively narrow pedicle from the anterior surface of the uterus near its fundus, while tiie smaller one, which distends the pelvic cavity, arises from the posterior surface of the uterus by a broad pedicle approximately 5 cm. in diameter.

Owing to the distension of the pelvic cavity by the smaller tumor and the upward traction exerted by the larger one, the uterus has been compressed against the symphysis pubis and has become greatly elongated and i-etroverted. From external os to fundus it measures 16 cm. in length. It is thinnest at the level of the upper margin of the symphysis and thickest just beneath the fuudal region, measuring 1 and 2.5 cm. in the two locations respectively. The external os is sharply marked and its anterior lip occupies a somewhat lower level than the posterior, lying approximately 5 mm. above the lower margin of tlie symphysis. The bladder has been compres.sed between the uterus and the symphysis pubis in such a nmnner that its lower portion is represented by a mere slit, while its empty upper half is transformed into an abdominal organ which extends 5.5 cm. above the upper margin of the symphysis. The rectum has been so markedly compressed that only its ampulla and lowermost portion are visible. The intestines have in great part disappeared, and are represented by three small loops, which occupy an irregularly triangular space between the myomatous masses and the fundus of the uterus, and by several loops of transverse colon lying between the stomach and the apex of the tumor.

Anteriorly, in close contact with the lower pole of the abdominal myoma and the upper portion of the uterus, is a small triangular mass, which represents a section through the lower end of the omentum which was adherent to the tumor just to the left of the midline.


Before the section thawed, the space between the abdominal wall, the anterior surface of the uterus, and the myoma was occupied by discolored ice, slightly yellowish in hue, and which represented frozen ascitic fluid. Above the upper pole of the tumor it is seen that the abdominal organs are markedly compressed and that all trace of the gastrointestinal tract has disappeared, except for the contracted stomach and four coils of transverse colon. The liver is in close contact with the under surface of the diaphragm, while the compressed contents of the thorax afi'ord striking evidence of the compression to which the body cavities had been subjected.

Figure 2, which represents the right side of the same section 3 cm. from the midline, shows essentially similar conditions, except that it gives a better idea of the manner in which the larger tumor arises from the fundus, and shows the abundant vascular supply and its extension into the lower pole of the abdominal myoma. In this section the uterus appears thicker than in the midline, and the small vertical slit in its center represents a portion of the uterine cavity. It will be noted that the pelvic cavity is still completely occupied by the pelvic myoma, and in addition that two small myomatous nodules ari.se from the posterior wall of the uterus and project into the space not occupied by the larger tumor.

Section 3 illustrates the extent to which the body cavity has been distended and the abdominal contents displaced by the tumor. It was drawn after removal of the tumor, which was effected by cutting thi'ough its connections with the uterus and separating the various adhesions which fixed its lower pole to the pelvic floor. It is seen that except for the compressed rectum, which is visible only in front of the last three sacral vertebra?, and for the uterus and bladder, which lie just behind the sym])hysis pubis, the pelvic cavity is entirely empty. Indeed the only structures which can be recognized on casual inspection are the external iliac vessels, which, roughly speaking, indicate the boundai-y of the superior strait, and the somewhat dilated ureter which extends obliquely downward and forward beneath the peritoneum covering the lateral wall of the pelvis to join the bladder in front of and above the external os.

It is apparent that the major part of the uterus has become an abdominal organ, and, with the few coils of intestines posterior to it, forms a diaphragm which partially divides the body cavity into two portions; the upper of which was occupied by the abdominal and the lower by the pelvic myomatous mass. In the upper cavity it is seen that the tumor was in direct contact with the anterior and lateral abdominal walls, and had displaced the intestines from their usual location, compressing them into an irregular triangular packet which occupies only the posterior and lateral portion of the abdominal cavity. The omentum is greatly elongated and thinned, and extends downward from the lower margin

of the transverse colon for almost the entire length of the abdominal cavity to become eventually attached to the lower-most portion of the largest myomatous mass, just to the left of the mid-line, and to contribute to its nutrition by means of numerous large vessels which it contains.

Figures 8 and t) represent the anterior and posterior aspects of the tumor, respectively. From these it is seen that the whole tumor, together with the excised upper portion of the utei-us, forms an irregular mass measuring 37X20X15 cm., and is made up of three main portions. The upper lobe, which in great part filled the abdominal cavity, i.s irregularly oval in shape, and measures 22X21X15 cm. in its various dimensions. It arises from the neighborhood of the fundus by a relatively small pedicle. Below it and arising from the right side of the uterus just below the cornu is a pedunculated kidneyshaped myoma, 12X16X4.5 cm. in its various diameters; while the third mass, which fills the pelvic cavity, arises from the upper third of the posterior wall of the right side of the uterus, with its pedicle extending only slightly to the left of the midline. This lobe measures 15X14X11 cm., being greatest in width at its upper portion, while its smaller rounded end was adherent to and distended the pelvic floor. At the junction of its upper and middle thirds a distinct furrow is visible, which has resulted from pressure upon the linea innominata and external iliac vessels. Above this depression the tumor expands on either side, probably as a result of the presence of smaller mj'omatous nodules within it.

Figure 9, which represents the posterior aspect of the tumcn', gives a good idea of the pressure which had been exerted by it, as well as of the compression to which it had been subjected by the various parts. In the upper third of the midline of the pelvic nodule a distinct depression is visible, which corresponds to the location of the promontory of the sacrum ; while below it there is a slight convexity which corresponds to the lateral concavity of the. hollow of the sacrum. Slightly to the right of this concavity is a broad vertical shallow grove, which has apparently resulted from pressure upon the i-ectum. Moreover, on the posterior surface of the larger tumor there is a vertical shallow depression which apparently has been produced by pressure against the second, third and fourth lumbar vertebrie, wJiile to the left of this is a narrower, but somewhat deeper, depression indicating the extent to which the aorta had been compressed.

The lower tumor had contracted numerous adhesions with the peritoneum lining the base of the pelvic cavity, which had to be severed before the tumor could be removed. Many of the adhesions were of considerable thickness and contained numerous small vessels, and at first led to some misapprehension when it was attempted to ascertain the relations of the pelvic fascia.


Upon inspecting figure 8 it is seen that the uterus had been deflected somewhat to the right, and had been divided by the sagittal mesial section in such a way that its vertical axis lay just median to the insertion of the left round ligament and the uterine end of the left tube. TVIoreover, it is apparent that its increased length was in great part due to hypertrophic elongation of the supravaginal portion of the cervix, while the cavity of its body was little, if at all, increa.sed in size.

Owing to the fact that the pelvic cavity was completely filled by the lower myomatous mass and that the uterus had been drawn far up into the abdominal cavity by the traction exerted upon it by the larger tumor, the tubes and ovaries have become abdominal organs, while the anatomical relations of the round ligaments have been profoundly altered. The median ends of both tubes are approximately normal. The lateral portion of the left tube is adherent to the external surface of the left ovary and is so compressed as to be recognized with difficulty. Its fimbriated end, however, is free and can be identified by a long ovarian fimbria, which is attached to the external surface of the upper pole of the coi'responding ovary. The right tube, except for its abnormal location, is apparently normal and at its lateral end is in contact with the flattened-out ovary. This measures 6.5X4.5X0.5 in its various diameters, and presents a convex external and a concave internal surface, as the result of its compression between the tumor and the abdominal wall.

The ovarian ligaments are normal in size and except for their upward course present the usual relations. Bnt for the fact that they are situated far above the pelvic brim, the infundibulo-pelvic ligaments, likewise, show nothing unusual except the presence of unusually large vessels. Upon laying back the peritoneum, the ovarian vessels were exposed, when it was found that the arteries were extremely tortuous and convoluted and measured 5 mm. in diameter. The veins were made up of numerous branches which had eventually united to form a single trunk. On the left side this occurred at the level of the fourth lumbar vertebra, above which the single vessel presented a diameter of 12 mm.

Owing to the distortion produced by the tumor the relations of the round ligaments on the two sides vary greatly. As shown in Fig. 8, the right ligament presents essentially normal relations, while the left has undergone great distortion. The former is comparatively short, and consists of a uterine portion 4 cm. long, and a terminal portion of about the same length, which occupies the right inguinal canal and terminates in the upper part of the labium majus. The latter, on the other hand, is greatly elongated and measures 22 cm. in length. Instead of extending upward, forward and outward to reach the internal ring, it extends downward and backward from its uterine origin to penetrate the peritoueum at a point 10 cm. posterior to the mitlliue aud 1.5 cm. above the level of the superior strait. It theu turns aud pursues a downward and forward course beneath the peritoneum, to disappear in the mons veneris, 1 cm. to the left of the midline.

As has already been indicated, the uterus has in great part become an abdominal organ, so that 11 cm. of its length lies above the level of the superior strait. Upon dissecting the broad ligament and the adjacent structures, it is found that the peritoneum forming the uterovesical sac can be separated from the anterior surface of the uterus to within 4.5 cm. of its fundus, above which further separation is impossible. As figure 1 shows that this is considerably above the apex of the bladder, it gives an idea of the extent to which further distention of that viscus could occur.

Our description thus far applies in great part to the general topography of the tumor and to the effect which it had exerted upon the economy of the woman. On the other hand, information of more particular gynecological and obstetrical interest nuiy be gained from the study of the .sections through the left side of the body which were made parallel to the superior strait.

Fig. 1 represents a retouched photograph showing the relations at the superior strait. The section passes through the top of the symphysis and the cartilage covering the promontory of the sacrum, and extends upward and backward to the skin of the back in such a way as to involve the body of the fifth, the arch of the fourth and the spinous process of the third lumbar vertebra. It corresponds to Hodge's first oblique itlane and shows clearly the extent to which the soft i)arts encroach upon the pelvic inlet.

The pelvic cavity is completely filled by the myomatous mass, which has pushed the uterus so far forward that it is separated from the posterior surface of th^ syml)hysis only by the compressed slit-like bladder. It is interesting to note how large a portion of the ilio-psoas muscle occupies the iliac fossa, as well as the fact that the external iliac vein follows practically the contour of the liuea innominata, but is in contact only with its anterior and |)osterior ends. It should be noted that the triangular space just lateral to the body of the first sacral vertebra is occupied by the hypogastric artery an<l vein, the ureter and by the obturator nerve. Anteriorly, it is seen that the uterine body is directly in contact with the bladder and so compressed that it does not exceed 1 cm. in thickness. The broad ligament extending from its margin is greatly thinned out and lies in contact with the anterior and lateral walls of the pelvis, instead of extending transversely across the middle of the pelvis, and dividing it into an anterior and posterior l)ocket. It should further be noted that the anterior face of the broad ligament lies in contact with the pelvic fascia, that the double fold of peritoneum which should


normally be present has disappeared; while the peritoneum covering its posterior face constitutes the peritoneal lining of the anterior portion of the pelvis. In other words, the myoma occupies the immensely distended pouch of Douglas, and has displaced and compressed everything in front of it.

Fig. 5 represents a .section parallel to, and two centimeters below, the superior strait. It bisects the symphysis pubis and the body of the first .sacral vertebra, while posteriorly it involves the arch of the fifth and the spinous process of the fourth lumbar vertebra. It will be noted that the bony ring of the pelvis is here complete, being formed posteriorly by the body of the sacrum, laterally by the iliac, and anteriorly by the pubic portions of the innominate bone.

The sacroiliac joint is well shown, and it is apparent that in this instance the sacrum does not form the keystone of an arch, but would prolapse into the pelvic cavity under the influence of the body weight were it not held in place by strong ligamentous structures. It is likewise apparent that the myoma completely fills out the pelvic cavity and has led to intense compression of the uterus, which at this level is represented by a crosssection of the flattened cervix. The broad ligament is much thinner than in the preceding section and lies in direct contact with the thinned-out lateral ligament of the bladder, which extends outward from the margin of that viscus.

Probably the most striking feature in this section is the presence of a small segment of the iliopsoas muscle, which has descended beneath the level of the linea innominata and actually encroaches upon the pelvic cavity. The distended ureter in its course towards the bladder has come to occupy the middle of the lateral pelvic wall and is separated by the pelvic cavity only by peritoneum and sub-peritoneal fascia. The various arteries and veins are well shown, and passing down over the anterior surface of the lateral mass of the sacrum is the lumbosacral trunk. In this section it is apparent that, while the ureter lies median to the pelvic fascia, the main vessels in the posterior segment of the pelvis are enclosed within sheaths derived from it. It is important to note that in this section there is as yet no sign of the obturator internus, the pyriformis or the levator ani muscles.

Fig. repre.sents the upper surface of the third parallel section and lies 2.5 cm. posteriorly and 3 cm. anteriorly below the section just described. This plane bisects the second sacral vertebra, while its anterior end terminates in the external genitalia half a centimetre below the lower margin of the symphysis. For practical purposes it corresponds with Hodge's second parallel plane and with Veit's main plane of the pelvis, and would be identical with the latter did its ends involve the lower margin of the first sacral vertebra and symphysis pubisi respectively. Here it will be noted that the bony wall of


the pelvis is no longer complete, its anterior portion being represented only by the descending ramus of the pubis, to whose left lie the ei-ectile structures just beneath the pubic arch ; while to the right is the obturator foramen, which is covered internally by the median end of the obturator internus muscle.

The sacroiliac joint is well shown, and it will again be noticed that the sacrum represents an inverted keystone of an arch, and that only a fraction of its thickness takes part in the formation of the joint. Laterally, where the innominate bone is sharply constricted and is devoid of cancellous tissue, is a plate of compact bone which corresponds to the apex of the sacrosciatic notch. In this section it is again evident that the pelvic cavity is completely occupied by the myomatous mass. Anteriorly, the uterus has disappeared and has been replaced by the vagina. Attention is directed to the fact that the thinned-out broad ligament of the previous sections has given place to its thick vascular base and to the perivaginal tissues. The fonner lies internal fo the lateral vesical ligament, and gradually fades awaj' as the middle portion of the pelvic wall is approached. Furthermore, it should be noticed that the uterine artery has receded from the midline, and, roughly speaking, occupies a position opposite the lateral margin of the obturator foramen.

Anteriorly and laterally, the obturator internus muscle is seen as a thin structure varying from 2 to 5 nun. in thickness, extending from the ischio-pubic ramus to the ischial spine, and giving no suggestion of the dimensions it will assume in the next succeeding section. In the l)osterior quadrant of the pelvic cavity, and lying in front of the sacroiliac joint, the sacral plexus may be seen approaching the sacrosciatic notch. It lies external to the pelvic fascia and is separated from the bone only by the superior gluteal vessels, and its location affords a ready explanation for at least some of the pain, which may be associated with relaxation of that joint. In the extreme anterior and median portion of the section the labium nmjus and minus are visible, while posterior to them lie the various erectile tissues associated with the clitoris.

Fig. 7 is very instructive and gives a conception of the topography of the pelvis which cannot be gained by the ordinary methods of study. This section, which is parallel to tiie preceding one, lies 2.6 and 2.8 cm. beneath its posterior and anterior ends respectively. Posteriorly it extends through the tip of the third sacral vertebra, while its anterior portion involves the external genitalia and is obliquely traversed by the urethra just beneath its upper surface. The tumor still occupies the greater part of the pelvic cavity, but the striking features of the section are afforded by the relations of the pelvic muscles, the sciatic nerve and the base of the broad ligament. At this level it is seen that the obturator internus has become transformed from the thin structure visible


in the previous section into a thick-bellied muscle, which extends from the descending pubic ramus to the ischial spine while external to the body of the ischium its strong tendon can be seen on its way to its point of attachment in the depression at the back of the trochanter.

The pyriformis muscle presents a striking picture and appears as a surprisingly large structure, which arises from the greater part of the anterior surface of the sacrum, and passes through and almost completelj- fills the sacro-sciatic notch on its way to its attachment to the femur. Internal to the obturator internus can be seen an oblique section through the levator ani muscle, which at this level varies between 3 and 4 mm. in thickness. It can be traced upward for about half a centimetre into the preceding section, where its insertion to the posterior surface of the symphysis and ischio-pubic ramus, to the white line, and to the ischial spine can be located. In this section the muscle is covered on either surface by fascia, while above the two layers fuse into the single layer of pelvic fascia above the white line. Under normal conditions in this location the peritoneum covering the internal layer of fascia over this muscle should line the entire pelvic cavity, but in the specimen under consideration a thick layer of abundantly vascularized pelvic connective tissue is interpolated between it and the pelvic cavity. In other words, the anterior segment of the pelvic cavity has become obliterated, while Douglas' cul-de-sac has become gi-eatly expanded.

In this section, likewise, we see the sciatic nerve in oblique section, which affords a vivid picture of its magnitude and teaches us how readily its median end can be subjected to pressure by an object distending the pelvic cavitj'. Just anterior to the nerve and almost in contact with the ischial spine can be seen the internal pndic vessels. On inspecting the anterior portion of the section, the vagina is represented by two slits, which are directed obliquely backward and are separated by a thick layer of highly vascularized tissue. This is the result of an oblique section through a fold of its mucosa, and by no means indicates that the canal is double. Posteriorly the sacroiliac joint is again seen, and once more it shows that the union at the sacroiliac Joint is such that, if the sacrum were not held in place by strong ligamentous structures, it would inevitably collajtse into the pelvic cavity.

This section represents almost the base of the ])elvic cavity; for in the next one, which lies 3 cm. beneatli it and extends through the tip of the fifth sacral vertebra, all that is left is a shallow oval area about 5..") cm. iu diameter, and 5 mm. in depth, lying immediately over the coccygeal vertebriv. Posteriorly, this depression is occupied by iieri-rectal tissue which is abundantly sup])lied witli vessels, while anterioi'ly and laterally the thinned-out margins of the levator ani can be distiu


guished, whose libers fuse with and disappear into those of the sphincter aiii. The greater portion of this last section, which is not figured, is made up of an oblique section through the gluteal muscle, outside of which comes a laj-er of subcutaneous fat which varies from 2 to 5 cm. in thickness.

Upon dissecting the lower surface of the fourth section from below, the ischiorectal fossa can be opened up as a large potential cavity bounded internally by the levator ani and its fascia, and externally by the layer of fascia covering the obturator internus, the pyriformis and the gluteal muscles, and reaching its apex at the level of the white line.

Thus far, we have studied the relations of the tumor to the pelvic cavity and to the structures within it. Incidentally, we have mentioned the enlargement of the ovarian vessels and those of the broad ligament, and we shall now study the changes which have occurred in the vessels supplying the pelvic organs. These are best sttidied in figure 10, which represents the lateral wall of the pelvic cavity after the removal of the tumor, as seen in a sagittal 'section through the right side of the body, ;{ cm. from the median plane. The striking features of this section are the relations of the ureter and of the l>elvic vessels. The former is dilated to several times its usual calibre and constitutes a hydro-ureter. Posteriorly tlie vessels present essentially normal conditions, while anteriorly they show numerous deviations from the usual relations which have resulted from the presence of the tumor and the consequent need for an increased vascular supply.

The median surface of the section involves the lowermost end of the common iliac vein just before it divides into the external and internal iliac veins. On the other han<l, the ])oint of division of the artery lay higher up and median to tlie plane of the section, so that the external and internal iliac arteries appear as separate vessels.

The external iliac artery and vein need not c'oncern us here, as they have already been considered in connection with Fig. i, where the latter formed the outer boundary of the superior strait. The internal iliac or hypogastric vessels are, however, of great importance. Shortly after entering the pelvic cavity the hypogastric artery divides into three main branches. The outer one continues forward, ujjward and eventually inward toward tlie umbilicus, as the obliterated hypogastric artery. The median branch is the uterine artery which is unusually elongated and enlarged. After passing beneath the ureter, it follows the pelvic wall as a vessel with many convolutions almost to the symphysis pubis where it enters the base of the broad ligament to supply it and the uterus, instead of entering it at about the middle of the pelvis. The posterior division is the largest of the three, and is much larger than under usual conditions.


After traversing the posterior portion of the pelvis for several centimeters, it divides into the inferior gluteal and the middle and inferior hemorrhoidal arteries.

Thus it is seen that except for a considerable increase in the size of all vessels and the striking elongation and tortuosity of the uterine artery, the arterial supply of the pelvis differs but little from normal. On the other hand, the venous supply has become extraordinarily expanded, and the section clearly shows that the lateral pelvic wall is covered by a number of large trunks, each of which approximates the external iliac vein in size, and which extend from the anterior portion of the pelvic cavity in order to carry away the blood from the enlarged uterus and its appendages. It is evident that they here form a plexus, which has attained immense proportions and covers the entire middle third of the lateral and anterior pelvic walls, and which drains the vesical and utero-vaginal regions. Posteriorly, just in front of the pyrifoi-mis muscle, the lumen of an immense vein is visible which has probably resulted from the union of the gluteal and hypogastric veins.

This picture was obtained by turning back the peritoneum and the subperitoneal fascia, and shows that all of the vessels in question were surrounded by their own fascial sheaths and lay median to the main pelvic fascia, so that, in their anterior portions at least, they were separated from the levator ani muscle by that structure. In the lower part of the section a small segment of the rectum is seen, whose anterior and npper wall has been deflected in order to expose the vessels beneath it. Tliis section likewise affords an excellent picture of the relation of the muscles of the pelvis. Anteriorly, the obturator externus and obturator internus muscles are seen upon the two aspects of the obturator fascia, respectively. Median to and in close contact with the obturator internus, the anterior half of the levator ani muscle is seen, which with its posterior portion forms a diaphragm concave above and convex below, which extends from the horizontal ramus of the pubis to the ischial spine. In this section the muscle scarcely exceeds 1 mm. in thickness and is considerably thinner than in Fig. 7; while in its anterior portion, immediately adjacent to the bone, it is almost fascial in character. Over-lapping its posterior extremity and lying external to it, a section through the coccygeal muscle is visible, which is only a fraction as extensive as the levator. Still further posterior and higher up, a section of the pyriformis is seen in the shape of a large triangular muscular mass lying between the anterior and inferior end of the sacral ala and the upper and anterior surface of the gluteus maximus muscle. In front of the pyriformis are the lower two, and above it and in contact with the anterior surface of the sacrum are the upper two, of the four .spinal roots of the sacral plexus; while at the uppermost por


356


[No. 380


tiou of the anterior surface of the sacrum a section of the lumbosacral trunli is visible. This is in marked contrast with the appearance of the sacral plexus as shown in Fig. 7, and illustrates the conditions more median to its point of origin. In front of the pyriformis muscle the internal pudic artery can be seen making its way toward the ischial spine on its course to the external genitalia.

This completes in a cursory way the description of what the various sections show, and it only remains to recapitulate in a few words the changes brought about by the presence of the tumor and to compare the behavior of the pelvic floor with what would have resulted had the pelvis been distended by a foetal head late in labor.

In the first place, the specimen gives an extraordinarily accurate idea of the extent of the abdominal and pelvic distension which may be produced by the presence of a large myoma. Owing to the fact that the nodule, which almost completely filled the pelvic cavity, arose from the posterior surface of the uterus, the distention has occurred altogether posteriorly to that organ, with the result that it together with the bladder has been compressed against the anterior pelvic wall. — In other words, the distention has occurred entirely within the pouch of Douglas. This has resulted in a remarkable displacement of the broad ligaments, so that they are in contact with the anterior and lateral wall of the pelvic cavity instead of dividing it into an anterior and posterior segment ; the former has become obliterated and the latter immensely distended. Owing to the forward displacement of the titerus and of the median ends of the broad ligaments, gi-eat elongation of the uterine arteries was necessaiy. This apparently has occurred to an even greater extent than was essential, as is shown by the fact that instead of entering the base of the broad ligament shortly after they have been crossed by the ureters, the arteries extend forward almost to the symphysis pubis before so doing, and at the same time have become unusually convoluted. Moreover, even though myomata are in general very poorly vascularized, the demands for the nutrition of the tumor have necessitated an increase in the blood supply, as is shown by the


unusual hypertrophy of the pelvic veins, which has attained a degree encountered only in full-term pregnancy.

Upon comparing the changes in the pelvic floor in this instance with those occurring during normal jjregnancy, several very important points of difference should be noted. In the first place, when the pelvic cavity is occupied by the fu-tal head late in the second stage of labor, the distension proceeds from a central point, with the result that the cervical canal together with the base of the broad ligament becomes expanded outwardly in all directions, to an equal extent, so that while the anterior and posterior peritoneal pouches are temporarily obliterated, no such change occurs as was noted in this instance. In it the pelvic tumor lay behind the uterus, and consequently, as it increased in size, the posterior pouch became immensely distended, while the anterior pouch was obliterated by the cervix being pressed firmly against the symphysis ])ubis. At the same time the broad ligaments were flattened out against the anterior and lateral portion of the pelvic wall, instead of involving its posterior segment as well. The distension has likewise led to comparable changes in the relations of the pelvic fascia and the structures beneath it, which, instead of being pushed centrifugally outwards from a common center, have become distended only posterior to the uterus, with the result that the levator ani muscle forms a continuous but thin pelvic diaphragm, instead of being perforated and pushed downward and outward to form the lower most portion of the birth canal.

Finally, particular attention is directeil to the unusual picture of the anatomical relations which have been obtained by the employment of oblique sections through the pelvis parallel to the superior strait, and which indicate the advantages which might well follow the use of similar sections in the study of the dislocation of the pelvic floor during labor, as well as in the study of the part played by the bases of the broad ligaments in the maintenance of the normal position of the uterus in the non-pregnant woman.



PLATE XLIV


PLATE XLV




Fic. 4.— Oblique section ttirough left half of body, showing relations at plane of superior strait, x 1/2.



Fw. 5. — Oblique section parallel to and 2 cm. below that shown in figure 4. To demonstrate absence of pelvic muscles in this area, x 1/2.



PLATE XLVI



Fig. 6. — Oblique section through left half of body, parallel to and 5 em. below that shown in figure 4. (Hodge's second parallel plane), x 1/2.



FKi. 7. — Oblique section parallel to and 2.5 cm. below that shown in figure 6. extending through the tip of the third sacral vertebra,. To illustrate the extent of the pelvic musculature, x 1/2.


PLATE XLVII


Fifi. 8. — Anterior view of tumor showing its relations to the uterus, round ligaments anrl tubes, x 1/3.


Fill. 9. — Posterior view of tumor showing indentations due to pressure exerted by the margins of the superior strait and vertebral column. X 1/3.



Fig. 10.— Sagittal section through right side of bod.v, 3 cm. from midline, adjacent to figure 2, showing relations of blood vessels, ureter and pelvic musculature. X 1/2.


THE CHANGES IN THE PARA-OCULAR GLANDS WHICH FOLLOW THE ADMINISTRATION OF DIETS LOW IN FAT-SOLUBLE

WITH NOTES OF THE EFFECT OF THE SAME DIETS ON THE SALIVARY GLANDS AND THE MUCOSA OF THE LARYNX AND TRACHEA

By SiiiNNosuKE Mori, M. D.

Chief of the Ophthalmological Clinic at the South Manchuria Railway Company Hospital, Dairen, Manchuria

(From the Department of Chemical Hygiene, School of Hygiene and Public Health, Johns Hopkins University, Baltimore)


In a previous paper ' I showed that the primary changes which occur in the eyes of rats on diets deficient in fat-soluble A are xerosis conjnnctivie, and xei'osis corneiP (xerophthalmia). These changes are identical with those of xerosis in human eyes which are believed to result from dryness of the tissue. The ulceration and destruction of the cornea wjiich follow xerosis corneie and conjunctiviP (keratomalacia i are due to the secondary infection of the cornea by micro-organisms. Since the primary change in these tissues is due to dessication, it would seem logical to look for the cause of the trouble in the organs whose secretions under normal conditions serve to moisten the conjunctival sac. The secretion of the lacrimal gland — the tears — is chiefly concerned with the moistening and cleansing of the surface of the anterior portion of the eye. Beside the secretion of the lacrimal gland the conjunctival sac receives that of the Meibomian (tarsal) glands in the lids. The Harderian gland in the orbit also pours its secretion into the conjunctiva, and the mucous cells in the membrane itself contribute also to the conjunctival fluid. All these secretions are mingled in the conjunctival sac, and the resultant mixture moistens and protects the eyeball. The anterior part of the eye is kept clean by this secretion in which there is a more or less constant current, running toward the nose, which serves to wash foreign material through the nasolacrimal duct. This fluid also checks the multiplication of micro-organisms, since the tears possess some bactericidal power. How effective this mechanism is in removing bacteria from the conjunctival sac may be judged by the fact that bacteria, after being sprayed into the conjunctival sac, may be recovered from the nose in five minutes.- The purpose of this paper is to report the changes which occurred in the above-mentioned glands from animals in various stages of experimentally induced xerophthalmia. Moreover, I have also attempted to correlate the changes in these para-ocular glands with those in the salivary glands, and the changes in the conjunctiva with those in the mucosa of the larynx and trachea.


Lacrimal gland. — The most striking change in the tissue of this gland in animals suffering from xerophthalmia is in shrinkage of the secretory cells of the acini. These cells become very much shrunken. They no longer show secretory zones. The nuclei take on irregular shapes, occupy the central part of the cytoplasm, which is very small and stains very poorly. The individual acini of the gland become so small that it is impossible to distinguish them with the low power of the microscope, and the gland appears to be a simple mass of nuclei. It is not to be expected that a gland in this condition can produce tears. In a certain number of animals the cytoplasm of the gland cells is occupied by very large vacuoles to such an extent that the bioplasm itself has almost disappeared. The nuclei of such cells are small, centrally located and deeply stained. Frozen sections stained with Sudan III and sections from material which has been fixed in rieming's solution show that some of these apparent vacuoles are in reality droplets of fat. Glands such as I have described are those which are most severely affected, but even in those that are most nearly normal, the cells show no evidence of pre-secretory or secretory activity; they are clear and small, and do not stain well. The poorly stained nucleus is situated in the basal portion of the cell, surrounded by small vacuoles in the cytoplasm, whose central portion consists only of a fine network. Although it is possible that such cells may produce some secretion it is quite certain that the production would be in very much diminished quantity.

The above findings indicate that the lacrimal glands of animals affected with xerophthalmia produce little, if any, secretion ; in any case it is difficult to believe that any secretion which they might produce could be normal. It is quite certain that some of these glands have ceased .secreting entirely.

Meibomian gland. — Sections of the lids of the eyes of animals suft'eriug from xerophthalmia very often show cystic dilatation of the ducts of the Meibomian glands. These cysts are filled with fat — the secretion of the gland cells. The epithelium of the margin of the lid shows


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evidence of a very marked xerotic process, aud the same change is found in the epithelium of the duct. It is very lilvcly that this xerosis may play a part in the occlusion of the duct and the formation of the retention cysts.

Harderian gland. — The lumina of the acini of this gland may be either very much dilated and empty, or narrowed by swelling of the secreting cells. Frozen sections of this gland show a remarkable diminution of the fat content of the cells and the lumen. The connective tissue about the gland acini is very often densely infiltrated with round cells.

The mucous cells of the conjunctiva are all entirely destroyed in the course of the xerotic process.

It is quite certain from these findings that the entire secretory apparatus of the eyes of these rats is in a state of dysfunction, that is, the secretion from these glands is either very much diminished or entirely lacking. The change.s in the lacrimal gland are the most important in the pathological picture of this disease, and in fact the changes in this gland would seem to be the cause of the lesions in the cornea and conjunctiva. The diminution or the lack of the secretion of the lacrimal gland would explain the dryness of the eyeball as well as the xerosis of the cornea and the conjunctiva. Tho failure of the lacrimal secretion would, moreover, explain the increase in number of the organisms found in the conjunctival sac.

Salivary glands. — Having found very evident changes in the lacrimal gland during the course of xerophthalmia, it seemed logical to study the salivary glands which both in nerve supply and in structure resemble the lacrimal glands very closely. I therefore made a comparative study of these glands in rats affected with xerophthalmia. The glands of normal and of xerophthalmic animals were fixed in 10% formalin, or in Fleming's solution, embedded in celloidin, or cut in frozen sections aud stained with hematoxylin-eosin, iron hematoxylin, safraniue, and Sudan III.

The submaxillary and parotid glands of the rat are serous ; the sublingual is a mucous gland. In many I'ats with xerophthalmia either all or some of these glands are either not secreting at all or secreting very little. The secreting cells become much shrunken. The acini are very small and show no traces of secretion. The epithelium of the intralobular ducts is shninken and the cells are irregular in size.

The epithelium of the principal ducts of these glands often shows a remarkable degree of cornification and desquamation of the thickened superficial cells, so that the lumen of the duct becomes narrow and is often occluded. The.se changes resemble the xerotic changes in the eye. In two cases the occlusion of the ducts of the sublingual gland was followed by the dilatation of the duct and the gradual atrophy of the gland cells. Dilatation of the small ducts occurs frequently in the parotid


gland. Coincident with the xerotic changes and those in the parenchymal cells, the ducts are invaded by bacteria and small abscesses arc formed in the gland. In other words, the findings in the salivary gland are of a nature similar to tho.se in the lacrimal gland, and the duct epithelium undergoes a change which is identical with those which occur in the conjunctival epithelium. The reason for the infection of the salivary glands in these animals probably explains also the parotitis which sometimes complicates typhoid fever. The glands are directly infected from a dry mouth which contains an abnormally large number of micro-organisms.

The other secretory organs, such as the liver, pancreas, bowel, kidneys, thyroid, showed no remarkable change in sections except in one case. In this animal the cytoplasm of a small number of cells of the pancreas was very much vacuolated. Tlie reproductive glands of rats on a xerophthalmia-producing diet do not function. Reproduction in these animals almost never occurs. Wason ^ has reported fibrosis of the testes in two animals which she examined after they had beeu on a xerophthalmia-producing regimen.

Clinically and at autopsy, the mucous membrane of the mouths of these rats was very dry. The animals thus afflicted ill the early stage of the disease sneeze and cough violeiitl.y, but later the cough subsides spontaneously and after a period of dyspnoea the animals die. Nevertheless, histological sections of the mucosa sliowed no more than the normal cornification of the epithelium. On the other hand, the mucous membrane of the larynx and trachea showed xerotic changes. The normal epithelium of the larynx and trachea is ciliated columnar in type, except that of the vocal cords, which are covered with stratified epithelium. The xerotic changes first apjiear in the epithelium of the larynx. The outer layer of cells in this region becomes cornified and stains deeply with eosin. The cells lose their nuclei, and become much flattened. In the second layer typical granules of kerato-hyaline appear. The epithelium becomes much thickened and is often infiltrated with pus cells. Gradually this xerotic change involves the tracheal mucosa. Xerosis then becomes complicated by an inflammatory process which often involves the bronchi and lungs and may terminate in a bronchopneumonia. Such a pneumonia was responsible for the death of most of the animals whidi were not sacrificed. The mucous cells in the mucous membrane are completely destroyed. Those which are embedded i" the subinucosa show evidence that their secretion is either diminished or that their secretory power is entirely destroyed. It is easily seen from the foregoing description how closely these changes in the mucosa of the larynx and trachea resemble those in the cornea and conjunctiva. If the same conditions obtain in the larynx and trachea as in the conjunctival sac, the bacterial content of the air passages should be much increa.sed


THE JOHNS HOPKINS HOSPITAL BULLETIN. OCTOBER. 1922


PLATE XLVIII


y^'^



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PLATE XLIX


THE JOHNS HOPKINS HOSPITAL BULLETIN. OCTOBER. 1922



C >



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mc



Fig. 7


Fig. 8


October, 1922]


359


because of the dryiug of the uuifosa and loss of motility of the cilia of the epithelium. At any rate just as xerosis of the cornea is followed by secondary infection and the formation of corneal ulcers, so xerosis of the larynx and trachea is followed by infianimatoiy changes which eventually spread and involve the bronchi in the lungs.

The secretion of tears is, as a rule, either scanty or absent in children suffering from xerophthalmia or keratomalacia even during crying. These children are, as a rule, veiy hoarse and the voice is never clear. As is the case in rats with experimental xerophthalmia, the larynx iu children is implicated in the pathological process, and like the rats the children usually die from bronchopneumonia. The pathological changes in rats explain the hoarse voice, the cough, bronchopneumonia and bronchitis, and the absence of tears and saliva which are seen in human cases of this disease.

If two per cent of cod-liver oil be added to the diet of xerophthalmic rats, the conjunctival sac becomes moistened, the number of bacteria becomes diminished, the xerotic change in the epithelium disappears. Coincidently with the healing of the xerosis of the eye the lacrimal and salivary glands become normal histologically and they show histological evidence of secretion.

The results of these studies make it seem probable that the effect of cod-liver oil, or fat-soluble A, is not exerted directly upon the tissues of the eyes and the lids. More probably the vitamin acts, either directly or indirectly, on certain of the secreting glands in the body to insure their normal function, which is held iu abeyance if the animal is deprived of his optimal intake of the vitamin. From this point of view it is necessary to consider the


clinical picture of xerophthalmia as a series of secondary symptoms which are indicative of hypo-function of a certain part of the secretory apparatus of the organism. In other words, xerophthalmia, as it is ordinarily seen, is the manifestation of a specific glandular lesion. It is one of a chain of symptoms, the other links of which are hoarseness, cough, lack of saliva, sterility, and general malnutrition.

BIBLIOGRAPHY

1. Mori, S.: Jour. Am. Med. Assn., 1922. 79, 197.

2. Maxcy, K.: Ibid.. 1919, 72, 636.

3. Wason, Isabel M.: Ibid., 1921, 76, 908.

DESCRIPTION OF FIGURES

Fig. 1. — Shows the shrinkage of the cells of the lacrimal gland (L) and the Harderian gland (H).

Fig. 2.— The retention cyst formed as the result of the occlusion of the duct of the Meibomian gland (M) of lid.

Fig. 3. — To show the shrinkage of the secreting cells of the submaxillary gland.

Fig. 4.— This figure shows the oornification (C) of the epithelium of the principal duct of the sublingual gland.

F^G. 5. — Atrophy of the sublingual gland cells and extrusion of mucus into the interstitial connective tissue caused by the occlusion of the principal duct.

Fig. 6. — Cornification (C) of the intra-lobular ducts of the parotid gland and the partial shrinkage of the secreting cells (SO.

B^g. 7.— Infection through the duct of the submaxillary gland (I). The epithelium of the duct is very much thickened and the glandular cells have become atrophic (C).

Fig. 8. — This figure shows xerosis of the epithelium of the trachea and the desquamation of the cornifled and thickened epithelium (E).


NOTE ON A MODIFICATION OF THE CHROMAFFIN RE ACTION, WITH OBSERVATIONS ON THE OCCURRENCE OF ABDOMINAL CHROMAFFIN BODIES IN MAMMALS

By G. B. WisLoCKi

(From the Anatomical Laboratory of The Johns Hopkins University j


That the cells of the medulla of the adrenal glands give a brownish reaction when fixed in a solution containing a salt of chromic acid was first observed by Henle ('<io|. This observation was extended by Stilling ('90), who showed that in the dog, cat. and rabbit there are groups of cells associated with the sympathetic nervous .system, that react similarly with chromium salts. These he called "chromophil cells." Kohn ('03) abundantly confirmed Stilling's observations, showing, in the cat and rabbit particularly, that "chromaffin cells," as he designated them, occur frequeutlj' in association with the sympathetic nervous system, either as microscopic clusters of cells embedded iu the ganglia, or as


bodies of macroscopic size located iu the sympathetic plexuses. These groups of cells he termed "paraganglia." He directed attention to a conspicuous strip of chromaffin cells situated ventral to the abdominal aorta and superior to the inferior mesenteric artery, which was I)resent in all of the mammals he studied and which he termed the "paragaiiglion aorticum abdominale." Finally, Zuckerkandl ('01 j discovered a group of paired bodies in the region of the inferior mesenteric ai'tery in human fetuses and newborns which gave the chromophil reaction. These are usualh' referred to as the "organs of Zuckerkandl."



The methods employed by all subsequent observers for demonstrating these bodies have been those recommended by Stilling and Kohu. The retroperitoneal tissue of the animal is exposed and cotton, saturated with 3.5 per cent potassium bichromate, is placed in the abdomen for from 6 to 12 hours. The reproperitoneal tissue is then excised and washed for several hours in running water and then examined for the presence of chromaffin bodies, which, when present, should stand out as brownish strands or patches again.st a lighter colored background. A modification of this method consists in immediately excising the tissue suspected of containing chromaffin bodies and immersing it in 3.5 per cent potassium bichromate for the same length of time, followed by washing. It is advisable to transfer the tissue finally to glycerine in order still further to bring out the chromaffin bodies.

AVhen subsequent microscopical examination of the tissue is desired, it is i-ecommeuded that the chromaffin reaction be performed by fixation in formalin-bichromate (formaldehyde 40 per cent, 10 c.c. ; potassium bichromate, 3.5 per cent, 90 c.c). In all of these methods it is of course desirable that the tissue be fixed as soon after death as possible, for in the course of 6 to 18 hours post mortem the reaction no longer occurs in the chromaffin cells.

That the technique is sufficient to demonstrate the chromaffin bodies in man, dog, cat and rabbit is evidenced by the observations of Stilling, Kohn and Zuckerkandl and the subsequent ones of Swale Vincent ('12) and Kahn ('12). Although the principal bodies are readily distingiiishable to the naked eye, in these animals, small scattered bits of chromaffin tissue cannot always be differentiated from clotted or extravasated blood without microscopic examination. Furthermore, Swale Vincent ('12) has been unable to discover any chromophil bodies in some animals, viz., monkey, pig, guinea-pig, rat, gopher, and squirrel. Kahn ('12) also reports the absence of macroscopic chromaffin bodies in four adult monkeys examined by him. One might assume that all of these animals lack macroscopically demonstrable chromaffin bodies. On the other hand, a suspicion might be aroused that the chromaffin reaction, as at present performed, is inadequate for their demonstration. The author proposed therefore to modify the technique, as heretofore used, and to investigate a number of animals for the presence or absence of chromaffin bodies. In the l)resent paper this modified technique is described, together with results obtained in the few mammals which it was possible to secure for this purpose.

Technique The tissue to be studied was excised from the freshly killed animal, washed as free of blood as possible in running tap-water and placed in a large volume of fixative consisting of 90 parts of 3.5 per cent potassium bichro mate and 1 part of 40 per cent formaldehyde (Kohn's


fluid). The tis.sue was not allowed to remain in this fixative over 3 hours, at the end of which time the chromaffin reaction was complete; it was then rinsed in tap-water and transferred for further fixation to 10 per cent formalin for 24 hours, after which it was washed thoroughly in running water and transferred successively to 60 and 70 per cent alcohol. The tissue was bleached by allowing it to stand for from 6 to 24 hours in sunlight in several changes of 70 per cent alcohol, prepared by adding 20 parts of hydrogen peroxide U. S. I', and 10 parts of water to 70 parts of alcohol. When completely bleached, the tissues, other than the chromaffin bodies, appear perfectly white. The chromaffin bodies stand out conspicuously (depending on the species of animal), as lemon-yellow to dark brown bands, threads, dots, or patches against a white background. The material may now be stored in 80 per cent alcohol, or portions of it embedded and cut. Excellent permanent gross preparations were obtained by dehydrating the material and clearing it in benzol and methyl salicylate according to the Spalteholz method. In these transparent preparations the chromaffin tissue stands out as a series of brown bodies whose position in reference to the blood-ves.sels is readily visible.

The observations of the earlier investigators concerning the size, shape, number, color, and distribution of the abdominal chromaffin bodies in the dog, cat, rabbit and in man have been amply confirmed by thi.s technique. Further, the opossum, guinea-pig, squirrel, rat, and monkey were examined and, with the exception of the rat, abdominal chromaffin tissue was demonstrated macroscopically in all of them.

Oposstnii (two adult females). — On bleaching the retroperitoneal tissues, from 8 to 10 lemon-yellow bodies, about 1 mm. in diameter, became visible, scattered irregularly in a plexus of nerves extending from the level of the adrenal glands to the bifurcation of the aorta. On section these bodies proved to be ganglia in which were embedded groups of from 10 to 30 cells whose cytoplasm contained dark brown stained material (Fig. 1).

Guinca-piy (three adult males and three adult females) . — In eveiy instance, on examining the bleached retroperitoneal tissue, a conspicuous oi-ange-colored stellate mass, measuring 2 mm. in diameter, was observed in the midline in the cceliac plexus, immediately below the superior mesenteric artery. On .section this proved to be a mass of cells possessing a distinct capsule and exhibiting a characteristic chromaffin reaction (Fig. 2). No cliromaffin bodies were demonstrable in the gross elsewhere in the abdomen.

Squirrel {one adult male grey squirrel). — After bleaching, an orange-colored mass, measuring 2 mm. in diameter and irregularly stellate in outline, became visible in the midline in the cffiliac plexus immediately below the superior mesenteric artery. On section this proved to be




Fig. 4


Plate, — Wislocki, G, B, A Modification of the Chromaffin Reaction, with Observation on the Occurrence of Abdominal Cliromaffin Bodies in Mammals,


I

I

I


I


October, 1922]


361


a characteristic group of chromaffin cells (Fig. 3). No chromaffiu tissue was visible to the uuaided eye elsewhere in the abdomen.

Rat (three adult males and three adult females). — After bleaching the retroperitoneal tissue, the material was carefully examined with the naked eye and with the aid of a lense, but no masses of chromaffin tissue could be discovered.

Monkey (one adult female macaque). — After bleaching the retroperitoneal tissue, a series of small yellow bodies appeared, extending in a brolien chain from the region of the superior to the inferior mesenteric arteries. The largest of these measured 2 mm. in length. On section these bodies proved to be typical masses of chromaffin cells (Fig. 4).

Summary

A modification of the technique for demonstrating chromaffin tissue is described, whereby the presence of chromaffin bodies of macroscopic size can be demonstrated in the retroperitoneal tissue of the opossum, .squirrel, guinea-pig, and monkey.


BIBLIOGRAPHY

Henle, K. : Ueber das Gewebe der Nebenniere und der Hypophyse. Ztschr. f. rat. Med., 1865, XXIV, 143-152.

Kahn, R. H.: Studien an Paranganglien. Arch. f. ges. Physiol., 1912, CXLVII, 445-472.

Kohn, A.: Die Paraganglien. Arch. f. niikr. Anat.. 1903, LXII, 263-365.

Stilling, H.: A propos de quelques experiences nouvelles sur la maladie d'Addison. Rev. d. m6d., 1898, X, 808.

Vincent, Swale.: Internal Secretions and the Ductless Glands. 1912.

Zuckerkandl, E.: Ueber Nebenorgane des Sympathicus ini Retroperitonealrauni des Menschen. Verhandl. d. Anat. Ges., XV Vers., 1901.

DESCRIPTION OF PLATE

Fig. 1. Sympathetic ganglia from the aortic plexus o( an opossum, showing a group of chromaffin cells, (x 400.)

Fig. 2. Chromaffin body from the coeliac plexus of a guineapig. (X 320.)

Fig. 3. Chromaffin body from the coeliac plexus of a squirrel, (X 290.)

Fig. 4. Chromaffin body from the aortic plexus of a macaque. (x200.)


ON THE CARDIAC COMPLICATIONS OF GONORRHOEA

By W. S. Thayer, M. D. {Baltiinore)


Tl>e old concept of gonorrhoea as of a local process in which complications arise largely through direct extension, or as a result of secondary infections, has long since been abandoned. The chills, prostration, leucocytosis, anaemia, which are associated with the common comi)lications of gonorrluea, epididymitis, cystitis, pyelitis, synovitis, arthritis, the fever and more or less severe general symptoms which may attend the onset of an apparently uncomplicated specific urethritis, are often, in their severity, quite out of proportion to the apparent gravity of the focal process. They are indeed manifestations which might lead us to suspect the existence of a septica-mia. And since tlie communication of Hewes in 1894, from various sources there has been accumulated abundant evidence that gonococci are often present not only in the focal lesions but also in the circulating blood. Furthermore, it has been shown that 'tis not only in tliose cases with grave general manifestations that septicaemia may be demonstrated. Gonococci have been found in the circulation in instances of mild continued fever in apparently uncomplicated gonorrhoea (Thayer, 1905).

The occurrence of endocarditis as a complication of gonorrhoea and its direct dependence on the diplococcus of Neisser has now been demonstrated by many observers.


Gonorrhoea, then, is an infection, focal at first, which spreads not only by direct extension, but also and not infrequently, through the blood stream — a general septicaemia associated with a variety of metastases, notably arthritis, synovitis, myositis and not infrequently endomyo- or pericarditis.

How frequently and under what guise do we meet with the cardiac complications of gonorrhoea?

This is a very difficult question to answer, and consul tation with the literature leaves one often in doubt be cause of the insufficiency of the criteria on which diag noses are made. The one source of accurate information at the outset, is the study of those fatal cases which have been confirmed by necropsy.

There is a considerable literature dating from the sixties and before, concerning gonorrhtfal peri- and endocarditis. At the outset these complications were generally regarded as secondary infections. Gradually, since the discovery of the gonococcus, and especially, since 1889, evidence has been accumulated which has demonstrated that the.se complications are in tlie beginning genei-ally pure gonorrhceal infections.

The consecutive steps by which this conclusion was reached were:

1. The demonstration of gonococci, microscopically in the focal lesions at necropsy.


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[No. 380


2. The association of this observation with the negative results of c\iltures talcen intra vitam on ordinary media.

j. The obtaining of positive cultures, intra vitam, and the demonstration of the organisms, microscopically and by culture, in the blood or valvular vegetations at the necropsy. 4. Reproduction of an acute urethritis in man by inoculation with gonococci obtained at the necropsy from the affected valves. In 1899, the writer had already collected thirty two instances of well-described ulcerative and vegetative endocarditis associated with gonococcal infection, with postmortem records, and fifteen in which the purely gonococcal nature of the infection might fairly be accepted.

The occurrence in the medical service of the Johns Hopkins Hospital, in the summer of 1920, of a case of gonorrhceal endocarditis of the pulmonary valves has turned our attention again to a subject in which we have been interested for years. Since then we have observed two additional cases. We have, accordingly, reconsidered our experience with gonorrhoeal endocarditis at the Johns Hopkins Hospital during the last thirty-three years.

At the outset it may not be uninteresting to record briefly the three recent cases above referred to.

Case I. Gonorrhwa. Ulcerative and. vegetative endocarditis of pulmonary valves — Cultures froiii the circulating blood, negative — Gonococci demonstrated in the valvular thromlii at necropsy and cultivated from the kidney.

E. R., (Med., 440S2; Path., 6319), a coloured boy of 19, between ten and eleven weeks before his death was seized with a stitch in his right side associated with a palpable friction rub. This was followed by fever, chills, sweats, cough, progressive weakness, increasing anfemia (R.b.c, 4,224,000; hb., 80%; — R.b.c, 2,528,000; hb.. 28%), high leucocytosis (20,000-28,000). There was moderate cardiac enlargement with some dullness in the first and second left spaces, and a loud to and fro murmur in the pulmonary area, well transmitted throughout the chest; no essential change in the aortic sounds. Irregular, intermittent fever (99.4-103.8°) ; albuminuria; pus and blood in the urinary sediment.

Gram-negative diplococei, morphologically gonococci, were demonstrated in the prostatic fluid. Cultures from the blood were repeatedly negative.

The patient failed rather rapidly. The transverse diameter of the heart increased in extent and a systolic murmur became audible in the tricuspid area. Severe diarrhoea set in with bloodstained stools. Eight days later, ten weeks after onset, the patient died.

The course of events seemed to be clear. Acute ulcerative endocarditis of the pulmonic valve: pulmonary embolism with infarction and pleurisy causing the first subjective symptoms; intermittent fever, chills, progressive ansniia, gonorrhoeal prostatitis, cystitis, pyelitis, nephritis, terminal colitis, death.

The necropsy wholly supported our clinical diagnosis. The heart was not remarkably enlarged. The aortic, mitral and tricuspid valves showed no abnormalities. There was an extensive ulcerative endocarditis with large, irregular, nodular vegetations on the pulmonic valve which showed no evidence of a pre-existing stenosis. There were two infarctions in the right lung, one older, in the right lower lobe, and partially organized, associated with pleural adhesions which occupied a position in the


right axilla corresponding to the area in which the patient had complained of pain at the outset; the other, fresher, in the lower lobe posteriorly. There was acute urethritis, haamorrhagic cystitis, and pyelitis. The kidneys were large, swollen, cloudy and dotted with small hffmorrhages.

Smears from the thrombi on the valves showed numerous Gramnegative, biscuit-shaped diplococei. Cultures from the kidneys revealed gonococci; blood and vegetations, negative. There was no evidence of a pre-existing pulmonic stenosis or indeed of any pre-existing valvular disease.

Case II. Acute vegetative and ulcerative aortitis with aneurysm formation — Cultures from circulating blood negative — Gonococci demonstrated in the focal lesions at necropsy.

A. S. (Med., 45333; Path., 6545), a coloured man of 28, with an indefinite history of an attack, five years previously, of pain about his heart followed by occasional sensations of palpitation on exertion, was seized, 8 weeks before death, with an arthritis in the right ankle and shoulder, and transient pains in the other joints. The symptoms of arthritis soon disappeared but a cough set in, so distressing as to interfere with sleep. The sputa were scanty, but on the 21st of March, three and a half weeks before death, were blood-stained. There was progressive weakness, sweating at night, and five weeks after the onset of his illness, the severity of the symptoms brought the patient to the hospital.

On entrance there was orthopnoea and severe paroxysms of coughing. There was fever. The heart was enlarged and showed obvious evidence of aortic stenosis and mitral insufficiency with failure of the right side — enlarged, pulsating liver, oedema of the extremities. There was grave anaemia (R.b.c., 2,832,000; hb., 47%) and a high leucocytosis (13,000-22,240). There was moderate albuminuria, and on several occasions blood in the urinary sediment. Irregular remittent and intermittent fever continued, 99° to 102. G°; persistent cough. Petechiae appeared in the conjunctivae. Death followed a sudden paroxysm of coughing, eight weeks after the onset of his illness.

Cultures from the blood during life were repeatedly negative.

The symptoms were regarded as indicating an ulcerative endocarditis of the aortic valve. The history suggesting pulmonary embolism (bloody expectoration) pointed to the possibility of involvement of the valves of the right side or thrombi probably in the right auricular appendage. The slow course of the case, eight weeks' duration, was suggestive of an infection with attenuated streptococci or gonococci, and the negative results of cultures supported the possibility of its being gonorrhoeal.

At necropsy, not obtained, unfortunately, until five days after death, there was found old chronic, fibrous aortic and mitral stenosis without acute changes. Somewhat above the aortic valves, however, as shown in the photograph, there was an acute ulceration of the aorta leading to an aneurysmal sac from which there were accessory dilatations. Around the edges of the sac and at its base were vegetations. The aortic wall had been torn through, and from the sac there exuded a thick gray fluid showing Gramnegative diplococei and a few Gram-positive cocci. Elsewhere the wall of the aorta was normal. Owing to the condition of the specimens, no cultures were made.

The overwhelming prevalence in the smears of organisms presenting the morphological and tinctorial characteristics of gonococci, when taken in connection with the negative blood cultures intra vitam. led us to regard the process as gonorrhoeal. There were no other evidences of acute gonorrhoea.

Case III. Vegetative and ulcerative endocarditis of aortic valves — Cultures from circulating blood, negative — Gonococci obtained in pure culture from the valvular lesions at necropsy.

M.M., (Med., 45977; Path., 6681), a healthy married woman of 21 was seized ten weeks before death, with arthritis in knees.


THE JOHNS HOPKINS HOSPITAL BULLETIN, OCTOBER. 1922


Ot^



Cam: ]I. a. S. Cuiioiorcal endaortitis with the formation of an aneurysm.


I


f


October, 1922]


363


ankles and hip, associated with irregular remittent and intermittent fever. The arthritis which was apparently at no time very acute, disappeared in 10 days, but the fever continued. Signs of aortic insufficiency set in with very collapsing pulse but without marked cardiac enlargment. There was pronounced dyspnoea. The patient became anaemic, was dull and confused mentally, and was brought to the hospital three weeks after the onset of her illness. In the hospital there was irregular intermittent and remittent fever (98 '-103.5°) ; progressively increasing ana?mia (R.b.c, 4.236,000 to 2,464,000; hb., 5S% to 28%), and a marked leucocytosis (13,520 to 39,080). There were no petechise and no obvious embolic phenomena. The pulse increased steadily in rate. Striking and rapid changes occurred in the heart sounds and in the character of the pulse which was at first very collapsing, later much less so, while the intensity of the diastolic murmur diminished and the first heart sound became strangely accentuated at the base.

Persistent delirium; fever; tachycardia. Progressive failure. Evidence of chronic passive congestion (enlarged liver). Blood cultures, repeatedly negative. Death ten weeks after onset.

At first it was thought that the case might be an instance of acute rheumatic endocarditis, but the remarkably sudden onset of an aortic insufficiency of so high a degree and the relatively small heart — for, in the young, hypertrophy soon follows an aortic insufficiency of any extent, suggested an ulcerative endocarditis. This was borne out by the subsequent course of the case — the remittent, high fever, the progressive anaemia, the high leucocytosis and especially the sudden changes in the character of the heart sounds.

As we put together the facts, we could not help being impressed by the possibility that this might have been an instance of gonorrhoea! endocarditis. In several similar cases in which cultures intra vitam were persistently negative, this has proved to be the case.

Necropsy showed an extensive vegetative and ulcerative endocarditis of the aortic valves, with large, white, crumbling vegetations, perforations of the valves, and an abscess extending from the valvular lesions into the muscle of the left ventricle. There was no evidence of preexisting valvular disease, one curtain remaining delicate and unaffected. There were fresh splenic infarcts and small hsemorrhages into the pulp. The kidneys were large. There were old adhesions between the omentum and tubes and round ligaments, and thrombi in the pampiniform plexus. Gram-negative intracellular diplococci were demonstrated in smears, and pure cultures of gonococci were obtained from the focal lesions on the valves.

Since the opei)iiig of the Johns Hopkins Hospital, nearly thirty-three year.s ago, there have come to necropsy

V27 instances of acnte eudocartlitis. In 133 cases the nature of the process was determined bacteriologically,* either before death by blood cultures, or at necropsy. To these may be added 16 fatal cases in which necropsy was not obtained, although the nature of the process was determined by cultures from the blood intra vitam, as well as 27 cases with positive blood cultures in which the patients were removed from the hospital unimproved, making 176 altogether.

Twenty, or 11.3% of these instances, were due to gonococcus.

There were others in which a bacteriological study was made, but the records and reports were destroyed in a recent fire in the Pathological Laboratory.


In -k of these 20 cases, there were terminal infections; in 2 with streptococcus; in one with streptococcus and staphylococcus, and in one with pneuniococcus. In all, gonococcus was evidently the primary cause.

Of the.se twenty cases, gonococci were cultivated intra ritam (10) or at necropsy in 11 instances.

(xonococci were demonstrated bacterioscopically at necropsy following negative cultures intra vitam or post mortem in 6.

There were two further cases which, clinically and anatomically, seemed to be gonorrhoeal, with negative cultures intra vitam in which, owing to a misunderstanding, no bacteriological examination was made at death. These we have included in some of our tables.

In addition, there were several other cases in which the endocarditis may well have been gonorrhoeal, although definite proof was wanting. One was a characteristic instance of ulcerative endocarditis appearing in the course of an acute gonorrhoea with negative blood cultures during life. No necrop.sy was allowed. These cases have not been used in the tables, although in the last instance the course of the illness, the negative blood cultures, and the sequence on gonorrhcea, render the diagnosis of gonorrhoeal endocarditis highly probable. The summary of this case may be found in the appendix.

This is a rather large material for one clinic, dependent, probably, on the circumstance that we have been on the lookout for the condition.

Age: Of these patients the age varied from !» to 12.

Sex: There were 16 males; 6 females.

Colour: Eleven were white, eleven were negroes — a large proportion of negroes, larger than was observed in other forms of endocarditis excepting pneuniococcus infectiom Time of onset in relation to the gonorrhoea: But little evidence could be obtained as to the relation of the onset of the symptoms to the stage of the initial local infection. Among our twenty-two cases the cardiac complications appeared almost with the onset of the gonorrhoea in one instance. In four they appeared about two weeks after the onset; in two more within a month; in the others (fifteen) the relation to the time of onset of the gonorrhoea was uncertain. In several instances all traces of the urethritis had disappeared.

Relation of the onset of cardiac involvement to the other complications of gonorrhcca: In like manner little information was thrown on the relation of cardiac involvement to the occurrence of other complications of gonorrha'a.

Of our twenty-two cases but seven or 31.1% had had arthritis in the course of the illness. This is in sharp contrast to the figures in cases reported by other authors. Among 54 instances of proven gonorrhoeal endocarditis collected from the literature in which there were notes as to the presence or absence of involvement of the joints, arthritis was noted in 37, or 68.5%. It is not impossible that this variance depends on the circumstance that where


routine bacteriological examiuatious are not made, the disease, in the absence of obvious urethral discharge or arthritis, is often overlooked.

Syrniitmns: The symptoms were uniformly those of a grave septicaemia. The onset was sometimes gradual, beginning with headache, lassitude, pains in the loins, or more sudden and initiated by a sharp chill. There was continued or irregularly remittent or intermittent fever, often associated with violent chills and sweating. Chills were present in the majority (63. G%) of our cases.

Blood: There was, as a rule, a rapidly developing antemia. The red corpuscles ranged from 5,467,000 to 1,248,000 per c.mm.; the hb. from 92 to 11%. The longer the duration of the case, the more marked, as a rule, was the anaemia.

In all cases there was a well-marked leucocytosis. In but two instances wei'e figures below 13,000 recorded and in each instance there was a leucocytosis later.

The leucocytes were over 15,000 in 19 of 22 cases.

The leucocytes were over 20,000 in 14 of 22 cases.

The leucocytes were over 30.000 in 7 of 22 cases.

The leucocytes were over 40,000 in 4 of 22 cases.

In one instance with a terminal pneumococcus pneumonia and septiciPuiia the leucocytes rose to 78,000 and 100,000.

Embolic phenomena were common, occurring in twothirds of the cases — in skin, kidney, spleen, lungs, brain, heart muscle, skeletal muscles.

Symptoms of these embolic phenomena were not uncommon during life. Pulmonary infarction, for instance, gave rise to the initial symptoms in the first of the three cases described at the beginning of this communication. Renal it^,iarction may be suspected by the transient presence of blood in the urine. Blood corpuscles were found in the urine of 10 of our cases. But occasional blood coiijuscles are not uncommon in the nephritis so frequently present. Pain and tenderness in the left hypochondrium may reveal splenic infarction. In two of our cases there was a partial hemiplegia:

Cutaneous hamorrlmges, usually in the form of petechias on limbs or trunk or neck or mucous membranes of the month or conjunctivae, were frequent. These were recorded in 6 of our 11 white patients — in but one of the negroes. Had the conjunctiva and mucous membranes been examined with greater regularity 'tis probable that these figures would have been dift'erent.

An acute or subacute nephritis was present in the majority of our cases. This was manifested clinically by the presence of a considerable albuminuria with casts, leucocytes, epithelial cells and often blood in the urinary sediment. In 4 cases of long duration there was anasarca and hydrops. In general, the renal manifestations were more striking in the cases of longer duration.

The duration of our gonorrhceal endocarditis varied from 3-4 weeks to 8 months, averaging 9.7 weeks. If one subtract four cases in which there was a terminal mixed infection, the average duration was 10.5 weeks. In the majority of instances the course of the disease was from 4 to 9 weeks. The shortest case, the duration of which was not over 4 weeks, was that of a little girl with gonorrhceal peritonitis in which death was not the result of the endocarditis. In many of our cases, the picture was that of subacute vegetative endocarditis similar to that seen in infection with streptococcus viridans.

A comparison of these figures with those of endocarditis of other origin is not uninteresting.

TABLE I Duration of fatal cases of acute bacterial endocarditis.



Pneumo 4d.-6ni. Av. 4.3 w.


St. aureus 3 d..6in. Av. 4.5 w.


Si. albus 3-11 w. Av. 6.8


Gono- Strep, all

3 w.-8m. fewd.-9m Av. 10.5 w.Av. 12.2 w.


Strep.

3 w.-i y. Av. 23.1


Inauenza

11-26 w.

Av. over

6in.


Under 13 weeks


21/22


20/24


4/4


18/22 , 37/60


4/22


1/3


9 ..


20/22


20/24


2/4


16/22


34/60


3/22



6 ••


17/22


20/24


2/4


8/22


31/60


3/22



3 •


11/22


14/24


0/4


.1/22


20/60


0/22



" 1 week


4/22 5/24




10/60




Pneumococcus, in our experience, produces an acute infection, one half of our Cases jiursuing tlieir course in less than 3 weeks. In but one of 22 cases was the duration above nine and a half weeks.

Staphylococcus aureus is not infrequently a terminal invader and in over half of our cases the process was rapidly fatal with a duration of less than three weeks. The duration was under 6 weeks in 83% of our cases. But while 'staphylococcus aureus endocarditis appears generally to be more acute and virulent than gonococcal, yet in 4 of our 24 cases the coni'se was subacute, lasting from 13 weeks to 6 months.

There were four instances of pure staphylococcus albus endocarditis, the duration of which varied from 3 to 11 weeks, the average coui'se being somewhat more chronic than in aureus infections.

In our group of streptococcus infections, because of tlie circumstance that our records go back through so long a series of years, during most of which no difi'erentiation of type was made, all strains of the organism are included. It is not therefore surprising to find a great variation in the duration of the disease. Thus 20 of 60 cases were peracnte, of under 3 weeks' duration, 11 of these running their course in a week or less. On the other hand 23 of the cases or over one-third lasted 13 weeks or over. Of the.se, all were probably instances of infection with streptococcus viridans.

In 22 cases in which S. viridans was iilentified bacteriologically, the duration ranged from 3 weeks to a year, averaging 23.1 weeks. In only 3 instances in whicli streptococcus viridans was isolated was the duration under 13 weeks, and in one of these instances it was a terminal- infection iu pernicious auiemia. It would aj)pear that htemolytic streptococci are associated generally with rapidly fatal infections.

There were three fatal cases of endocarditis due to organisms of the B. influenzi* group. These were all chronic, lasting from 11 to 53 weeks.

Gonococcus seems to give rise to an endocarditis of a malignancy between that depending on staphylococcus and i)neuniococcHs on the one hand and the slower viridaus and influenzal infections on the other. The rapidity of the course and malignancy of bacterial endocarditis rises, in our experience, in the following sequence:

1. B. influenzie

2. Streptococcus viridan.s

3. Gonococcus

i. Staphylococcus albus 5. Staphylococcus aureus (5. Pneuniococcus 7. Streptococcus ha>niolyticus

Anatomical Lesions: The anatomical lesions were those of a vegetative and ulcerative endocarditis, sometimes spreailing to the walls of the ventricles or upwards into tlie sinuses of Valsalva, associated often with extensive destruction of valves and aneurysm formation, sometimes intiltrating the adjacent muscle as suppurative myocarditis. In general, the vegetations formed large, rather brittle, yellowish gray masses.

The only instance in which the vegetations were not large was that of a girl of D who died of a general gonococcal infection with peritonitis. Here the lesions were on the wall of the aorta above the valves and were of short duration — not over four weeks — probably somewliat less. In one other instance — that of A. S., described at the beginning of tliis communication — the lesions were tliose of a vegetative and ulcerative aortitis.

The abundance and size of the vegetations were commonly iu direct relation to the duration of the case.

Mural endocarditis: The vegetations extended onto the wall of the heart in 8 of 20 cases, involving the left ventricle, papillary muscles and chord;? tendinete in 5 cases, the right side in 1, the left auricle in one.

Tegctative and iilccratirc a(^rtiti,^: Two of the twenty cases coming to necropsy showed a vegetative and ulcerative aortitis alone. In two other cases, in addition to the valvular lesions, there were vegetations on the aortic wall.

Acute pericarditis occnrre<l in four of the cases.

In one ca.se there was a peri-aortic suppuration in the loose connective tissue just above the valves. There was pleurisy in two cases, in one of which gonococcus was cultivated from the fibrinopurulent fluid.

Our figures, as might be expected, show that the left side of the heart is affected more often than the right, but the percentage of right-sided involvement is remarkably high, considerably higher than in those cases of gon


TABLE II Anatomical distribution of the lesions.



Gonococcu.


St. aureus 24 rases


St. albus 4 cases


Pneumo. 25 cases





J. H.H.

20 rases


Literature 60 rases


Combined 80 rases


52 cases



A


6


30


36


6


2


9


9



M


2


15


17


7


1


4


17


•5,


AM


2


8


10


4



6


16


.


Aorta, ^


mural


2



2

12-60%


53-88.3%


65-81.2%


17-70.8%


3-7 5%


19-76%


42-80.7%



P


4


1


5


1



1


1


•S


T


1



1


5


1


1


2


I


PT



1


1








EC



5-25%


2-3.3%


7-8.7%


6-25%


1-25%


2-8%


3-5.7%



AP



1


1 AT


2


1


3


1



1




MT


1



APM



1


1








« 


APT


1



1






1


<s


AMT



1


1





3


3



A MPT



1


1






2




3-15%


5-8.3%


8-10%


1-4.1%



4-16%


7-13.4%


orrhanil endocarditis that we have collected from other sources. This high proportion of aft'ection of the right side was also found in staiihylococcus endocarditis, and is in rather sharj) contrast to that which is shown by the figures for jineumococcus and streptococcus. In general, however, it is in accord with the well-recognized relative frequency of acute ulcerative lesions of the right side of the heart.

TABLE III Distribution of the affection among the individual valves.


20 c


80 c


24 <


25 t


52 (


13 c


A 13-65%* 56-70% 12-48% 2-50%|19-76% 32-61.5% 8-61.5% M 4-20% 30-37.5% 11-44% 1-25% 13-52% 39-75% 11-84.6% P 5-25% ;10-12.5%, 1-4% 1 I 1-4% , 4-7.6% ; 1-7.6%

T 4-20% 8-10% ! 6-24% 1-25% 6-24% 9-17. 3%i


' Including two instan


Of the individual valves the aortic was attected with greatest frequency; the pulnioiiary next, iu a quarter of our cases. This is somewhat striking and again in contrast to the usually accejited figures which show a somewhat larger percentage of aortic invidveinent — 1Q% in the combined 80 cases of reasonably proven gonococcal endocarditis that we have collected.

One striking point, however, that stands out in these figures is the greater frequency of the involvement of the mitral valve in streptococcal infections than in gonococcal, staphylococcal and pneumococcal endocarditis.

The probable explanation of this divergence is found when we consider:



(1) The relative frequency of pi-e-existing valvular disease so far as could be determined by the histories and the anatomical changes.

(2) The relative frequency with wiiich previously unaffected valves were involved.

TABLE IV Evidence of pre-existing valvular disease.


4/20 or 20%jl0/24 or 41.6% 1/4 or 25% 9/25 or 36% 35/52or67.3%

There was evidence of pre-existing valvular disease in only 20% of our gonorrhoeal cases; in 41.6 of the staphylococcus aureus cases; in 25% of the four albus cases; in 36% of the pneumococcus infections as against 67.3% for our streptococcus cases. This is a striking contrast. 'Tis clear that acute streptococcus endocarditis was commoner in patients with chronic valvular disease, whereas the contrary was true in infections with the other organisms.

If, now, we consider, among the valves themselves, the relative frequency in which the att'ection was found on previously undiseased valves, it appears that the valves involved were, so far as could be determined, previously unatfected in the following percentages of the several commoner types of infection.

TABLE V

Per cent in which the acute process involved previously

unaffected valves.


Conococcu,.


Staph, aureus


1 Staph. albu»


Pneumococcus


Streptococcus


91.2%


65.5%


1 75%


65.7%


39.2%


• Not Includine the mural aortitis.

In other words, in more than half of our cases, 61%, Strei)tococcus attacked previously diseased valves — in sharp contrast to the behaviour of the other chief causal agents of acute endocarditis, notably of Gonococcus. But the prevalence of mitral involvement in chronic endocarditis is iccll knoii-n. If, as seems probable, Streptococcus attacks, generally, previously diseased valves, its predilection for the mitral valve may well depend largely on the circumstance that chronic mitral disease is very common.

Further light on this question might be obtained by a consideration of the relative percentage in which the individual valves are involved among those ])rimarily affected.

TABLE VI

Relative percentage of involvement among those valves

primarily affected.


Gonococcus


Staph, aureus 1 Staph, albus


rnenmocoecDs


Streptococcus


A 10* 45.4%


4-21% 1 1-33.3%


13-52%


12-37.5%


M 4 18.1%


8-42.1% 1 1-33.3%


9-36%


14-43.7%


P 5 22.7%'


1-5.2%


0-0


1-3.1%


T 3 13.6%


6-31.6% 1-33.3%


3-12%


5-15.6%


• Not including the


two mural aortitides.




The figures would appear to show a tendency toward aortic involvement in gonococcal and pneumococcal infection, towai'd involvement of the mitral in infections with streptococcus and staph, aureus. But the number of ca.ses on which the table is based is so small as to render it practically worthless.

In sumnuiry, then, the cardiac complications of gonorrhoea, as we have seen them, appear usually in the form of an acute or subacute vegetative and ulcerative endocarditis which conies on at varying periods in the course of the infection, often in the absence of other apparent complications. The onset may be sudden or more gradual, but is generally rather acute. There are usually chills and high remittent or intermittent fever, rapidly developing anipmia and considerable leucocytosis.

The constitutional symptoms, those common to all grave general infections, are usually early and profound. There is often a petechial eruption. Embolic phenomena of various sorts are common. There is albumin, casts, often blood in the urine, and, in the longer cases, anasarca and hydrops give evidence of a grave nephritis.

The duration is generally from 5 to 9 weeks, but longer subacute cases may occur.

The aortic valve is that most conmionly involved but, as in all acute ulcerative endocarditides, the right side apjiears to be affected more often than in the chronic socalled rheumatic valvular disease. In our experience involvement of the pulmonary orifice has been rather common. Gonococcus usually involves healthy, previously unaffected valves. Mural endocarditis is common. Suppurative myocarditis, as described in 1893 by Councilman, may occur. Pericarditis is not infrequent.

Gonorrhoeal endocarditis is generally a malignant process pursuing a progressive and fatal cour.><e. In its virulence it occupies a position between the slower and more subacute viridans and influenza infections and the more virulent albus, aureus, pneumococcus or s. ha?molyticns endocarditis.


But is gonorrhceal endocarditis always fatal? May recovery occur ?

If no, what is its frequency and what is the prognosis?

GonorrlKeal septicaMuia with every evidence of endocarditis is not necessarily fatal. Characteristic instances of acute endo- or pericarditis have been recorded in which gonococci were cultivated from the blood during life with subsequent recovery. Good examples are the cases reported by Silvestiini, AYitliington, Dieulafoy. and ilarfan and Debr^.

Silvestrini records the case of a man, 20 years of age, who acquired gonorrhoea a month before entry into the hospital. He was treated with injections of wine — an unworthy abuse of a noble fluid. Eleven days after apparent recovery, anorexia, nuilaise, chills and sweats set in. On the third day there were pains in the joints of the spine with swelling and redness of the accessible joints. The patient's appearance was typhoidal. In the snbclavicnlar fosssB there appeared erythematous patches with central lupniorrhages. The cardiac impulse was in the fifth space in the niammillary line. There was an intense systolic murmur in the mitral area, and a doubled second sound. A pericardial friction rub was audible. The spleen was enlarged. There was a well-marked leucocytosis. Gonococci were cultivated from the blood, joints and urethral discharge. The pericardial friction disappeared and the patient gradually recovered.

The heart diminished in size but a harsh systolic murmur at the apex and a reduplicated second sound persisted. The patient left the hospital with ankylosis of several joints, and returned nine months after the onset of his trouble, still showing evidence of chronic mitral disease with systolic and presystolic murmurs.

Withington had the opportunity of observing a man of 2G who acquired gonorrhea and, two weeks later, mea.sles. Soon after convalescence from measles, prtecordial pain set in, associated with chills, irregular fever and orthopuoea. There was cardiac enlargement, galloj) rhythm and evidence of solidification at the bases of the lungs. There was a left femoral thrombo-phlebitis. A pericardial friction rub appeared and a short systolic murmur was heard in the fourth intercostal space to the left of the sternum. Ciiltures from the blood showed gonococcus.

Gradually the patient recovered, the temperature reaching normal about three weeks after the onset of the syni])toms pointing to cardiac involvement. The heart returned almost to its normal size but a systolic murmur persisted in the second left space.

Dieulafoy reports the case of a man of 23 who was admitted to the hospital with headache, diarrhoea, high remittent fever and sweating. Two days later a mitral systolic murmur appeared, soon becoming loud and rough. There was an urethral discharge in which gonococci were present. Cultures fi-om the blood, intra vitum, showed gonococci. Tlie patient was very ill. There was bronchopneumonia. After treatment by rest and gonococcal vaccine, the patient gradually recovered and left the hospital well. The evidence of a mitral lesion remained.

Marfan and Debr6 report the case of a little girl, aged 101/2- who, about a week before entry into the hospital on 24/VI, began to suffer from pain in the abdomen, fever and exhaustion. There was obvious pelvic peritonitis. The heart was somewhat eidarged with a slight systolic thrill and an intense, rough, systolic murmur transmitted outwards into the axilla. Five days after entry there was a pericardial friction murmur. High, irregular fever. Cultures from the circulating blood showed characteristic gonococci.

Treatment with gonococcic vaccine and anti-meningococcic serum. Gradual recovery after about 3 weeks, with


persistent signs of mitral insufficiency and adherent pericardium. Ten months after the beginning of her illness the child still showed signs of adherent pericardium and mitral insulliciency.

Goiirvich, in 1807, in an excellent study of the literature which has passed almost unnoticed, brought together a considerable number of instances in which transitory cardiac manifestations, interpreted as endocarditis, had been observed in the course of gonorrlicea. Many of these are suggestive but most consist of the description of systolic murmurs at the apex of the heart appearing during the height of the process and disappearing with recovery. Similar cases have been reported by other observers since the article of Gourvich.

The recognition of mild, early endocarditis is often very difficult, indeed impossible. Systolic murmurs at the apex and pulmonary areas are commonly heard in the course of any acute infection, and, in themselves, offer little basis for the diagnosis of endocarditis. In nmny instances of rheumatic fever 'tis only through long continued observation of the patient that a positive diagnosis of endocarditis is reached, and then only on the strength of the later mechanical effects of scarring and retraction of the valves in the production of pernument stenoses and insufficiencies.

The very frequency of gonorrhoea contributes to the difficulty of ascertaining its relation to endocarditis. Few genitourinary surgeons, probably, examine the hearts of their patients as a routine procedure, and without knowledge of the condition of the heart at the time of onset of the gonorrhoea, it may be difficult or impossible to reach a conclusion as to the relation of the malady to a chronic valvular disease which is recognized later.

Our impression, and it must be acknowledged that it is but an impression, is, that the analogy between the cardiac complications of gonorrhu'a and those occurring in pneumococcus and staphylococcus infections is close ; that while mild infections with a favorable cour.so may occur, gonorrhceal endocarditis is generally a serious affair, in no way comparable to the acute, verrucous, so-called rheunuitic endocarditis of uncertain origin, which affects so commonly the mitral valves, and tends toward recovery with scarring, deformity, and the production of chronic lesions which are notoriously a seat of predilection for later streptococcal invasion with the development of acute and subacute vegetative and ulcerative lesions.

How often endocarditis occurs in gonorrhoea we have no clear idea. This, however, we do know. Gonorrho'al endocarditis is not a very infrequent nmlady, forming upwards of 11% of our fatal acute en<locarditides, the bacterial nature of which has been determined at necropsy.

Summary and Coxclitsioxs The cardiac complications of gonorrhoea occurring in thirty-three years at the Johns Hopkins Hospital have been reviewed. Twelve hitherto unreported cases have been added to our series * aud considered iu connection with sixty cases collected from the literature.

In 176 instances of acute endocarditis of determined origin 20, 11.3%, were gonococcal.

Oonococci were cultivated intra ritum (10) or post mortem in 14 instances; they were demonstrated bacterioscopically following negative cultures intra vitam or poHt mortem in 6 instances.

There were two additional cases, in all probability gonorrhopal, with negative cultures during life in which no bacteriological examination was made at necropsy, and several other instances of more or less characteristic endocarditis occurring in the course of acute gonorrhoea with negative blood cultures but without necropsy ; these latter cases were not considered in our tables.

But little evidence could be obtained as to the relation of the time of onset of the cardiac involvement to the appearance of the disease or other complications. Arthritis occurred in but 11.1% of our cases but in 68.5% of 54 cases collected from other sources.

The cardiac complications of gonorrhoea, as we have seen them, appear usually in the form of an acute or subacute vegetative and ulcerative endocarditis which comes on at varying periods in the course of the infection, often in the absence of other apparent complications. The onset may be sudden or more gradual, but is generally rather acute. There are usually chills and high remittent or intermittent fever, rapidly developiug an;emia and considerable leucocytosis. The constitutional symptoms, those common to all grave general infectious, are usually early aud profound. There is often a petechial eruption. Embolic phenomena are common. There is albumin, casts, often blood in the urine, aud in longer ca.ses, anasarca and hydrops give evidence of a grave nephritis.

The duration is usually from 4-0 weeks but longer subacute cases may occur.

The aortic valves are those most commonly involved but, as in all acute endocarditides, the right side appears to be atfected more often than in the chronic, so-called rheumatic valvular disease. In our experience, involvement of the pulmonary orifice 1ms been rather common. .Cronococcus usually implicates liealthy, previously undiseased valves. Mural endocarditis is frequent.

The cardiac involvement is not restricted to the endocardium. Endaortitis, pericarditis and suppurative myocarditis are not unusual.

Gonorrhieal eiulocarditis is in general, a malignant process pursuing a progressive and fatal course. In its virulence it occupies a position between the slower and more subacute S. viridann aud B. influenza' infections and the more virulent Staph, albtm, Staph, aureus, Pneumocoeeus or S. hcpinolyticus endocarditides.


See appendix.


Milder instances of gonorrhoeal endocarditis or pericarditis with recovery may occur. How frequent these are is not clear. It is our impression that they are relatively rare.

Gonorrhoeal cardiac infections as a whole are by no means very unusual.

TABLE OF RBFERENCES

The table of references is arranged in alpliabetical order. Beside tlie references to tiie authors mentioned in the text there are included references to the articles describing the eighty cases of gonorrhoeal endocarditis from which some of the tables have been made.

Babes and Sion: Arch. d. sc. med. de Bucarest, 1896, 1, 505.

Berg: Med. Record, N. Y., 1899, IV, 602.

Bielogolovoi: Vrach, St. Petersb., 1S99, XX, 50.

Bonnamour, S. and Gauthier, P.: Lyon med., 1910, CXII, 11521158.

Carageorgiades, H. J.: De I'endocardite gonococcique. 8°, These de Par., 1896, no. 79.

Councilman, W. T.: Am. J. M. Sc, Phila.. 1893. n.s., CVI, 277-285.

Danila, P.: Arch, des nial. du coeur, des vaisseaux et du sang. Par., 1917, X, 436.

Dauber and Borst: Deutsch. Arch. f. klin. Med., LVl, 1896. 231.

Dieulafoy, G.: Intel nat. Clin., Phila., 1909. 19 s. Ill, 59-70.

Dwyer: J. Am. Med. Ass., Chicago, 1920, LXXV, 1643-1644.

Finger, Ghon and Schlagenhaufer: Arch, fiir Dermat. u. Syph.. 1895, XXXIII, 141; 323.

von Frendl, B.: Wien. klin. Wchnschr., 1903. XVI. 789-791.

Fressel: Inaug. Diss., Leipzig, 1894.

Fritz: Med. Corr.-bl. d. Wiirttemb. artzl. Ver.. Stuttg., 1904, LXXIV, 339-342.

Gallavardin, L., Croizier, L.. et Rey, C: Bull. So.^. med. d. hop. de Lyon, 1912, 311-315.

Ghon and Schlagenhaufer: Cf. Lucas. L'endocardite blenorrhagique. 8°. These de Par., 1908, p. 132. L. gives full summary of this article but no references.

Gourvich: Rousskyi Arch. Patol.. Bakt.. i klin.. Med., St. Petersb., 1897, III, 329.

HaI16, Serrand, Sagot: Cf. Sagot. L'endocardite gonococcique. 8°. These de Par., 1920, no. 325.

Harris, N. MacL. and Dabney, W. M.: J. Hopkins Hosp. Bull.,

1901, XII, 68.

Harris, N. MacL. and Johnston, W. B.: ,1. Hopkins Hosp. Bull.,

1902, XIII, 235.

Hewes, H. F.: Boston M. and S. J., 1894. CXXXI. 515.

Higgins, M. E.: U. States Nav. M. Bull., Wash., 1915, IX, 436.

His: Berl. klin. Wchnschr., 1892, XXIX, 993.

Horder, T. J.: Tr. Path. Soc, Lond., 1906, LVII, 53-57.

Huebschmann: Ztschr. f. Hyg. u. Infektionskrankh.. Leipz., 1912, LXXIII, 1-12.

Hunter. William: Brit. M. J., 1905, I, 525.

Klemperer: Deutsche med. Wchnschr., 1909, XXXV, 459.

Krause. P.: Berl. klin. Wchnschr., 1904, XLI, 492-494.

Kulbs: Wien. klin. Wchnschr., 1907, XX, 11-13.

Lartigau: Am. J. M. Sc, Phila., 1901, CXXI, 52-59.

Lenhartz; Berl. klin, Wchnschr., 1897, XXXIV. 1138.

Leyden; Deutsche med. Wchnschr., 1S93, XIX, 909.

Loeb: Arch. f. klin. Med., LXV, 1900, 411-420.

Lucas: L'endocardite gonococcique, 8°. These de Par., 1908, 132.

MacCaskey: Med. Record, N. Y., 1900, LVIII, 1005.

Marfan et Debre: Cf. Sagot who gives an incomplete reference but a full quotation.

Miehaelis: Ztschr. f klin. Med.. XXIX, 1896, 556.

MineUi, S.: Gazz. med. ital., Torino, 1908, LIX, 251-254.

Miiller: Deutsche toilitaraitzl. Ztg., 1904, XXIII, 141.

Oettinger and Marie, P. M.: Bull, et mem. Soc. med. d. hop. de Par., 1914. 3 s., XXXVII, 295-305.

PolozofE, A. A.: Morsk. Vraich, Petrogr., 1914, 631.

Prochaska: Arch, fiir path. Anat. etc., 1901, 164; 492.

Prochaska: Deutsch. Arch, fiir klin. Med., 1905, LXXXIII. 183-188.

Rendu and Halle: Bull, et mem. Soc. med. d. hop. de Par., 1897, 3 s., XIV, 1325.

Reye: Jahrb. d. Hamb. Staatskrankenanst., 1905, H»mb. u. Leipz., 1906, Pt. 2, 223-244, 1 pi.

Rothmund: Endocarditis ulcerosa. Inaug. Diss., Ziirich, 1889.

Rothrock, J. L.: Northwest. Lancet, Minneap., 1903, XXIII, 71. Also; St. Paul M. J., St. Paul, Minn., 1911, XIII, 494-499. (Same report except for photograph.)

Sagot: L'endocard4te gonococcique. 8". These de Par., 1920, no. 365.

Scherer: Bull. Soc. m6d. militaire frangaise. Par., 1909, III, 180.

Siegheim: Deutsche med. Wchnschr., 1897, XXIII, VereinsBeilage, 94.

Silverstrini, R.: Riv. crit. di clin. med., Firenze, 1903. IV, 385; 401; 417.

Simmonds: Munchen. med. Wchnschr., 1909, LVI, 1207.

Smith: Am. J. M. Sc, 1921, CLXI, 824-S30.

Stengel: Univ. Med. Mag., Phila., 1897, IX, 426.

Stocker, Q.: Ueber endokarditis gonorrhoica. Inaug. Diss., Bonn, 1900.

Thayer, W. S. and Blumer, G.: Arch, de med. exper. etc., Par., 1895, VII, 701. Also: J. Hopkins Hosp. Bull., 1896, VII, 57.

Thayer, W. S. and Lazear, J. W.: J. Exper. Med., 1899, IV, 81-116.

Thayer, W. S.: Am. J. M. Sc, Phila., 1905, CXXX, 751.

Tyree, J. E.: Tr. Chicago. Path. Soc, 1903-6, VI, 364-368.

Ugolini, R.: Bull, della R. Accad. med. di. Roma, 1904-5, XXXI, 296-303.

Wassermann: Deutsche med. Wchnschr., 1901, XLVII, 298-300.

White, W. Hale: Lancet, Lond., 1896, I, 533.

Widal and Faure-Beaulieu: Bull, et m^m. Soc. med. d. hop. de Par., 1905, XXII, 613.

Wilson: Cf. Harris and Johnston who give incomplete references.

Winterberg: Festschr. z. 25 Jaehr. Jub. d. Vereins Deutscher Aerzte zu San Francisco, 1894, 8°, 40.

Withington, C. F.: Boston M. and S. J., 1904, CLI, 99.

Zawadski and Bregman: Wien. med. Wchnschr., 1896, XLVI, 313; 351.



NOTES ON NEW BOOKS

The Heart, Old and New Views. By H. L. Flint. Cloth. •$4.00. (New York, Paul B. Hoeber, 1921.)

Among the many shorter texts dealing with the cardiac irregularities we know of none which presents the subject in the interesting way so successfully attained by the author of this volume.

Written with the two-fold purpose of appealing to those who may have but little time to sift critically the exhaustive literature, as well as for those who desire to turn to the original sources, its value is greatly enhanced by the historical treatment of the subject and by the marginal references to almost all, if not all, the really important literature. The first part of the volume dealing with the heart in antiquity comprises a delight


ful review of the knowledge of the time of Hippocrates, Galen and Vesalius, and concludes with a study of the period beginning with the discovery of the circulation by Harvey down to recent times. It includes such names as Malpighi, Loeuwenhook, Von Helmont, Descartes and Stenson, and among a host of other equally conspicuous investigators reference is made to the work of James Keill (1673-1719), who was among the first to apply mathematics to the problems of circulatory hydraulics. Part II takes up the development of instruments and instrumental methods, and here again the author is equally successful in his critical analysis and in his appreciation of the fundamental importance of physiology. The various methods for determining the blood pressure, blood velocity and output of the heart are referred to brietly. The use of the clinical polygraph, the physiologj- of the venous curves and the interpretation of the polygraphic records are dealt with in detail, while the subject of electrophysiologj- and the interpretation of the normal galvanometric curves is soundly discussed and lucidly described. The chapter devoted to a consideration of the five functions of the heart muscle is a concise and clear presentation of the modern conception of cardiac physiology as originally enunciated by Gaskell, and presents a very clear analysis of the much discussed doctrines dealing with the neurogenic and myogenic theories of cardiac function. If we were to venture any criticism of the author's treatment of this phase of the subject, it would be that we fear lest, by the very necessity of the method followed in his analysis of the various functions of cardiac muscle, lie has conveyed the impression of an apparent interdependence of physiological function which actually does not exist to the degree suggested by the text.

The cardiac irregularites are discussed in a very satisfactory way and are well illustrated by both polygraphic and electrocardiographic curves, a large number being taken, as the author states, from the writings of Lewis and Mackenzie, to which are added many of his own records.

Short chapters on the systolic murmur and the principles of treatment conclude the volume, the value of which is not a little enhanced by a most satisfactory index.

This is a work which it is a genuine pleasure to commend, quite as much for the way in which the subject matter is presented as for the soundness of the views expressed. It is interesting, as illustrating the rapid advances of our knowledge, that even in this book,- though bearing the date of 1921, there is no allusion to the modern theory of the "circus movement" in the description of auricular flutter and fibrillation.

We are confident that a second edition will soon be called for in which the more recent contributions of Lewis and his coworkers will be incorporated; and may we venture to add the hope that reference to the work of Einthoven and Hugenholz and to that of Arbeiter. in connection with the subject of electrical and mechanical phenomena, may find a place in any subsequent revision.

E. P. C.

Tiiherculosis in Infancy and Childhood. By J. Ci..\xton Gittings, M. D., Fr.\xk Crozer Kxowi.es. M. D. and Astlet P. D. Ashhurst, M. D. Cloth, $5.00. (Philadelphia and London, J. B. Lippincott Company. 19.i;i.) The authors approach the subject of tuberculosis from the point of view of the pediatrician. In most text-books on pediatrics, a proportionately large space is devoted to tuberculosis, but in such books the really detailed information that is often required is apt to be missing. This volume fills a definite need in furnishing such Information in a concise but comprehensive manner, and in presenting impartially a summary of our present knowledge of tuberculosis, as it occurs in infancy and childhood. In several respects it illustrates the changing attitude of pediatricians in regard to the prognosis in tuberculosis, in pointing out that the mortality in infancy, although very high, is distinctly lower than was formerly believed. This change in attitude is due largely to the increased use of well defined methods of diagnosis, chiefly the intradermal tuberculin test. The chapter on the use of Tuberculin in diagnosis is particularly well presented. The authors stress the point to which all pediatricians bear witness; namely, that until in the most advanced stages tuberculosis in infants often does not interfere noticeably with the general nutrition.

The book is well arranged. The few illustrations are very good, but add little to its value.

B. T.


The Blood Supply to the Heart in its Anatomical and Clinical Aspects. By Louis Gro.ss, M. D., C. M. Cloth, $5.00. (.Paul B. Hoeber. New York, 1921.)

This monograph presenting the results of the author's researches upon the structural evolution of the circulation of the heart, based upon an exhaustive study of one hundred hearts of all age periods, is a very valuable contribution, and illustrates in a striking way the important clinical application of a study primarily undertaken as a purely anatomical investigation. After describing briefly the technical methods used by previous investigators, the author discusses the diflSculties involved in any attempt to inject the coronary circulation, and describes in detail the technique followed in his method of procedure. He then discusses the vascular supply to the auricles and ventricles and summarizes the tirculation to the heart as a whole, emphasizing the fact that, although there can be no hard and fast line of demarcation between the distribution of the right and left coronary arteries, which, as he has shown, have profuse and abundant anastomoses, it is quite possible to adopt an arbitrary division, with certain overlapping territory, which permits of an accurate description of the distribution of the coronary circulation in the average heart. He classifies by means of tables and text all the most important deviations from the usual in his series of hearts.

We have been particularly interested in the account of the blood supply to the neuro-muscular tissue, and have been greatly impressed by the evidence adduced to show that there exists a specific vascular supply for the sino-auricular node from the Ramus ostii cava' superioris of the right coronary — occasionally arising from the left — and that there is no evidence for the opinion that anastomoses exist between the vessels supplying the sinoauricular and the auriculo-ventricular nodes; a view entirely confirming and amplifying the previous observations of Keith and Flack and of Koch. The author is of the opinion that a distinct and specific blood supply exists for both the sino-auricular and auriculo-ventricular nodes, the main stem of the His bundle, the first portion of the left branch and a large part of the right branch of the specialized conduction tissue — an anatomical difference of the greatest practical significance in our interpretation of lesions arising from any functional disturbance due to changes In the vascular supply. This is further emphasized by a brief discussion of a few instances from the series of cases reported by Moenckeberg. No more appealing brief for the necessity of an accurate correlation of our clinical and galvanometric observations with careful anatomical and injection studies could be offered than this chapter upon the blood supply to the neuromuscular system. The all too ready assumption of post hoc propter hoc plays often far too large a part in many utterly uncontrolled conclusions.

The question of the blood supply to the heart valves has been without doubt one of the most controversial fields of cardiac vascular morphology. IMviding the prevailing conflicting evidence into three main groups, the author concludes, as a result of experimental work and his interpretation of the literature, that each of these conflicting views may be in a measure correct, if not accepted in entirety, and that they can all he coordinated into a logical and reasonable explanation for the genesis of many eases of acute valvular endocarditis. In this connection the following paragraph may be quoted in full:

"If the literature on the mechanism of acute valvular endocarditis is now reviewed in this light, one is at once struck with the fact that the occurrence of endocarditis bears a strikingly close relationship to that of the existence of musculature and of blood vessels in valves. This becomes even more striking when the incidence of endocarditis is considered from the point of view of the frequency with which it occurs on the left and right sides as well as on the individual valves and cusps."


The author's premises in this connection based upon his experimental data, as given on page 71, are very interesting and suggestive, and impress one as being sound. We wish that we might quote at length from his discussion of the subject of anastomoses between the coronary arteries, but this chapter must be read in its entirety to be justly appreciated. His general conclusion that the heart is, perhaps, the richest organ in the body as regards capillary and precapillary anastomoses between branches of the same coronary artery, as well as between branches from both coronaries, seems to be abundantly supported on the basis of his available evidence and exhaustive study, and can but reinforce our conception of the marvellous provision for the maintenance of the normal anabolic and katabolic functions.

We know of no text which can in any sense be said to approach the careful detailed and concise outline covered by the author in his description of the venous circulation of the heart. The concluding chapter, dealing with the age period changes in the blood supply and their pathogenic relations, furnishes a most stimulating and suggestive discussion based upon a broad biological consideration of all the factors at work in the postnatal evolution of the individual. The volume is illustrated throughout with really remarkable plates and its value as a work of reference is tremendously increased by the exhaustive bibliography and by an index of authors, as well as an unusually satisfactory index of subjects. E. P. C.

Obstetrical A ursing. A Text-book on the Nursing Care of the Elxpectant Mother, the Woman in Labor, the Young Mother and Her Baby. By C.^roly?* Conant Va^ Bl-\eco.\i, R. N. (The Macmillan Company, Netc York, N. Y.) ($3.00)

Examination of many of the text-books of obstetrics for nurses shows that most of them present the subject inadequately and mjake little attempt to elevate the art of midwifery to the science of obstetrics. Some are so antiquated and scientifically inaccurate that they are of little value. In many instances the attempts at brevity so limit description of the methods employed in the various clinics as to restrict their use to the hospitals from which they originated.

It is a pleasure to welcome a book which has overcome most of these objections. Miss Van Blarcom takes the stand that the obstetrical nurse is more useful if she understands more than the nursing care of her patients. Therefore, the anatomy, physiolog>- and pathology of pregnancy, labor, the puerperium and the newborn child are adequately and simply explained. The author has familiarized herself with the obstetiical procedures of the leading hospitals of this country and Canada, and this broad view is reflected in her book. It gives the nurse a much clearer understanding of the subject and prepares her for the varj'ing practices with which she may meet in her work. The nursing care of the mother and child is presented in a manner quite different from usual. While the various procedures employed in the better hospitals are described in detail, it is recognized that many women are delivered at home and that the nurse must often work with inadequate equipment. To meet this need, therefore, the author has simplified, as far as possible, the routine hospital procedures.

The author is to be commended especially for including in her book many of the modern ideas concerning prenatal care, the mental hygiene of the expectant mother and the nutrition of the mother and child. These chapters are among the best in the book and add much to its value. Incidentally, much of this subject matter might well be incorporated in the texts for medical students and practitioners.

Throughout the Iwok sustained effort is made to impress the nurse with the fact that she is dealing with an individual rather than with a "case," that her patient is going through the most eventful period of her life, many of whose phenomena she does not understand, and that thoughtful sympathy on the part of the nurse is as much her duty as the actual nursing care of the patient.

The book is written in an interesting and simple style, and the text is clarified by numerous excellent photographs, drawings and charts. It adequately fills a real need in the teaching and training of nurses. J. W. H.

Management of the Sick Infant. By Langlet Poeter, M. D. and William E. Caeter, M. D. Cloth, $7.50. (St. Louis, C. V. Mosby and Company, 19:li.)

The authors have prepared a book that deals exclusively with the management of diseases peculiar to children and infants. The commoner symptoms of disease, such as convulsions, vomiting and so forth, their causes and methods of treatment are taken up in order. Following this, the disease entities of the different systems are similarly treated. At the end of the book there is a long list of prescriptions, which the authors have found useful. The chapter on the preparation of various food substances is particularly well arranged and should prove very helpful. The book is of use particularly to the practicing pediatrician and should be of value as a book of reference for any physician in his management of sick children.

There is nothing revolutionary or startlingly new in the irolume. The authors have presented, in an exceptionally complete way, detailed directions for the management of all emergencies. B. T.

An Introduction to the Study of the Protozoa, with Special Reference to the Parasitic Form.s. By B. A. Minciii?j. Second Impression. Cloth, $8.50. (London, Edward Arnold. lOii.)

Professor Minchin, whose researches have added many important contributions to the knowledge of zoology, was elected Professor of Protozoology in the University of London in 1906. While there, he wrote this book, which was recognized at once to be a masterful. lucid, and readable presentation of all the main facts about the structure and classification of the protozoa.

Since its publication in 1913, the book has been of great use to all students of systematic protozoology and also to those more directly concerned with the medical aspect of the subject. It attempts to determine the position of the Protozoa in nature. To guide the student through the technicalities of the study, all technical terms are defined and explained. While the mutual affinities and interrelationships of these organisms are expressed in as rigid a classification as possible, the author discusses philosophically those parts of the subject where Protozoa "throw great light on some of the fundamental mysteries of living matter — as for example, sex — and a special chapter dealing with the physiology of the Protozoa has been added."

The author states in his preface that as he is "not a medical man. . . . purely medical problems. ... as the symptoms and treatment of diseases caused by the Protozoa — are not dealt with in this book. The needs of the medical men have, however, been specially kept in view, and the author hopes that the book will succeed in supplying them with useful information, at least from a general zoological or biological standpoint." From this point of view, the reviewer finds that the section of the book dealing with the trypanosomes and allied parasites is the most satisfactory.

In 1915, Professor Minchin died. The volume just issued from the press is an unrevised reprint of the first edition published in 1913. It lacks, therefore, the completeness which it would have had if its author had lived to add to it the knowledge of the protozoa which has been established during the last nine years. In spite of that deficiency, it remains the best book in English on this subject. S. B. J.


CONTENTS

  • The Influence of the Treatment of Syphilitic Pregnant ' Women upon the Incidence of Congenital Syphilis. By J. Whitridge Williams 383
  • The Use of Large Reverdin Grafts in the Healing of Chronic Osteomyelitis. (Illustrated.) By Mont R. Reid 386
  • The Morphological Theory of Monochorionic Twins as Illustrated by a Series of Supposed Early Twin Embryos of the Pig. (Illustrated.) By George W. Cormer
  • Development and Growth of the Metanephros or Permanent Kidney in Chick Embryos. (Eight to Ten Days' Incubation.) .(Illustrated.) By William' Francis Riexhoif, Jr., M. D. . . 392
  • Biometrical Studies in Pathology. II. Pathometrlc Index Numbers: Preliminary Account of a Quantitative Method of Evaluating Pathological Phenomena. By Raymond Pearl 406
  • Vaso-Motor Reactions. By W. M. Batliss, M. A., D. Sc, F. R. S. . . 412
  • Notes on New Books 416

The Influence Of The Treatment Of Syphilitic Pregnant Women Upon The Incidence Of Congenital Syphilis

By J. Whitridge Williams


In a paper upon The Value of the Wasscrmann Reaction in Ohstetricf!, which appeared in October 1!)20, and which was based upon the study of 4547 women whose Wassermann reaction had been determined during the four years ending December 31st, 1919, I considered the significance of the Wassermann reaction during pregnancy. At that time it was shown that in our material syphili.s constituted the most important single factor concerned in the causation of foetal death, and was responsible for 31.4 per cent of all deaths occurring between the period of viability and the expiration of the first two weeks of the puerperium. It was. furthermore, shown that the results of treatment were highly satisfactory. When no treatment was instituted 48.5 per cent of the children manifested signs of syphilis, as contrasted with 39.2 per cent and 6.7 per cent, when the treatment was inefficient or efficient, respectively.


Intensive work along these lines has been continued, and has led to a great reduction in the incidence of foetal syphilis in our service. Moreover, in the routine histological examination of the placenta syphilitic lesions are now relatively rarely encountered, instead of very frequently as in the past. In the two years following the completion of the series just referred to, that is, from January 1st, 1920 to December 31st, 1921, 9G of the women included in it have passed through 113 pregnancies in the service, and it has seemed to me that it might be interesting to follow their outcome, especially in regard to the effect of treatment; more particularly as no one has as yet studied a series of syphilitic women who have been under continuous observation for a number of yeai-s. Furthermore, the conclusions reached should be especially valuable, as it has been attempted to follow as far as possible the history of all the children concerned. In April and May of the current year every child that could be traced was visited, so that we have notes on the condition of a large number of the children 4 to 28 months after their birth.

It might be added that in both series the Wassermann tests were made and the treatment instituted in the Syphilitic Department of this Hospital. In talking with Dr. Albert Keidel, concerning the results obtained, he suggested, and I agree with him, that in the former article the differentiation between efficient and inefficient treatment was based upon somewhat uncertain premises and possibly led to erroneous conclusions. In order to insure greater accuracy I have, at his suggestion, classified the treatment under one of the five following groups : A. The administration of at least six doses of diarseuol, followed by mercurials and iodides, and repetition of the treatment until the Wassermann became negative and remained so for at least one year after the cessation of treatment. B. I'atients in whom the Wassermann remained persistently negative for six months after the last treatment. C. Patients in whom it did the same for three months after the last treatment. D. Patients in whom a negative Wassermann was attained only shortly before the birth of the child; while Group E includes all patients in whom the treatment was notoriously insufficient, or was not followed by the conversion of a positive into a negative Wassermann, no matter how long it was continued.

Upon analyzing the 113 pregnancies occurring in the 96 women during the two years under consideration, it was found that 9 had ended in abortion and 6 in premature labor. In the former the children were not viable, while in the latter they were either born dead or died during the puerperium, thus leaving 98 pregnancies for consideration. Such an incidence seems high, and at first glance one might be tempted to regard it as a result of syphilis, were it not for the fact that current clinical statistics teach that in normal women at least every fifth pregnancy ends in abortion, which is higher than the incidence here noted. Moreover, upon studying the abortions more critically, it was found that one was induced on account of severe chronic nephritis in the mother. Three occurred in the early months before the effects of syphilis could become manifest, while five occurred between the fifth and sixth month. In three of the latter, autopsy showed no signs of syphilis, and the placental and X-ray findings were likewise negative; while in the other two, autopsies were not obtained but the i)lacenta! were normal on histological examination. In four of the six premature labors, the autop.sy and placental findings were negative; while in the other two an autopsy was not obtained but the placental findings and maternal Wassermann were negative. Accordingly, while it cannot be categorically stated that syphilis played no part in the production of any of the fifteen abortions or I premature labors, it nevertheless seems permissible to assume that it did not.

Of the 98 children remaining, four died from accidents of labor — one premature separation of the placenta, one prolap.se of the umbilical cord and two craniotomies. In each instance, autopsy showed no sign of syphilis. On the other hand, four other children were born dead or died during the puerperium and showed signs of congenital syphilis, thus leaving 90 living children which were discharged from the clinic in ai)parently good condition.

During the months of April and May (1922) strenuous efforts were made to locate these children, and owing to the persistence of Dr. John G. Murray and our prenatal nurses all but nine of them were found, and were subjected to careful physical examination and a specimen of blood was withdrawn for a Wassermann. This must be regarded as a surprising achievement when the character of the clientele of the service is taken into consideration, and it is recalled that many of the patients had moved several times since leaving the clinic.

Of the 81 children which could be traced, 71 were found to be living and well, manifested no signs of syphilis, presented a negative Wassermann, and were apparently in excellent condition. In another instance, the child could not be traced after the end of the third month, but as it pre.sented a positive Wassermann at that time, as well as at birth, it must be regarded as syphilitic. Of the remaining 9 children, five were living and apparently well, but their mothers would not allow blood to be withdrawn for a Wassermann ; while four others had died — three from pneumonia at periods varying from 7 to 14 months after birth and one from gastroenteritis at the age of IIV2 months. From these figures, it is apparent that of the 94 children who were born alive, 5 or 5.3 per cent showed demonstrable evidence of syphilis, while 76 were living and well, 9 had been lost track of. and four had died.

The results obtained are graphically shown in Table I which represents an analysis of the 113 pregnancies. In the first five lines in the second column the type of treatment employed is indicated, while in the last two the notation "no treatment" or "spontaneous cure"' is made. In the former it may be stated that 13 pregnancies occurred in 12 women, and a word of explanation is necessary concerning the lack of treatment. Ordinarily, it was due to the fact that the patients had registered so late in pregnancy that labor occurred before treatment could be instituted, and sometimes even before the result of the Wassermann test became known ; while in other instances the patients had neglected or refused treatment. The eleven pregnancies under the caption of spontaneous cure occurred in nine women, who were not treated for the reason that the Wassermann was already negative, and the histoiy indicated that the disease had disappeared. In these women, after the birth of a varying number of syphilitic

children, the Wassermann had become negative without treatment and subsequently all children were normal. In one patient in the series, this had occurred as early as l'J12, while in others the negative Wassermann dated from 1915, 191G, 1917 and 1918 respectively. Such spontaneous cures substantiate the observations of the older writers and indicate how cautious one must be in attempting to evaluate the results of treatment in a given case.

TABLE I. Analysis of Treatment In 113 Pregnancies.



(The figures in parentheses refer to the serial numbers of the cases.)

The results obtained by the varying types of treatment are graphically shown in another way in Table II. Here it will be noted that the 49 women, who had received type A to D treatment, had gone through 257 pregnancies in the two series. Of these, 172 occurred before and 85 after the institution of treatment, and resulted in the birth of 40.7 and 89.4 per cent of live children, respectively. In this connection it should be noted that we have classified abortions as well as premature labors as dead children. Before the patients came into our hands we had no means of determining the cause of fa'tal death ; so, if similar allowances are made in each group, it seemed that gross figures would give a more correct idea of the results before and after treatment. It should also be noted that a number of these women were treated only in the previous series and received no treatment subsequent to 1920, others were treated in both series, while still others were treated only after January 1920. In either event the results are striking, and indicate that the incidence of dead children and abortions was six times less after than before treatment.

Moreover, comparison of the results recorded in Tables I and II indicates that type A to C treatment gave almost


ideal results, while in Group D they were surprisingly and unexpectedly good. Furthermore, the results noted in Group E were in some respects even more startling, as they show that, from 29 pregnancies occurring in 18 women, 19 normal children were obtained, as demonstrated by examination four to twenty-eight months after birth, notwith.standing the fact that the treatment had been notoriously inefficient when judged by the usually accepted standards. Indeed, in several instances it was noted that the administration of only two or three doses of diarsenol had apparently sufficed to produce a negative Wassenuann and to result in the birth of live children.

TABLE II. Results of Treatment — 9 6 Mothers.


Type

of

Treatment.


Number

of Women.


Pregnancies

before Treatment.


Deaths

before

Treatment.


Pregnancies

after Treatment.


Deaths

after

Treatment.


A


12


47


26


20 1 3


B


11


39


27


15



c


8


28


20


14


2


D


18


58


19


36


4



49


172 102


>85 3




59.3%


10.6%


E


26


103 46


^48 6


No



44.6%


12.5%


treat.


12


,66 2^ +


1 syphilis alive


Spont.



43.9%



cure.


9





96



What conclusions can be di-awn from the data just adduced? It must at once be admitted that it is difficult to make any precise statement, except that our figures clearly indicate that almost ideal results follow anything like efficient treatment of .syphilitic pregnant women, and that surprising results may sometimes follow what would ordinarily be regarded as altogether inefficient treatment in men or in non-pregnant women, which would seem to indicate that pregnant women are unusually amenable to anti-syphilitic treatment. On the other hand, too roseate a view of the possibilities of treatment should not be indulged in; for, while surprising results have been obtained in many patients, others were encountered who were extraordinarily refractory to treatment; — as for example, Case 23. to whom 30 doses of diarsenol were administered without bringing about a negative Wassermann. Notwithstanding this, the child showed no evidence of syphilis and when examined nine months after deliveiy presented a negative Wassermann.

Such favorable results as we have recorded appear to offer another exam])le of the extent to which syphilis, as it occurs in women during pregnancy, differs from that occurring at other times, as well as in men, and indicates that tlii're must be something about the pregnant condition which mitigates the virulence of the disease and predisposes to spontaneous cure. Observations tending to support such a proposition were made by Keidel and Moore in studying their patients with neurosyphilis, when it was noted that extension to the nervous system was less frequent and much more benign in women who had had children than in uulliparous women or in men. Furthermore, all who have had experience in dealing with syphilitic pregnant women, have come to believe that the disease is much more benign in them than in others, and tends in a certain measure to spontaneous cure. Moreover, experiences of this character are in line with the observations of Wade Brown that in experimental animals the pregnant female is very refractory to inoculation with syphilis, while non-pregnant controls are readily infected by the same virus, and tend to indicate that there is something about the pregnant female which seem,s to interfere with the extension of the spirochete.

At this time it appears futile to discuss the factors which may be concerned in such reactions, but it is apparent that a large field is opening iip, which offers unusual opportunities for experimental study to those fitted for it. On the other hand, the practical lesson is that the results already attained afl'ord abundant evidence that the syphilitic pregnant woman is unusually amenable to treatment, and this fact should encourage all who are interested in prenatal work to make every efl:"ort to recognize the existence of syphilis and to treat it during pregnancy, as by so doing a normal child may not only be obtained, but the mother can apparently be cured with far greater ease than at any other time.

In this connection a few words may be said concerning the diagnosis of syphilis in pregnant women. From my experience with my own assistants, as well as from what I gather elsewhere, it must be admitted that there exists a general tendency to base the diagnosis almost exclusively upon the presence of a positive Wassermann i-eaction and to neglect the clinical study of the patient. In my previous papers I pointed out that the presence of a persistently negative AVassermann on the part of the mother did not preclude the existence of .syphilis and the possible birth of syphilitic children, and I have insisted upon the necessity for the greatest care in taking the history and searching for stigmata of the disease. Unfortunately, however, the results obtained in this respect are not very encouraging; as in the 96 women under consideration in this report only 12 presented a history of frank syphilitic infection, while in five others a suggestive history was obtained. Consequently, with the exception of these few cases, we were obliged to base the diagnosis either upon the existence of a positive Wassermann or upon the autopsy findings in the child.

Within the last two years our diagnostic facilities in the case of the newly-born have been greatly enriched by the work of Shipley and Pearson on the use of the X-ray, and we have had abundant opportunity to confirm its value. Naturally, if an autopsy can be obtained and is (lone by a competent man, the X-ray findings are merely confirmatory, but if an autopsy is not permitted, or the child is still alive, they offer a very important means of diagnosis, as in our experience the characteristic shadow of \A'egner's bone disease at the junction between the diaphysis and epiphysis of the long bones must be regarded as pathognomonic.

In conclusion, I am aware that the present paper is based upon obsei-vations made upon a series of women value, but at the same time the results which we have obtained are highly suggestive and are interesting from two main points of view. First, because our study is based upon observations made upon a series of women with whose past history we have long been familiar, and secondly, because the results obtained are extraordinarily stimulating from the standpoint of treatment and of the great hope which it offers for the future.

THE USE OF LARGE REVERDIN GRAFTS IN THE HEALING OF CHRONIC OSTEOMYELITIS

By Mont R. Rbid, Cincinnati, Ohio (From the Sitrgica} Department of the Johns Hopkins Hospital)


The many methoils that have been introduced for treating bone cavities following operations for chronic osteomyelitis, attest to the difficulty of dealing with this surgical problem. In general they have been based upon the principle that obliteration of the bone cavity is desirable, and may be grouped under three headings :

(]). Autoplasfic operations with the use of soft tissues. This idea perhaps originated with Neuber who, in 18S6, described a method of treating bone cavities by turning into them flaps of skin and other


soft tissue. This principle has been widely employeil. (2). Autoplastic oix^rations with the use of bone. This procedure is essentially the same as the preceding, except that the overhanging edges of bone are used together with the fascia and skin to fill the cavity. (3). Obliteration by the use of rarimis fiUinff 7naterials. Schede introduced the method of healing by means of the moist blood-clot ; MosetigMoorhof used a paste; Senn employed decalcified bone chips, and various surgeons have used fresh detaelied pieces of fascia, bone, fat and muscle.


November, 1922]


387


The purpose of this paper is to introduce another method of treating certain bone cavities, remaining after operations for chronic osteomyelitis, which the above procedures too often fail to cure. It consists essentially of epithelializing the walls of bone cavities by the use of large Eeverdin or pinch grafts. J. P. Lord, in 1902, employed a Thiersch graft in a similar way, but so far as I know, the use of pinch grafts in the treatment of bone cavities has not hitherto been reported. The value of the two methods of grafting will be discussed after the report of two illustrative cases.

Case 1. — Surgical No. 50382. A white man, aged 37, was admitted December 29, 1919. He complained of swelling, pain and lameness in the left leg.

The infection, beginning acutely in the upper end of the tibia, had been present for twenty years. During all this time, periods of quiescence of the infection with healing of the sinus were followed by periods of pain and swelling of the leg which were relieved by the discharge of pus and sequestra.

Operation, December 31, 1919. The cavity in the upper end of the tibia, measuring two inches in length and half an inch in diameter, was found filled with granulation tissue. This was removed with a curette. An attempt was then made to obliterate the cavity by cutting away its bony walls, but its position in the tuberosity of the tibia and the proximity of the knee-joint made this impossible. The operation terminated, therefore, in an attempt to secure healing by Schede's moist blood-clot method.

After three weeks, during which the healing of the wound seemed quite satisfactory, there developed signs of a mild infection. This subsequently necessitated the opening of the wound and the removal of the infected blood-clot. The cavity was then treated by the Carrel-Dakin method. When it became lined with healthy, relatively sterile granulation tissue it was epithelialized by the use of thick pinch-grafts removed from the thigh.

Since the patient's discharge from the hospital March 21, 1920, there has been no further trouble with his leg (vide Fig. 1).

C-A-SE 2. — Surgical No. 49232. A white man, aged 55. was admitted August 6, 1919. He complained of "bone disease in the right leg."

The infection of the bone had begun when the patient was 9 years old, and for forty-six years his leg had many times discharged pus and fragments of bone. Periods of incapacity had varied from several months to a year. During the many years of his affliction the only surgical treatment of the disease had consisted in the incision of superficial abscesses.

Fio. 2 shows the great increase in the size of the right leg, the numerous scars of previously discharging sinuses, and, what has often been noted in long-standing cases of osteomyelitis, the increase in the length of the leg. In this instance the right tibia measured 3 cm. longer than the left.

Operation, August S. 1919. Numerous small abscesses each containing a tiny sequestrum were present throughout the diseased portion of the tibia. The process was so extensive that the operation resulted in the production of a large boat-shaped cavity which extended from the tuberosities to the malleolus.

From August S until September 10 the wound was treated with Dakin's solution. The granulating surface was then covered with thick pinch-grafts which promptly grew and covered the surface of the cavity with epithelium.

February 17, 1920 [vide. Fig. 3). The cavity was well lined with skin which showed no tendency toward maceration. The photograph, unfortunately, does not show the limits of the upper


end of the cavity which extended far into the tuberosities of the tibia.

Technique of skin, grafting. — The bone cavity is treated with Dakin's solution until it becomes lined with clean firm granulations. Without the use of Dakin's or some other antiseptic solution the granulation tissue becomes oedematous or sea-weedy," and forms an unhealthy base for the growth of grafts. Two hours after the last irrigation with Dakin's solution large thick pinch grafts, half a centimeter in diameter are placed closely together upon the surface of the cavity. The grafted wound is then exposed to the air for from six to eight hours. This short drying period serves to fix the grafts firmly to the granulation tissue. The grafts are then covered and held in place with a single layer of gauze which is firmly secured to the normal skin so that the moistening and changing of saline compresses during the next two days will not displace the grafts. After two days the grafts have taken and the use of Dakin's solution instead of salt solution is begun. This is applied by laying wet compresses directlj' against the wound (the thin layer of protecting gauze having been removed) every two hours during the day and everj- four hours at night. After about five days the Dakin's solution is discontinued and the wound is dressed with rubber "protective" or old linen. Invariably the grafts grow quickly and cover the granulation tissue with epithelium in from ten days to two weeks. Should the granulations become high (usually from the oedema of infection I caustics should not be used; Dakin's solution reapplied for one or two days will reduce the swollen granulations to the level of the grafts, and will not injure the growth of new skin.

Indications for skin-grafting. — The cure of chronic ostemyelitis when the disease is confined to the shaft of a long bone is usually not difficult, for the surgeon can frequently remove bone enough to prevent the formation of a cavity or else to permit soft tissues to fall readily into a shallow cavity. But in the ends of long bones, particularly the upper and lower ends of the tibia and occasionally the lower end of the femur, the successful treatment of a bone cavity is made difficult by the presence of the joint which limits the amount of bone that may be chiseled away. In such locations the surgeon has always to face the problem of dealing with a deep cavity, and it is in such cases that we have for the past four years made use of thick pinch-grafts.

Again, when the shaft of the tibia is greatly enlarged, a thorough removal of all sequestra and foci of infection may often result in a cavity that cannot be obliterated by the removal of bone or by the infolding of the scant soft tissues that cover this bone. One occasionally encounters a similar condition in the humerus. Whenever, therefore, we have a large bone-cavity that is difficult to treat by other methods, the use of pinch-grafts makes it possible


388


[No. 381


to secure a rajjid epithelializatiou of the wound.* (vide Figs. 2, 3.)

We have adopted the use of pinch-grafts for several reasons. The epithelial covering which results is thicker and more durable than tluit obtained by the use of Thiersch gi-afts. In deep cavities there is always a ten dency toward the formation of moisture which will macerate delicate epithelium, and in the bone cavities of which we have been speaking, we have found that the thick pinch-grafts are tough enough to withstand this tendency to maceration, whereas Thiei-sch gi-afts often appear to melt away after taking.f

Another factor in favor of the use of piuch-grafts is the certainty of their taking. On a surface properly prepared with Dakin's solution practically every graft will take. Thiei'sch grafts, on the other hand, do not grow as constantly, for the impossibility of sterilizing completely a granulating surface will often result in the accumulation of pus which may lift these grafts from their base.

Changes in tlie size of the grafted bone cavity. — The fate of the epithelialized bone-cavity has been the subject of an interesting study. For several years we have been interested in watching the changes that take place after grafting deep wounds of the soft parts. Ample opportunity for this study has been afforded by the cases of carbuncle which it has been bur practice to excise completely and graft within a few days. In this way, we have


Another application of the method has been useful In a few cases of extensive acute ostemyelitis of the tibia when, at the time of the primary operation, it has seemed necessary to split the periosteum throughout its length. In each instance the periosteum quickly retracted and the wound presented the appearance of extruding the shaft of the tibia. After from four to six weeks the removal of the sequestered tibia left a relatively flat involucrum covered with granulation tissue. These wounds were treated with Dakin's solution and then epithelialized with the aid of large Reverdin grafts. The results have been the substitution of flat bones for the normal tibitP. In the few cases that have been followed these new-formed buues have tended to become round.

t Reverdin's original grafts were very small. He describes them as epidermic grafts. However, he points out that his grafts included the whole epidermis and very little of the dermis. By preparing a more nearly sterile granulating surface with Dakin's solution we can at the present time successfully transplant very large Reverdin grafts. In the center of these large grafts the entire thickness of the skin is included. Though differing from Reverdin's grafts in that they are larger and thicker, the principle remains the same and the present day pinch-grafts should still, I think, be called after Reverdin.


produced many epithelialized depressions of the soft tissues. Kature corrects to a remarkable degree this cosmetic blemish, for after several months the normal contour of the body is re-established to a surprising degree. Tills subject will be dealt with more fully in a paper on wound healing and wound contraction. It is mentioned here because of a similar process which takes place in epithelialized bone cavities. The patient upon whom Lord operated wrote after returning home that the cavity in his bone was much smaller. Ignorant of this observation, Dr. W. F. Rienhoff and I began studying the changes in the size and shape of the bone cavities in our cases. This has been done by making plaster casts of the cavities before grafting and at frequent intervals afterward. In Fig. 5 is shown a photograph of a series of casts from one patient. The leveling of the sides and the decrease in the depth of these cavities form a striking example of nature's attempt to obliterate a cosmetic defect. Whether or not epithelialized bone cavities may be completely obliterated has not yet been determined.

With such a method of treating chronic osteomyelitic bone cavities the time required for curing the most stubborn cases is usually from four to six weeks. This means a great economic saving. Considering the chronicity of this disea.se and the difficulty of curing it, the resultant deformity is of no moment to the patient. In a recent case in which the patient had suffered for thirty years and the ankle joint had become completely ankylosed, I grafted a large cavity running entirely through the ankle (vide Fig. 6).

Everyone is aware of the liability to recurrence of the infection after any of the other methods of treating chronic osteomyelitis. In the method I am proposing there should be but a relatively slight chance for recurrence ; but that it may not recur must for the time being renutin an undetermined point.

REFERENCES Lord. ,1. P.: .lour. Am. Med. Assn., May 31, 1920, vol. 38. p. 1433.

Neuber, G.: Arch. f. klin. Chir., 1896, LI, G83. Nichols, E. H.; Jour. Am. Med. Assn., Feb. 13, 1904, vol. 42, p. 438.

Senn: Amer. Jour. Med. Sci., 1899, N.S. XCVIII, p. 219. Mosetig-Morrhof (von): Deutsche Ztsch. f. Chir.. 1904, LXXI, 419.

Reverdin: Bull. d. e. Soc. imperiale de chir. de Paris, Dec. 1869, pp. 493-511.

Arch. gen. de m6d.. 1872, pp. 27G, 555, 703.

Gaz. med. de Paris, 1S71, 3 s., 26, p. 554.

Bull. g#n. de th^rap.. etc., Paris, 1872, LXXXII, 71-74.


THE JOHNS HOPKINS HOSPITAL BULLETIN


The Hospital Bulletin contains details of hospital and dispensary practice, abstracts of papers read and other proceedings of the Medical Society of the Hospital, reports of lectures, and other matters of general interest in connection with the work of the Hospital. It is issued monthly. Volume XXXIII is in progress. The subscription price is $4.00 per year.

(Foreign postage, 50 cents.) Price of cloth-bound volumes, $5.00 each.


THE JOHNS HOPKINS HOSPITAL BULLETIN. NOVEMBER. 1922


PLATE LI!



i%'


Fig. 1. — Case 1. Surgical

No. 503S.2. Johns Hopkins Hospital.

On the thigh are the scars that resulted from cutting the pinch grafts. In the bone cavity the grafts are still visible. Photograph taken fourteen months after the patient's discharge from the Hospital.



Fic. 2. — Case 2. Surgical No. 49232. Johns Hopkins Hospital.

Duration of the disease 4 6 years. Note tlie increase in size and length of the right tibia.


Fic.


-Case


Several months after epithelialization of the bone cavity. The extent of the cavity into the ends of the tibia is not shown in the photograph. The grafts can be seen.


PLATE Llll


THE JOHNS HOPKINS HOSPITAL BULLETIN. NOVEMBER, 1922



Fre. 4.— Surgical No. G2S12. Cincinnati General Hospital. Bone cavity is shown completely epithelialized two weeks after being grafted with large pinch grafts.



-Surgic


il .\(i. .'>i1lM;) Jolins Hopkins Hospital.



— riurgical No. 5303 Hospital.


Johns Hopkins


Plaster molds of an epithelialized bone cavity; 1 day before. 5 days after, 18 days after. 42 days after and 10 months after skin grafting. The decrease in the size of the cavity is plainly shown in the molds.



The joint was ankylosed by a chronic osteomyelitis of thirty years duration. The arrow shows an epithelialized cavity extending through the destroyed joint.


Fio. 7. — Surgical No. 55STS. Johns Hopkins Hospital.

Bone cavity three weeks after grafting it with large pinch grafts.



The Morphological Theory Of Monochorionic Twins As Illustrated By A Series Of Supposed Early Twin Embryos Of The Pig

Corner GW. The morphological theory of monochorionic twins as illustrated by a series of supposed early twin embryos of the pig. (1922) Johns Hopkins Hospital Bulletin 33: .

By George W. Corner

(From, the Anatomical Laboratory of The Johns Hopkins University)


The subject of identical twins is of such iuterest from many biological viewpoints that conjecture as to the mechanism of their origin has far outrun our actual information. Almost the whole mass of discussion now current in the literature of embryologj- and genetics is pure hypothesis constructed by reasoning backward from the observed anatomy of late stages, chiefly from the dispo sition of the foetal membranes at birth in cases of twinning. Even the term "single-ovum twins" is an assumption, when applied to mammals.

So far as is knowTi to the present writer the extant specimens of monochorionic twin embryos, in stages early enough to be of value for morphogenetic study, are (1) the blastodermic vesicle of the sheep, about 7 days old, and a similar embryo of the ferret (Putorius), described by Assheton ('98), in each of which two inner cell masses are found in one blastocyst; (2) the human embryo "Mateer" described by Streeter ('19a), in which a similar arrangement is supposed to exist at a slightly later stage of development; (3) two human specimens, each containing twin embrj'os about 12 mm. long, recently described by Arey ('22) ; and (i) the complete series of enibn,'os of the nine-banded armadillo (a species in which singleovum quadruplets are produced at every birth) which we owe to the work of Newman and Patterson ('10, '13). With Asshetou's specimens and his own as the basis for hypothesis, Streeter ('lOb) has constructed a very plausible series of diagrammatic stages of human single-ovum twin-formation, in which this phenomenon is represented as due to the production of two embryonic areas of inner cell masses on the inside of a single blastocyst, before the formation of the amnion. Such a hypothesis fits in well with the usual disposition of the foetal membranes at birth ; and it would also readily account for the appearance of monochorionic twinning in any mammalian spe cies, since all mammalian embrj^os, whatever their later divergencies of form, pass through an essentially similar blastocyst stage. Such a view of twinning would furthermore accord with the fact of general occurrence of polyembryony in the animal kingdom, and would give added philosophical value to the experimental production of uniovular twins and double monsters in fishes and amphibians.

A different view has been proposed by Newman in his interesting book "The Biologj' of Twins" ('17), upon the basis of his studies of the armadillo. In this species development proceeds as in those mammals which undergo so-called "inversion of the germ-layers," until the amnion is formed ; then the embryonic area divides into four embryos, and each embrjo migrates down a meridian of the chorion, dragging with it the surrounding portion of the amnion, until finally a single chorion contains four embryos, each enclosed in a portion of the original amnion, now drawn out into four sacs with a common central connection. It is obvious that this form of polyembryony cannot take place except in mammals in which the formation of the embryo proper is delayed until after the appearance of the amniotic cavity; and since in man a similar proigenesis of the amnion occurs, Newman has been led to believe that in man also monochorionic twin-formation follows the same scheme as the polyembryony of the armadillo. Furthermore, if in mammals monochorionic twinning is indeed dependent upon proigenesis of tlie amnion, then it cannot occur in species in which the amnion is produced by rolling up of the chorion over the embryo. Newman has been willing to make this assumption, and in his book has ventured the prediction that single-ovum twins ^^ill probably never be found in ungulates.

The three specimens of earl}' enibiyos of the pig which are to be described in the following pages all came into the author's hands within the space of a few weeks, at the packing house of Joseph Stern and Company, in New York City.* It is remarkable that all three were found among about 500 pregnant swine examined in this one abattoir, whereas similar specimens have never been seen in many hundreds of sows at other places. The packinghouse in Baltimore which has furnished pig embryos to the Johns Hopkins laboratories for thirty years has never yielded, as far as the writer knows, anything resembling monochorionic twins except one double monster and one specimen to be mentioned later, in which it was not certain whether one or two chorions were present.

Tlie following descriptions and illustrations will be clear to the reader who is not familiar with the specific embryologj' of the pig, if he will recall that the blastocyst


The writer was at the time working under the auspices of the Department of Experimental Evolution of the Carnegie Institution of Washington, at Cold Spring Harbor, Long Island. To the Director, Dr. C. B. Davenport, thanks are due for providing support and assistance.



does not remain spherical, but is rapidly elongated to 30 or more centimeters, so that by the fifteenth day the embryo itself is a small thickening upon a very narrow but immensely elongated cylindrical chorion. The chorion does not develop villi, as does the human gestation sac, but remains smooth, and can at all times be readily peeled out of the uterus. The allantois reaches such size as to fill a large part of the cylindrical chorionic cavity, and ultimately becomes adherent to the inside of the chorion, giving rise to a difl'use allantoic placentation.

Specimen 1. — The uterus of sow C-15 5 contained 14 single embryos and the twins to be described. Eight of the single embr>'os were normal at the stage of 33-34 somites (i. e. of the 3rd week). Six showed abnormalities as follows: Two were retarded, without gross defects; one was retarded, with extreme flexure; one was slightly retarded, with undistended allantois. In another the embryo itself appeared normal, but its allantois was entirely cut off from the embryo by the amnion; the embryonic stump of the allantois was closed over, and the extra-amniotic part of the allantois which was adherent to the chorion had upon it a small highly vascularized vesicle. Finally, one embryo was represented merely by an almost amorphous chorionic mass. Since there were 16 corpora lutea, it seems that one of the embryos was totally missing.

In the twin specimen (Fig. 1), two embryos lay about 18 mm. apart within one continuous chorion of dimensions quite similar to those of the normal embryos in the same uterus. Fluid injected into the chorionic cavity dilated the whole space; that is. there was no septum between the embryos. When the cylindrical chorion was slit open, it was found that both embryos were abnormal as Illustrated. In one, the allantois was entirely cut off from the embryo, and was growing in an almost unexpanded conditiou upon the chorion adjacent to the embryo. In the other, the allantois had been converted into a cluster of small irregularly dilated vesicles. The yolk-sac vessels of this embryo had not grown, as normally, out along the yolk-stalk onto the sac, but remained as a convoluted nodule on the yolk-sac near the umbilicus. That portion of the yolk-sac (or sacs) lying between the embryos had become curiously twisted and vesiculated, as shown in Fig. 1, so that it was unfortunately Impossible to determine whether the two embryos had origina.lly possessed a common yolk-sac. The two amnions seemed perfectly normal; the enclosed embryos were slightly smaller than the normal single embryos in the same uterus.

Specimen 2. — The uterus of sow C-392 contained 10 normal embryos of 11 mm. crown-rump length, and one pair of twin embryos enclosed in a single chorion (Fig. 2). There were 14 corpora lutea; three ova were therefore missing, a proportion not much above the avei^age loss of ova in this species. The twin embryos appeared quite normal, but one of them occupied, with its allantois, about two-thirds of the available chorionic cavity, leaving but one-third for the other. The embryo with the larger allantois was 9.5 mm. crown-rump length, the embryo with the smaller allantois was 8. .5 mm.; there was no significant difference in the development as indicated by external form. Injection of fluid into the allantoic cavities showed that the two were not continuous. When the chorion was opened, it appeared that the yolk-sacs, which at this stage in the development of the pig are beginning to be obliterated by pressure of the allantois, were in the twin specimen very much twisted. It was thus again impossible to determine the original relation of the yolk-sacs, for although they were entirely separate in this specimen, the separation might conceivably have been produced by torsion, as the figure clearly shows.


Specimen 3. — The uterus of sow C-66 contained 7 normal embryos of 23 mm. crown-rump length and a pair of twin embryos. There were 15 corpora lutea. Fig. 3 shows that the arrangement was somewhat different from that in the other two specimens, for here the two embryos were not placed end to end in the chorionic cavity, but side by side. The allantoides were expanded so fully that they came entirely into contact with the inner chorionic surface, obliterating the chorionic cavity. Each amnion was now surrounded by its allantois, as usual at this stage, so that each embryo with its amnion appeared to occupy a separate cavity divided from the other by a septum. The cavity was obviously the allantoic cavity and the septum was formed by the apposed walls of the two allantoides. The areas of attachment to the chorionic wall were so near together that the two umbilical cords appeared to spring from almost the same point, in the line of reflection of the allantoic septum. A section taken through this line showed clearly (Fig. 4) that the chorion was continuous over the surface, not dipping between the allantoic walls; that is, there was a single chorion containing two allantoides. Obliteration of the yolk-sacs had proceeded so far that their relation to each other could not be made out; they seemed to be separate. The embryos occupied approximately equal parts of the chorionic cavity. One was 16 mm. crown-rump length and the other 15 mm. These embryos were sufficiently advanced to permit determination of their sex by the criterion described by Spaulding ('21), which (as Dr. Spaulding assures the author) can be applied to the pig as well as to human embryos. In both these twin embryos the urethral groove runs on to the glans portion of the phallus, and they are both therefore presumably of the male sex.

Discussion

The specimens which have been described and illustrated may obviously be explained by the assumption that each is an example of twins arising within a single blastocyst, or by the alternative supposition that a deceptive appearance of twinning arose bj' the fusion, in each case, of two originally separate blastocysts. This latter possibility seems unlikely ; one would expect to find, in addition to specimens like these, other and more numerous examples not so deceptively perfect. Such fusion is at least not common in early stages of development of the pig; the present writer has never seen it during the study of hundreds of embryos of the first three weeks. It is true that after mid-pregnancy the adjacent necrotic ends of two chorionic sacs occasionally adhere or invaginate in a more or less complete way, but in such cases the larger total dimension of the combined chorions,, the considerable distance between the foetuses, and the presence of a chorionic necrosis or invagination at the region of fusion gives sufficient evidence of the dual origin of the ftetus. In the two earlier specimens under consideration there was no evidence, to the eye or under the dissecting microscope, of any chorionic septum between the embryos, and in the third .specimen there was microscopic evidence that the chorionic space was single, i.e., that the septum was formed by the internal membranes alone, without participation of the chorion. On the whole, the theory that the specimens were monochorionic twins seems more



Pig. 5.

Explanation of Pig Twinning.

Single Twin


Fig. 6.

Hypothetical Diagrams of Human Monochorionic Twins

Duplication before Duplication after

formation of formation of

amniotic lavit.v amniotic cavity



probable than that they were examples of accidental fusion of separate blastocysts with obliteration of the intervening trophoblastic (chorionic) wall. It should be mentioned at this point that in a specimen from a local packing-house, studied through the courtesy of Dr. G. L Streeter, which appeared at first sight to be another case of monochorionic twinning, in mid-pregnancj' (fcetuses 130 mm. long), dissection revealed evidences hinting at a possible origin from two separate chorions, through the extensive invagination or penetration of one by the other. The impression gained from all of these specimens taken together is in accord with the recent views of Arey (1922) formulated after study of twin tubal pregnancies as recorded in the Carnegie Embryological Collection, that the appearance of monochorionic twinning may, at times be due to the fusion of two chorions, but that true polyembryony may also occur.

Assuming that our specimens are indeed monochorionic twins, it is possible to reconstruct the stages of their formation by means of a series of diagrams involving but one or two hypothetical steps. In Fig. 5, I to IV, such a series is placed in parallel with the actually known stages of development of the single pig embryo. Stage I in the twin series is borrowed from the sheep ol Assheton's description, stages II and III are hypothetical, and stage IV is specimen 1 of the author's collection. In this scheme it is assumed that twinning is first made evident by the appearance of two embryonic areas (inner cell-masses) on the inner surface of a single blastocyst. Following this stage we may suppose that an amnion is formed about each embrj'onic area exactly as in single embryos. Meanwhile the endoderm has been spreading around the inside of the blastocyst. At the same time the blastodermic vesicle has been undergoing the great elongation which is characteristic of the pig and allied ungulate species. Whether the endoderm would usually form a single inner lining of the blastocyst, and thus provide both embryos with a single common yolk-sac, or pinch off into two yolk-sacs, is a matter for conjecture, but in preparing the accompanying diagrams the author has preferred to follow his actual specimens and to represent the yolk-sacs as doubled structures. The result, as shown in Fig. 5. stage IV, (also. Fig. 1, specimen 1| would be a single chorionic vesicle bearing separate embryos, each enclosed in its own amnion, with a yolk-sac which, for all we know at present, might be either doubled or common to both embryos. Each embi-yo would thereafter produce its own allantois, and when the allantoides had expanded to a moderate degree we would have the stage represented by Fig. 2 (specimen 2). Next the allantoides would expand so voluminously as to surround the amniotic membranes completely, as in Fig. 3 (specimen 3), in which we have a single chorion containing two allantoic cavities with the amnions invaginating the allantoic walls and the yolk-sacs passing out through the necks of the invaginations to be between the allantoic walls and the chorion. Finally, as the expansion of the amniotic cavities brought about complete fusion of the three foetal membranes, the chorionic cavity would be lined at all points by both allantoic and amniotic tissue, and would be divided into two cavities by the abutting portions of the respective allantoic-amniotic membranes.

It thus seems apparent that monochorionic twinning in ungulates is not to be considered impossible merely because in mammals of this order the embryonic area is well differentiated before the amniotic cavity arises. Proigenesis of the amnion may no doubt influence the morphology of twins, but the occasional o^ irrence of monochorionic twins is not thereby limited to any special kinds of mammals.

In conclusion, we may permit ourselves to leave the more or less solid ground afforded by our three specimens, and to indulge in a brief speculation regarding the morphogenesis of human monochorionic twins as suggested by our studies of polyembryony in the pig. We have shown the probability that, in the pig, twinning must already be apparent before the formation of -the amnion, while in the amiadillo, as investigated by Newman and Paterson, the polyembryonic budding of the four embryos awaits the formation of an amniotic cavity, which is thus common to all the embryos of one ovum. It may also be pointed out that the human embryo, like all other mammalia, must first pass through a stage of simple blastocyst structure with an inner cell mass, before it proceeds to the splitting open of the amniotic cavity. It is true, as Newman points out, that at this latter stage the morphological relations are much like those at which in the armadillo multiplication of the embryonic areas begins ; but it is also true that at the earlier blastocyst stage there is opportunity for twinning as in the pig, by the appearance of two inner cell masses instead of one. Our hypothesis, illustrated diagrammatically in Fig. 6. is that human single-ovum twins may be of two types. One sort of twinning, (Fig. 6, A. B. C.) arising by duplication of the inner cell mass before formation of the amniotic cavity (pig type) would typically give ri.se to two embryos in a single chorion with two independent amniotic cavities. The "Mateer" embryo as interpreted by Streeter illustrates this relation, and it is this scheme of twin-formation which is propounded in Streeter's contribution of 1919. A second kind of twinning, occurring by duplication of the actual embryonic areas after formation of the amniotic cavity (armadillo type), would produce two embryos within a single amnion (Fig. 6, D. E. F.). There would probably be intermediate and unclassifiable stages, because the moment of separation of the embryos might be intennediate in time, (t. e., during the opening of the amniotic cavity) and also because in the first type the embryos might lie so near together that the separate amniotic cavities would fuse. Our theoiy thus covers, perhaps too easily, the known variations of arrangement of the foetal membranes of twins in pre-natal stages and at birth. (For collections of such data see O. Schulze, 1897, and Wilder, 1904). The conception that there may be a series of types of human single-ovum twins accords well with the fact that there is a rather wide variability in the degree of resemblance between so-called "identical twins;" but caution suggests that we leave these concluding notions to await the further test of actual specimens.

LEGENDS OF FIGURES

F*iG. 1. — ^Specimen 1. Embryos of the third week, showing the cylindrical chorion cut open to expose the embryos. Points indicated by the letters A-A were adjacent before the yolk-sac was cut in opening the chorion, as were points B-B. Note abnormalities of the allantoides and yolk-sacs as described in the text. (X 7.)

Fig. 2. — ^Specimen 2. Normal embryos 8.5 and 9.5 mm. long, enclosed in a single chorion, (x 2.5.1

Fig. 3. — Specimen 3. Semi-diagrammatic. The embryos lie side by side within a single chorion. In this stage the amnions have invaginated the allantoides. The yolk-sacs appear to be separate, as in the previous specimens, (x 2.5.)

Fig. 4. — Diagram of a section taken through the chorion and allantoic septum near the point of attachment of the embryos of Specimen 3, showing that the chorion does not participate in the formation of the septum.

Fig. 5. — Explanation of Pig Twinning. Single Twin


Fig. 6. — Hypothetical Diagrams of Human Monochorionic Twins.

Duplication before Duplication after

formation of formation of

amniotic cavity amniotic cavity

PAPERS CITED

Arey, L.: 1922 a. Direct proof of the monozygotic origin of human identical twins. Anat. Rec., XXIII, 245.

1922 b. Chorionic fusion and augmented twinning in the human tube. Anat. Rec, XXIII, 253.

Assheton, R.: 1S9S. An account of a blastodermic vesicle of the sheep of the seventh day with twin germinal areas. Jour. Anat. Physiol., XXXII, 362.

Newman, H. H.: 1917. The biologj- of twins. University of Chicago Press.

Newman, H. H. and Patterson, J. T. : 1910. The development of the nine-banded armadillo. Jour. Morph., XXI, 359.

Patterson, J. T.: 1913. Polyembryonic development in Tatusia novemcincta. Jour. Morph., XXIV, 559.

Schultze, O. : 1897. Grundriss der Entwickelungsgeschichte des Menschen und der Saugethiere. Leipzig.

Spaulding, M. H.: 1921. The development of the external genitalia in the human embryo. Contributions to Embryolog)% vol. 13, Carnegie Institution of Washington, Publication No. 276, p. 67.

Streeter, G. L.: 1919 a. A human embryo (Mateer) of the presomite period. Contributions to Embryology, vol. 9, Carnegie Institution of Washington, Publication No. 272, p. 389.

1919 b. Formation of single-ovum twins. Johns Hopkins Hosp. Bull., XXX, 235.

Wilder, H. H.: 1904. Duplicate twins and double monsters. Amer. Jour. Anat., Ill, 387.

Development And Growth Of The Metanephros Or Permanent Kidney In Chick Embryos (Eight To Ten Days' Incubation)

Rienhoff WF. Development and growth of the metanephros or permanent kidney in chick embryos (eight to ten days' incubation). (1922) Johns Hopkins Hospital Bulletin 33: 392-405.

By William Francis Rienhoff, Jr., M. D.

From, the Department of Surgery of the Johns Hopkins University, and the Department of Embryology, Carnegie Institution of Washington.


The story of the development of the metanephros, the organ which forms the permanent kidney in the higher mammals, occupies a volumiuous literature and is based on the work of many competent obsenei*s extending over a long period of time. Herring (1900), Schreiner (1902), Stoerk (1904), Huber (1905), and Felix (1914), in their classical contributions to the subject, have given a very complete resume of the work of previous observers whose investigations, made by means of fixed sections and reconstructions, were continued until this method could yield no further results. However, with the perfection of the tissue-culture method it became evident that by this means of approach new results might be obtained in this field. Carrel and Burrows (1910) were the fli-st to make cultures of the kidney. They observed, after five or six days, that tubes had grown into the plasma for a short distance and that there seemed to be a lumen limited by epithelial like cells. These tubes had, they thought, the appearance of renal tubules. Champy, in 1914, also cultivated renal tissue, but he claimed that a dedifferentiation of the entire renal anlage into an indifferent epithelium took place. In 1920 Atterbun- grafted the metanephric anlage of chick embn'os (6 to 7 days' incubation) in the allantois, with the result that the already formed epithelial tubules proliferated and the less differentiated nephrogenic tissue acquired an epithelial arrangement. Hematopoiesis also occurred and glomeruli developed.

In the hope of clearing up some of the controversial points in the development of the kidney, a study of the metanephros by means of cultures, as well as by fixed serial sections and spreads of living tissue, was undertaken. The results obtained in regard to the earliest stages of development of the metanephros, the anlage and early developmental stages of the renal evagination and nephrogenic tissue, and the later development of the iiriniferous tubules, confirmed those of Schreiuer and Huber and therefore need uo further discussion. Only the observations that differ materially from those of previous observers, such as the growth of the collecting tubules, differentiation of the anlage of the uriniferous tubules, and development of the glomerulus, together with the formation of the blood-vascular system, will be discussed in this paper.

I am indebted to Dr. and Mrs. Warren H. Lewis, not only for advice and assistance, but also for the technique employed, which made possible the cultivation and observation of the renal tissue.

Material and Methods

Cultures were made by explantiug small pieces of chick embrj'os after from 8 to 10 days' incubation. Tlie pieces to be explauted were chosen with the aid of the dissecting microscope from the most inferior medial pole of the mesouephros and the superior medial pole of the metanephros, because, as is commonly known, the differentiation of the mesonephrovS proceeds antero-posteriorly, whereas in the metanephros it is postero-anteriorly. For this reason the degree of differentiation at any age depends to some extent on the part of the renal body from which the specimen is taken. The areas chosen were the least differentiated embryologically and the most isolated anatomically. The tissue was cut up into pieces as thin as possible and placed on sterile coverslips, in a small drop of Locke- Lewis solution (85 c.c. Locke's solution plus 15 c.c. chicken bouillon plus 0.5% dextrose). The coverslip was then inverted so as to form a hanging drop, sealed onto a ring of vaseline, and incubated at 39° C. The cultures were observed at varying intervals in a warm box at 39° C. Some of them, after varying periods of growth, were fixed either by means of iodine vapor or of Zenker's fluid and stained. Serial paraffin sections 5 micra in thickness were cut, stained with hematoxylin-eosin, and mounted separately. The entire metanephric body of both injected and non-injected chicks was studied at different ages in fresh spreads.

Morphology

The urino-genital system of chick embryos from 6 to 10 days' incubation can be dissected with considerable ease without the microscope. When the abdominal cavity is opened and the stomach and intestines have been removed, the mesonephric bodies appear as two greenish-red structures filling up a large part of the remaining space in the abdomen. They are situated one on each side of the vertebral column and extend from just below the attachment of the liver to a point immediately above the bifurcation of the abdominal aorta. They are more or less bilaterally symmetrical and seem to be connected across the mid-line by only a thin sheet of mesothelium. Their dimensions vary according to the age of the embi-j'o; between 6 and 10 days' incubation they average about 1 mm. in diameter and about 3 mm. in length. They are somewhat wedgeshaped pyramidal bodies, diminishing in width toward the caudal pole. The color is characteristic and is in marked contrast to that of the metanephroi and other abdominal viscera. Over the glistening surface can be seen many pin-point red spots, the glomeruli. At the upper (cephalic) poles are the gonads, two opague white tubular-like bodies, which, starting above and behind the pointed upper poles of the mesonephric bodies, continue across the uppennost anterior surfaces and course caudalward along the antero-medial margins, ending in a point corresponding to one-half the length of the mesonephric bodies. From the dorsal surface, and continuing below the lower pole of each mesonephric body, is the mesonephric or Wolffian duct, which serves as the excretory duct for the mesonephric body or embryonic kidney. These small white ducts approximate each other in a downward course and end in the cloaca. The branching connections of the Wolffian duct with the mesouephros, the mesonephric ureters, could be distinctly seen by elevating the lateral margins of the mesonephric bodies. Along the vertebral column, dorsal and also immediately posterior to the mesonephric bodies, lie two bilaterally symmetrical translucent structures, the metanephroi. The genitourinary apparatus is entirely covered by a thin layer of cubical epithelium which is known as mesothelium or coelomic epithelium. In the youngest ages, the metanephros is completely obscured by the overlying mesouephros, which reaches its greatest volume at about the eighth day. From this time on, however, the metanephros increases rapidly in size, with the result that in a 10-day embryo the mesouephros appears to be a relatively small body situated on the anterior surface of the metanephros. The supply of blood to the metanephric body between these ages is very small, while the blood-flow through the mesouephros is quite abundant, a fact which accounts for the difference in the color of the two bodies. The shape of the metanephros changes constantly during the growth and development of the embryo, so that no definite shape can be described as typical for all ages. However, in general it may be said that the shape approximates that of a dumb-bell, being broad at both poles and Harrow in the middle. This is well shown for older embiyos (18 days) by Minoura (1921, Fig. 8). At the age of 6 days the metanephric body has a smooth exterior surface, with no lobulations. A little later, however, the bodies begin to show definite lobulations, which by the tenth day have become discrete lobes completely covered by coelomic epithelium.

The entire metanephros was dissected out and placed on a slide, thus making it possible to study the fresh tissue at different ages with the aid of the microscope.


In the 6-day embryo it was found that the metanephric tubule, which sprouts off the Wolffian duct, had already grown into the metanephric body, completely traversing the undifferentiated tissue from the posterior to the anterior pole. Along the entire length of the metanephric tubule the primary collecting tubules had budded off in three main planes, lateral, dorsal, and ventral, and there seemed to be an enlargement of the lumen of the metanephric tubule at the anterior and posterior poles, at which places the metanephric body is broader. Each of the primary collecting tubules, although they had grown only a slight distance from the metanephric tubule, had just divided dichotomously into two secondary collecting tubules. Xo further division, that is, beyond the secondary tubules, was ever observed at this age. Immediately about the primary- and secondai-y collecting tubules was a sharply defined covering, consisting of a single layer of endothelial cells. This structure will be described in detail later. Between the bases of the primarj collecting tubules, near the posterior pole of the metanephric body, sinuses had differentiated out of the tissue surrounding the ba.scs of these primary collecting tubules. These spaces or sinuses were lined with flat endothelial cells which gave rise .to blood elements. This was the fii-st appearance of sinus formation observed in the metanephrogenic body.

At 7 days the branches of the primary collecting tubules, namely, the secondary tubules, had again divided dichotomously into tertiaiy tubules. The branching appeared always to occur in a forked manner, the tubules growing toward the peripherj-. There was a marked increase in the length of the primary and secondary collecting tubules during the sprouting of the tertiary tubules. The apparent dilatation of the lumen of the metanephric tubule at the anterior and posterior poles had disappeared by this time, but in these areas there was a much more rapid growth of the collecting tubules than in the intermediate region. This growth was perhaps somewhat more pronounced at the posterior than at the anterior pole, for, as is well known, the metanephros differentiates poster o-anteriorly. In the body of the metanephros the formation of sinuses proceeded hand-inhand with the elaboration of the collecting tubule tree, replacing the undifferentiated tissue about the bases of the primary and secondary collecting tubules. Towards the peripheiT of the metanephric body, strands of mesenchymal cells, arranged in fe.stoons, divided the cortex into a .series of lobes which corresponded roughly to the secondary collecting tubules. The definition of this lobulation was more pronounced in the 8-day embiyo, constituting complete separation of the metanephrogenic lobules into distinct lobes surrounded entirely by a layer of cubical cells and arranged always in relation to the secondary collecting tubules. The length of the primary, secondaiy, and tertiary tubules increased greatly with this lobulation, the tertiaiy tubules being entirely intralobar, while the primary, as well as the secondarj' tubules, were extralobar. In the anterior and posterior poles of the metanephros the tubules grew to a greater length and gave off more sprouts, resiilting in a gi-eater number of secondaiw and tertiary branches in these regions. As has been stated above, the lobes were establi.shed in the 8-day embryo, but an intralobar lobulation was obsen'ed to begin immediately following the lobe fonuation. This subdivision of the lobe into smaller lobules persisted until the elaboration of the collecting tubule tree was complete. In the 9-day embryo it corresponded to the branching of the quarternaiw tubules or the first intralobar division of the tertiar}^ tubules. Up to this time no sign of the secreting or convoluted tubule had appeared. About these quarternary tubules, however, differentiation of the future convoluted tubule had occurred and from now on, with each succeeding division of collecting tubules, there, was a simultaneous formation of convoluted tubules (including glomeruli, etc.) in the angles of the branching collecting tubules. Eventually, each branch of the collecting-tubule tree, with its corresponding convoluted tubules, was surrounded by mesenchymal tissue which formed a sort of capsule about it as a unit. In the kidney, therefore, as in the lung, liver, etc., there is a definitive lobule, which is the elementarj- unit of the entire excretory apparatus. This unit con.sists of the terminal branch of the collecting tubule, the convoluteil tubules, including the glomenili, and the surrounding endothelial and mesenchymal tissues. Owing to the lobulation, the growth and division of the collecting-tubule tree inside the lobe was much more elaborate in the 10day than in the 9-day embrj-o. The distribution of the lobes in the 10-day embryo corresponded exactly to the distribution of the secondaiy collecting tubules. As a result, the greatest number of lobes were found composing the caudal pole, while the next greatest number constituted the cephalic pole. The lobes making up the intermediate region were evenly and regularly distributed in all planes, so that all the lobes at any given level were practically at the same stage of development regardless of their distance from the original metanephric tubule. Differentiation of the lobe always proceeded from the base towards the periphery. The most developed convoluted tubules were associated with the quarternary branches of the metanephric tubule. From this base or center the cycle of development was repeated for each generation of the renal units as we approach the periphery of the lobe towards the cortex and also towards the future columns of Bertini, not only in the longitudinal diameter but also in the transverse diameter. The development of the systems of sinuses obeyed the same general principle, that is to say, they were formed first at the base of the lobe and later at the periphery.


In conclusion, it may be stated that, although the posterior portion of the nietanephric body grew and developed more rapidly than the anterior portion, nevertheless differentiation was always from the nietanephric duct towards the periphery, not only in the lobes and lobules but also in the blood-vascular system and in the supporting tissue; so that even in the older embryos the most mature elements were located near the base of the lobe, while the immature structures occupied the cortical regions.

Growth and Development Within the Explant

In cultures of chick embryos of from 8 to 10 days' incubation the evolution of all the elements making up the excretory unit of the permanent kidney could be followed in detail throughout their differentiation and development. Usually the explauts contained at least one lobe with its lobules, in different stages of development according to the age of the embryo from which the piece was taken. Observations on the inesonephros were made for a comparison, but these will be described only where they differ in some fundamental way from those made upon the metanephros. The component parts of the excretoi-y unit in cultures of different ages will be described in the order of their differentiation and development : i. e., collecting tubules, nephrogenous tissue (secreting or convoluted tubules and glomeruli), and endothelium, including the blood-vascular system. The growth and development within the explant, as well as the growth extending out from the explant, were studied.

Eight-day Embryos

The living explant was slightly yellowish and so transparent that the structure of the metanephros could be readily observed. The most striking characteristic of the explant was the collecting tubules, which at this age have undergone extensive growth from the nietanephric ureter into the undifferentiated metanephrogenic tissue and become branched like a tree, as was mentioned when speaking of the morphology. It is in the growth of this element of the excretory unit that the inesonephros and metanephros differ most \^^del3^ In the inesonephros the collecting tubule, from the beginning of its growth from the primary excretoiy duct, is a single uubranched tubule, twisted upon itself, opening individually into the excretory duct, and destined to become connected with one convoluted tubule. It is coiled from the beginning, and becomes more so as it ajiproximates maturity. The characteristic tree like formation observed in the metanephros was never found in the inesonephros.

In the explauts from 8-day einbiyos masses of undifferentiated metanephrogenic tissue surrounded the ends of the collecting tubules and, as there were no other differentiated elements of the excretory unit, the collecting


tubule tree (primary, secondai-y, and tertiary tubules) stood out strikingly because of the marked contrast between the differentiated and undifferentiated tissue (Fig. 1) . After the culture had grown for 20 hours, the explant became flattened out and attached to the cover-slip, owing to the migration of connective-tissue cells, neiwe filaments, endothelium, and tubules. Coincident with the migration was a division of these cells, thus forming an extensive area of new marginal growth (Figs. 3, 13, and 14). The migration of cells from within the explant to form the marginal growth left the collecting tubule intact and sharply defined (Figs. 2 and 3). The definition was aided by a single layer of highly refractive endothelial cells which completely enshrouded each collecting tubule and its branches, even covering the terminal ends of the tube (Figs. 2, 6, and 18). The endothelial cells formed a vei-y thin sheet, through which the bases of the columnar cells forming the tubule could be seen as triangular or prismatic cellular bodies. The collecting tubules grew in a comparatively straight line from the tertiary tubules and branched out in a characteristic and regular manner, i. c, always from the base towards the periphei-y of the lobe (Figs. 1, 2, and 8). There may be a slight, gentle curvature to the course of the growth of either the main trunk or its branches, but never was there seen the bending and intertwining observed in the tubules of the niesonephros or later in the convoluted tubles. There was a regular gradation in diameter from the tubules making up the central trunk to those constituting the peripheral arborization. The distance between the branches along the main stem was the same, although the branches came off in any plane (Fig. 2). The most common type of branch formation was dichotomous or trichotomous (Fig. 1), although there was occasionally a branching into fours. The branches always came off from the main stem at angles greater than ninety degrees, and grew in a lateral or peripheral direction. However, it must be stated that, while there was no constant direct trunk or axis of the tree growing through the center of the lobe with the branches coining off laterally, yet there was always one main branch, which maintained a general peripheralward course of growth and remained approximately the axis of the lobe (Fig. 2). The lumen of the collecting tubule was established at this age and patency was maintained by a fluid-like medium in which granules and cellular detritus were seen floating about. The lumen was bordered by a high, non-ciliated, columnar epithelium (Figs. 12 and 24) . About the ends of the tubules the undifferentiated metanephrogenic tissue was observed in the foi-m of a cap of oval cells arranged radially. These were packed together quite closely, forming a layer about four cells thick, which was, as a general rule, more dense and thick immediately over the ends of the tubules and tapered around all sides of the tubules for a short distance. Peripheral to this cap was another layer of undif ferentiated metanephrogenic tissue, the cells of which were less densely packed, contained fewer granules, and had no characteristic arrangement. These two regions were first described by Schreiner (1902) as the inner and outer zones, respectively, of the metanephrogenic tissue. Beyond this was undifferentiated mesenchyma, which followed the same arrangement as the cap and, by dipping down between the ends of the branches of the collecting tubules, marked out what would eventually be the lobulation of the permanent kidney (Fig. 2) .

In cultures 48 to 144 hours old, the explant had become so transparent that the actual growth of the collecting tubule was easily followed. This growth took place not only by mitotic division of the cells all along the length of the tubule, but also from the ends of the tubule, the latter being the more active of the two and the more effective in increasing the length of the tubule. In the end of the tubule in which growth and branching were about to take place, there occurred a symmetrical bulbous or ampullar formation, due to a general proliferation and change in shape of the cells making up the the ends of the tubule. In areas of active proliferation and rapid growth, there seemed to be a metamorphosis always to the spherical type of cells with a subsequent reversion to the adult type. As a result of this proliferation, there occui'red, coincideutally with the bulbous expansion, bud like projections from the blind end of the tubule. Usually these projections were situated at opposite poles of the tubule ; occasionally there were three or even four of them. Their rate of gi-owth was approximately the same. At first they appeared to be growing out horizontally to the cortical surface of the lobule; they soon altered their course, however, one growing almost perpendicularty and continuing in a general way the peripheralward growth of the tree, while the other or others tended to grow out more horizontally, making up the surrounding arborization. These bud-like projections were at first solid sprouts, but by continued proliferation and reshaping of the spherical cells into a columnar type the bud increased in length, breadth, and thickness and by a rearrangement of the cells a lumen was formed, which in a very short space of time extended almost the entire length of the sprout ; thereafter, the lumen formation progressed handiu-hand with the growth of the tubule. This phenomenon was of a different type from that described by Dr. Sabin (1920) in the formation of the lumen of the blood-vessels, for in the collecting tubule no vacuolization of the cells took place; on the other hand, a small but gradually widening cleft within the sprout, was formed by a rearrangement of the cells and a transformation in shape to that of the columnar type. The new collecting tubule branches continued to grow with a simultaneous formation of a lumen, until a certain length had been attained, when a new branching occurred. The endothelial sheet covering the tubule proliferated and grew simultaneously


with the growth of the branches, and the collecting-tubule tree was thus covered throughout with a shroud of endothelium. The undifferentiated metanephrogenic tissue was plainly visible in the cultures and its growth and development were closely associated with that of the collecting-tubule tree. Felix (1912) described this as forming a circumferential sheet which surrounded the ends of the ingrowing collecting tubules and was pushed peripheralward by the growth of the tubules after each succeeding layer of newly formed convoluted tubules and glomeruli had been formed. This, however, was not found to occur in the chick. Here the undifferentiated metanephi-ogenic tissue surrounded each individual collecting tubule that grew into the lobe; as the tubule branched, this metanephrogenic tissue was split up, some of it being carried forward by the growing branches, the rest remaining behind in the angle.s between the branches. The portion of the metanephrogenic tissue carried forward over the end of the tubule extended to cover the new branches of the tubule, partly by proliferation and partly by a peripheralward migration, as was evidenced by a slight distortion of the cellular mass.

The deposition of the undifferentiated tissue in the angles of the branching tubules was found not only in the cultures and spreads but also in the sections of the metanephros (Figs. 10, 13 and 16), and it was from this tissue that the future convoluted tubules and glomeruli always developed. This constant relation of the undifferentiated metanephrogenic tissue to the ingrowing collecting tubule was especially striking in the cultures. No matter to what extent the collecting tubule may have grown inside the explant or out into the margin, as will be shown later, there was alwa,vs the same relation maintained between it and the undifferentiated metanephrogenic tissue (Figs. 8 and 10). Since the convoluted tubules always developed in the same position with relation to the collecting tubules, and always from the metanephrogenic tissue of the inner zone and never elsewhere in the mass of undifferentiated tissue, it may, I think, be deducted that the metanephrogenic tissue of the inner zone is made up of cells that are predestined to become the anlage of these convoluted tubules. This anlage always appeared as a mass of undifferentiated tissue, quite distinct from the surrounding nephrogenous tissue of the outer zone and the collecting tubule (Figs. 10 and 15). The cells constituting the inner zone, which formed a cap-like covering over the blind end of the ingrowing collecting tubule, were at first quite similar in form. However, within 24 hours, in cultures of the 8-day embryos, morphological changes, and also changes in the general arrangement of the cells, were observed. The cap became sharply defined by three and often four layers of cells which completely ensheathed it (Figs. 15 and 18). The cells making up these layei-s were rather long and narrow, approximating the endothelial cell in shape, and stood out in marked contrast to the adjacent tissue. Between the two outermost layers appeared a space, which likewise encircled the anlage and served to define it still more sharply (Figs. 16, 18, and 24). Simultaneously with the differentiation of this endothelial sheath, there occurred not only a change in the morphology of the cells making up the cap but also a change in the form of the cap itself. The cells proliferated rapidly, becoming more spherical in shape and somewhat less granular. There was also an increase in the size of the individual cells. Thus there was foniied, as it were, a solid central core of spherical cells approximating the epithelial type (Figs. 15, 16, 18, and 19) . The difference in the polarity of these two distinct types of cells was quite striking. In the spherical cells there was a tendency toward a radial arrangement about a central core. The axes of these cells were perpendicular to the axis of the central core, while the axes of the endotheliallike cells about the periphery of the central core were at right angles to the axes of the spherical cells' (Figs. 16, and 18). In twenty-four hours' growth, before any suggestion of tubule formation, there was thus differentiated in situ two distinct types of cells, endothelial and epithelial, from one common mass of undifferentiated tissue. Furthermore, the entire anlage had become surrounded by a space lined with endothelium (Fig. 18).

Concomitantly with the differentiation of these two types of cells, the inner cell mass, composed of spherical epithelial-like cells, was observed to become shaped like an inverted comma (Figs. 19 and 20). (This comma shape was first mentioned by Ribbert in 1899. ) The head of the comma was always to the side of the ingrowing collecting tubule, while the tail covered its blind end (Figs. 16 and 19). By continued growth the comma-shaped mass increased in size and underwent a gradual transition into a solid S-shaped tubule or core. This transition required about 24 hours. At first its differentiation and growth were uniform throughout, that is, the tail end of the comma appeared quite as early and was as far advanced as the head-pole (Fig. 19). However, during the transition the most rapid growth was seen to occur in the head pole of the comma, or what I shall call the distal pole, where the spherical cells became arranged in what appeared to be a solid, sphere-like dilatation (Fig. 21). Immediately above this sphere the growth was almost as rapid, causing an angulation which resulted in the formation of the first curve of the future S-shaped tubule (Fig. 21).

Differentiation beyond this stage was not observed in the metanephrogenic tissue of the inner zone in the 8-day embryos. In the outer zone, however, growth and differentiation did occur. The future connective-tissue septa appeared at the periphery of this outer zone of the metanephrogenic tissue and with their appearance there were laid down the interlobular septa constituting the microscopic lobulation. Throughout the growth of the collecting tubule tree, lobulation was observed to keep pace with the formation of branches of the tree.

Nothing has jet been said concerning the region about the main stems and branching collecting tubules. Concomitant with the growth and development of the lobule, there was constantly left behind, in the path of differentiation and peripheralward growth, a cellular mass of tissue wTiich surrounded the trunk of the collecting-tubule tree for that lobule. This tissue constituted a supporting framework containing the blood capillaries, for the developing excretoi-j' unit. Simultaneously with the differentiation of the excretory unit, and well advanced before the appearance of the convoluted tubules, there was formed progressively a system of sinuses. As stated under the description of the morphology, the process of differentiation and development of endothelial sinuses was first observed in the undifferentiated metanephrogenic tissue between the bases of the primary collecting tubules in the 6-day embryo. The presence of a sinusoidal circulation in the mesonephroa was first mentioned by Minot (1892), but nothing was said about the existence of a sinusoidal circulation in the metanephros or the method of development of these sinuses. In cultures of the 8-day embryo all stages of development of the sinuses could be seen. The most fully developed were near the base of the lobe, while the least developed ones were found about the anlage of the excretoiy unit, toward the periphery. This formation of sinuses followed, so to speak, in the wake of the growing collecting-tubule tree (Figs. 1, 13, 14) and, as in the differentiation and development of all the other elements of the lobe, the progression was always from base to periphery. In the cultures the sinuses were sharply defined from the surrounding tissue and they also were readily seen in fixed sections, although much shrunken and distorted (Fig. 16). About the base of the lobes the sinuses were quite large and filled with a fluid medium, in which blood elements were commonly found (Figs. 5, 13, and 17). The walls of the sinuses consisted of a single layer of endothelial cells, which at times were in apposition to the endothelial layers covering the tubules, and at other times were connected with these layers of endothelium by endothelial sprigs or offshoots (Figs. 12, 16, 17, 24). The larger sinuses connected freely with each other and also with a capillary network of the lobe, so that in an embryo of this age there were two distinct blood-vascular systems, capillary and sinusoidal. Blood-islands were commonly seen attached to the inside of the walls of the sinuses and from these hemoglobin-containing cells, as well as white bloodcells of lymphocyte type, were differentiated. Sinuses also were differentiated in situ from the undifferentiated tissue making up the bulk of the metanephric body. These sinuses had their origin in the vacuolization of cells quite similar to angioblasts. These cells were rather conspicuous, clue to the fact that there were always two and sometimes three in juxta-position, making them appear as deeply pigmented multinucleated giant cells, joined together by very minute protoplasmic processes (Fig. C). In each cell there appeared a vacuole, which constantly enlarged until there remained only a rim, of cytoplasm and an eccentrically placed nucleus. The vacuolization of these cells was complete in 24 hours and fusion of the two or three original cells occurred, resulting in a small space surrounded bj' flattened out cytoplasm and eccentrically placed nuclei. This space increased in size by multiplication of the flattened-out cells and was held patent by a fluid medium. These sinuses were of different shapes and sizes and were connected with each other, as well as with the capillary network, and with the endothelial spaces about the anlage of the convoluted tubule, by means of direct sprouting of the larger sinuses. Throughout the entire life of the culture differentiation of capillaries in situ and direct growth after differentiation by sprouting was observed. Thus, the sinusoidal and the capillary systems develop in situ at the same time from a totally undifferentiated mass of tissue making up the bulk of the metanephric body. The two types of circulation were verj' similar in their differentiation and development.

In old cultures the macrophages appeared to ingest and digest tissue left behind in the wake of the developing lobe (Fig. 1-4) as well as injured tissue. Inside these macrophages red blood-cells and other forms of cellular detritus were observed. The macrophages were much more numerous in the base of the lobe.

Nine- and Ten-day Embryos In the explants of the metanephros, made from 9- and 10-day embryos, one had, of course, a more completely developed excretory unit to begin with than in the explants from 8-day embryos. Notwithstanding the fact that there Avere growth and differentiation in cultures from the latter, these were always much retarded as compared with an embryo of corresponding age. In the explant of the metanephros from 9- and lOday embi^os, the collecting-tubule tree had become much more branched and complex throughoiit the lobe. The main trunk had increased not only in length but also in diameter, and its primary branches had already divided into secondary, tertiary, and quarternary branches. The dichotomous branching of these tubules contiiiued with a lobulation or division of the lobe into smaller lobules by the rearrangement of the mesenchymal septa, corresponding to the branching tubule tree. The sinuses and blood-vascular elements formed and grew just as in cultures from the 8-day embryo, so that the essential difference between the growth of the explants in the two series lay in the differentiation and development of the secreting tubules. All stages, from completely differentiated to entirely undifferentiated metanephrogenic tissue, could be studied in the explants of 9-day embryos, as there was constantly occurring a repetition of the cycle of differentiation and growth of the complete excretory unit towards and in the future cortical portion of the lobules. Between the 9th and 10th day of incubation seemed to be the optimum age for a study of the development of the metanephros. Whether this was due to the fact that metanephrogenic tissue was of a greater vitality than at 7 and 8 days, or whether the undifferentiated cells were more nearly on the verge of differentiation and therefore less affected by the change in enviionment, I cannot say; in any event, within twenty-four hours all stages of differentiation from a comma-.shaped body to an £-shaped tubule could be observed. The main areas of growth during the transition were the sphere like group of cells at the distal pole and, immediately superior to this, the portion which \5'as to form the first knuckle of the future 3-shaped tubule (Fig. 21). At these two points proliferation was most active. The continued growth of the sphere at the distal pole, immediately under the rapidly proliferating first curve, caused a slight flattening of the sphere with a subsequent bulging about the sides and to some extent the ends of the overhanging knuckle of the first-formed cun'e. The growth of the sphere and that of the large knuckle (first curve), being parallel and in the same direction, caused an acute flexion of the solid core immediately posterior to the sphere, thus forming the second curve of the S (Fig. 22). Owing partly to the relatively slow growth of the cells making up this second curve, and partly to the pull of the rapidly growing sphere and first curve, the second curve became very small in diameter and acutely Hexed (Figs. 6, 23). The solid S-shaped mass, therefore, developed in situ, and not, as heretofore thought, from a sphere into an S-shaped tubule. The entire solid core developed from cells already present, and assumed an 2-shape because of more rapid growth in some regions than in others.

The endothelial sheath, together with the sinus formation, progressed hand-in-hand with the development of the S-shaped core, i. e.. the S-shaped core was completely covered by a layer of endothelial cells and in addition to this the entire anlage was completely surrounded and delimited by a space or sinus lined with endothelium (Figs. 12, 16, 24). The core was thus sharply outlineil. A marked difference in the refractivity of the endothelial and epithelial cells further aided in the definition of the tubule so that it was easy to distinguish changes taking place within the tubule from those in the tissue without. The close approximation of the glomerular pole to the under surface of the first curve of the S might give the false impression of a cleft formation, as stated by Ribbert (1899) and Huber (1905) instead of a solid core doubled upon itself (Figs. 21, 22). The potential space between the distal pole and the under surface of the first cuire contained the endothelial layers covering the solid tubule and those fomiiug the wall of the sinus (Figs. 17, 21, 24), so that the bud of an endothelial sac was thus placed between the glomerular end of the tubule and the under surface of the first curve. This slight indentation, however, approximated in no way the cup formation described by other investigators, notably Huber, Schreiner, and Felix.

After the comma-pattern had developed into an 3shaped tubule, there was a continued growth throughout the entire tubule. Proliferation of the cells in the glomerular end occurred more rapidly than elsewhere in the S-shaped tubule until that end became dilated and bulbous, forming a tuft of epithelial cells (Figs. 2, 6, 11, 22). At this stage no capillaries or blood elements were ever observed in this tuft, all the cellular proliferation being inside the endothelial sheath, and therefore within the tubule. The cells causing the dilatation of the glomerular end were at first all more or less round ; however, a, differentiation of these cells took place in situ along with their continued proliferation, resulting in the fonnation of flat, endothelial-like cells, and spherical epithelial cells, thus constituting the pre-glomerular tuft (Figs. 17, 23 ) . The endothelial cells, because of their shape and greater refractivity, could be traced winding about amongst the epithelial cells (Figs. 23, 24). The latter, however, formed the greater part of the cell mass producing the dilatation of the glomemlar end of the tubule. By 72 to 96 hours, collap.sed endothelial spaces could be seen winding through the epithelial tuft and everywhere covered with epithelial cells (Fig. 24). Thus the presence of endothelial cells intimately intermingled with epithelial cells wa.s observed in the bulbous glomerular end while the future secreting tubule was still in a solid state and as yet no connection with any type of circulatory system could be seen. No evidence of a growth from the endothelium covering the exterior of the tubule down into the glomerular end was observed. The cellular proliferation in the glomerular end proceeded rapidly, the dilated bulbous end being completely filled and much distended in comparison -nith the remainder of the tubule. The curve of the tubule immediately proximal to the glomerular end became reduced to a very small tube whose walls were formed by a rather low cuboidal epithelium one cell in thickness, covered by an endothelial sheath. In the loop of the £ immediately proximal to the glomerular end the formation of a lumen was first observed (Fig. 23 1. This followed the same course as in the collecting tubules, i. e., the lumen formed by a rearrangement and separation of the cells and not by vacuolization, as was observed in the vascular system.

The cells abutting against the lumen were the first ones to become cuboidal in shape. The rearrangement of the cells about the lumen progressed distally and proximally with about equal rapidity. The lumen formation began about the inferior half of the spherical tuft of cells, then progressed to the superior half, until the central tuft was completely separated from the walls of the glomerular end of the convoluted tubule, except at one pole, usually distal to the point of transfonuation of the secreting tubule into the glomerular end (Figs. 6, 23, 24).

This lumen formation in the glomerular end was the anlage of the capsule of Bowman, the cells bordering the lumen constituting the layers of the capsule (Fig. 24). The flat epithelial cells formed the parietal layer, while the cuboidal cells made up the visceral layer. Immediately before the fornuition of the lumen the spherical tuft of cells attained its maximum size, decreasing from that time on until the glomerulus began to function, when it again enlarged. With the completion of the lumen formation and the changes in shape of the bordering cells forming the anlage of Bowman's capsule, the appearance of an invagination of the remainder of the spherical tuft was obtained. However, the etched-like outline of endothelial cells enabled one always to separate that which occurred inside the tubule from that which occurred outside. Any invagination or cup formation that involved the entire glomerular end of the secreting tubule would of necessity cause a disturbance of the contour outlined by the ensheathing endothelial covering. If sections of merely the cuboidal cell layer of Bowman's capsule constituting the most distal margins of the glomerular end of the tubule are reconstructed, then one obtains an incorrect impression of a tubular invagination.

A connection was now established between the newly formed space or sinus surrounding, the secreting tubule and the larger sinuses already foi-med. This connection was by means of direct sprouting oft" of endothelial strands from the larger sinuses which became confluent with the sinuses differentiated in situ about the anlage of the secreting tubule. Thus the endothelium-lined space between the superior surface of the glomerular end and the second curve of the 2-shaped tubule was in direct communication with the larger sinuses. A continuation of endothelium was theu established from the endothelial elements within the glomerular epithelial tuft through the sinus about the secreting tubule by way of endothelial sprouts into the larger sinuses about the collecting tubules (Figs. 2, 24). The endothelial sprouts connected the glomerulus with the sinuses always through the attachment of the glomerular tuft of ceHs to the wall of the tubule ; that is, at the point where the capsule of Bowman was reflected, which usually lay distal to the urinary pole. At this point the visceral and parietal layers of the capsule were confluent. In the older cultures it was found that the sinuses became connected with the capillaries that developed in their neighborhood.

Hematopoiesis occurred in the spherical tuft of the glomerular end with the formation of red blood-cells, plasma, and white blood-cells having the appearance of


IvDiphocytes (Figs. 17, 23, 24). The forruatiou of the blood-elements was usually first noted in that portion of the tuft adjoining the secreting tubule and progressed from this point throughout the glomerular portion. It occurred at about the same time the connection with the sinuses took place. It must be borne in mind, however, that in the cultures no circulation was present. At no other point in the entire excretory tubule did hematopoiesis occur, a fact which also indicates the endothelial nature of the cells inside the spherical tuft Lumen formation by thinning out of the cells, characteristic of the blood-vascular .system elsewhere, was found to obtain in the glomerular tufts. The blood elements made the definition of the endothelial channels much more pronounced and the latter could be traced with ease, winding among the epithelial cells and covered by a single layer of these cells.

After the differentiation, the convoluted tubule con tinned to grow in exactly the same manner as the collecting tubule, that is, by proliferation of the cells along the entire tubule or at the end nearest the collecting tubule. The convoluted tubule grew always toward the collecting tubule, until finally the proximal end of the former abutted against the side wall of the latter. This contact usually, though not always, was in the immediate region of a newly foi-med branch of the collecting tubule. The anastomosis of the convoluted and collecting tubules could be observed in cultures of the 9-day embryos after 24 hours' growth. In general, it required about that length of time to effect complete continuity of the convoluted and collecting tubule lumina. The continued growth of the secreting tubule against the wall of the collecting tubule exerted sufficient pressure to indent the latter and cause a distortion of its constituent cells (Fig. 23). The secreting tubule grew into the collecting tubule at an angle of about forty-five degrees. The cells in the wall of the collecting tubule formed a sort of arciform arrangement about the ingrowing blind end of the convoluted tubule. There was a rapid proliferation of the cells constituting the latter and the cells reverted to a spherical type, as was observed in the growth of the col lecting tubule. As a result of this proliferation, the end of the convoluted tubule protruded like a wedge through the wall of the collecting tubule into the lumen. This was not due merely to pressure from the outside against the wall of the collecting tube; there was an actual growth of the convoluted tube inside the wall of the collecting tube, the cells dividing and making room for the increased number. An interruption in the continuity of the wall of the collecting tubule was thus effected (Fig. 24).

Lumen formation in the secreting tubule followed upon the proliferation of the proximal or blind end, so that about two hours after the secreting tubule had grown through the wall of the collecting tubule the lumina of


the two had become continuous (Fig. 24). This continuity was proved by the coursing of fluid containing granules back and forth from one tubule into the other. After the anastomosis of the convoluted and collecting tubules, there was continued growth of the former throughout its entire length, as evidenced by mitotic figures. The greatest growth activity, however, occurred in the convolutefl portion next to the collecting tubule, which in the adult kidney would correspond to the distal pars convoluta.

Developjient in Marginal Outgrowths The outgrowths from explants of the metanephros (of embryos of 8 to 10 days inclusive) were made up chiefly of four types of tissue — mesenchyme, renal epithelium, endothelium, and nervous tissue. The marginal growth began immediately after implantation. In from two to four hours after explanting, proliferation of mesenchymal cells about the margin and growth of nerve-fibers into the marginal zone could be made out. Growth of the four elements of the marginal zone increased steadily throughout the life of the culture up to 5 days. In the beginning it consisted of only a thin sheet about one cell in thickness. The most advanced edge of the outgrowing margin always remained quite thin and attenuated, but as the growth increased in width, so did it increase in depth. The different elements composing the marginal outgrowth proliferated and grew with different degrees of rapidity, but there was little difference ultimately in the extent to which they grew.

Usually the first tissue to grow was the mesenchyme; it was also the most hardy and prolific of all the tissues (Fig. 5). In appearance, these cells were typical fibroblasts connected with one another by long branching proces.ses, which were direct extensions of the cytoplasm (Fig. 7). At first there was no definite arrangement of the mesenchyme cells; they simply grew out in a flat spreading sheet with a centripetal tendency (Figs. 4, 14). However, after 48 hours, when the g:i'o\\i:h near the explant had become thicker in different areas, definite patterns and arrangements of cells could be seen, although tlie attempt of the cells to assume their normal formation was often abortive and resulted in bizarre disturbances of organization (Fig. 14). This interference with the tissue in its effort to acquire its predestinetl form was due, no doubt, to surface tension and adhesion to the coverslip.

The renal epithelium grew out readily, but like the mesenchyme, in different forms in the various cultures. Proliferation and growth began immediately at the margin. Arising from the excretory unit (the collecting tubules, convoluted tubules, glomeruli, and undifferentiated nephrogenous tissue) growth proceeded in three fundamentally different ways: (1) with no particular formation, as in a flat sheet (Fig. 5) ; (2) as a perfectly organized element, carrying with it undifferentiated tissue, which later differentiated in the marginal outgrowth (Figs. 8, 9) ; (3) out into the margin where an abortive attempt at formation or organization was made (Figs. 3, 4, 14). Wherever there was an end of a collecting tubule or convoluted tubule near or immediately at the margin of the culture, there was always an outgrowth of renal tissue, which appeared quite as early and grew as rapidly as the mesenchymal tissue. When growing out hand-in-hand, as it were, with other tissue of the margin, the renal tissue always appeared as a flat sheet of cubical cells closely associated with one another. This sheet was usually one cell thick and often bounded on all sides, except towards the periphery of the growth, by outgrowing mesenchjnual and endothelial cells. In these flat outgrowths there was never any attempt at organization or difl^erentiation ; the outgro^rth consisted simply of a multiplication of already differentiated cells.

In the event that a well-advanced marginal growth had preceded the outgrowth of renal tissue by a few hours so that there was some depth of cells into which the renal tissue could grow, then the outgrowth and differentiation occurred exactly as they did inside the explant. In other words, the collecting tubule grew into the marginal zone as a tubule intact, just as the collecting tubule grew inside the explant, carrying with it undifferentiated nephrogenic tissue which went on to complete differentiation in the margin (Figs. 8, 9, 11). The excretory tubule maintained the same relation to the collecting tubule, with regard to position and growth, that it maintained inside the explant: when it differentiated, it always did so in situ, i. €., in the region or at the site of the angle formed by the branching of the collecting tubule, afterward becoming S-shaped by direct growth of the already differentiated cells. There was never a long, straight, direct outgrowth of the convoluted tubule such as there was in the collecting tubule.

When the marginal gi'owth of cells was not thick, the attempts at organization were abortive, although differentiation occurred. In all marginal growths there was a certain point beyond which the entire margin grew out flat and "went wild," that is, all formation or organization was lost and the individual elements broadened out into a flat sheet. This transition could be followed in most of the cultures that sunived long enough.

The third element in the marginal growth was the endothelium. As was the case with the renal tissue, the endothelium grew out in different ways. In the first place, there was a sheet-like growth, with greater intervals between the individual cells, however, than occurred in the mesenchyme or renal tissue (Fig. 3). In fixed preparations the endothelial cells were fibrillated and connected by long branching processes. No formation of blood-vascular elements was seen in this type of outgrowth. On the other hand, capillaries grew out in arciform loops from other capillaries already formed in the explant. The growth of the.se could be watched in detail.


The development in situ of sinuses, blood-islands and capillaries occurred in the marginal outgrowth wherever the necessary undifferentiated tissue had been carried out from the explant. Among the tissue of the explant and marginal outgrowth, at all ages, long nerve-fibers, also ganglia, were sometimes observed. In the marginal growths, therefore, there occurred differentiation and organization of the renal parenchyma, as well as of the other basic tissue elements making up the renal body.

Differentiation, followed by a marked distortion due to the attempt of the cells to assume their usual organized positions, afforded an excellent opportunity for study. One could find places in which epithelial cells had differentiated in small flattened circular areas. Among these were highly refractive endothelial cells, intimately associated with the epithelial cells, being interwoven among them in exactly the manner noted in the development of the glomerulus. As this association of endothelium and epithelium was not observed anywhere else in the marginal growth, although there were many outgrowths of renal epithelial cells, the logical inference is that these small translucent circular areas were probably made up of cells destined to develop into the glomerulus.

Discussion

Most observers agree on the growth and division of the collecting-tubule tree and the coincident splitting up of the nephrogenous ti.ssue, thus affording a cap or covering for each branch of the collecting tubules. As far as I have been able to find, however, none of the previous observers have described the endothelial covering of these tubules, although several figures given by Herring show it.

In regard to the nephrogenic tissue, all observers agree that an S-shaped tubule is formed from a renal vesicle situated in the angle made by the collecting tubules. Ribbert (1899), was the first to call attention to the fact that a comma-shaped body preceded the formation of the tubule. Huber (1905, p. 17, Fig. 2) presents a drawing of a .section of a human embryo, showing a collecting tubule, on each side of which a comma-shaped body is clearly defined. In the inferior pole of each of these bodies can be seen a renal vesicle, which Huber states differentiates out in situ. He does not specifically refer to the main body or the tail of the comma except to say that the renal vesicle becomes separated from the nephrogenous tissue and becomes bordered by columnar cells. The S-shaped tubule is then formed by constant growth and elongation of the renal vesicle produced by active proliferation and mitotic division of the cells. In the figure referred to above the arrangement of the cells will be found to coincide exactly with the description of j my observations, the cells making up the tail and body of the comma being arranged with their axes perpendicular to the axis of the central core. Huber mentions only the sphere of cells at the lower pole and disregards

the remaintler of the comma. He, as well as all other previous observers, admits that the renal vesicle develops in situ, but though they show in all their figures that the remainder of the comma also undergoes a definite rearrangement of cells and difi'erentiation, still nothing but the spherical end of the nephrogenous tissue has been heretofore considered. In my observations the entire comma-shaped mass was seen to differentiate in situ from the undifferentiated cap of metanephric tissue and to form the S-shaped tubule.

In the formation of the convoluted tubule, from one type of tissue there are differentiated, therefore, cells of very diverse biological activity. . These inherent differences are evidenced in the embiyo by a difference in the growtli rapidity in the several regions of the tubule anlage, and foreshadow the different functions of these respective areas in the adult.

The modus operandi of the union between the secretory and collecting tubules has never been fully described, although Schreiner hypothesizes that it occurs in given regions of the collecting tubule which are recognizable by the presence of mitotic figures. This theory is not surprising, in view of the fact that previous observations have been made on sections. In the study of the living tissue, it was possible actually tO' watch the uniting of the convoluted and collecting tubule, and it was seen that the convoluted tubule definitely grows into the collecting tubule.

Another very interesting fact, brought out in the cultures, was that, no matter to what extent undifferentiated tissue was displaced from the explant, it assumed within certain limits its predestined shape and activity. Nephrogenic tissue developed into secreting tubules and alwaj's became convoluted, while the reverse was true of the collecting tubules, which alwaj's grew in a comparatively straight line, regardless of the absence of pressure from surrounding structures or other physical factors that occur nornuilly in the embryo. This, it would seem, shows that this tissue has been given a stimulus to assume a cei-tain form and activity which will be accomj)lished, unless the tissue reverts, by a state of extreme activity of growth, to a spherical type of cell. This was shown in the development in the exi^lauts and also in the growth comprising the margin.

Bowman's capsule is formed from the end of the secreting tubule, which previous investigators claim invaginates to receive the coincident ingrowth of capillaries to form the glomerulus. While it might be possible to arrive at such a conclusion from an examination of sections, the study of the living tissues, either in spi-eads or tissue-cultures, shows that it is not the correct one. In the first place, the endothelium that differentiates in situ around the convoluted tubule, coincidentally with the growth and differentiation of the latter shows, from the earliest appearance of the bulbous enlargement of the glomerular end, that the wall adjacent to the overhanging first curve of the S-shaped tubule, remains intact throughout the development of the glomerulus. In the second place, in the earliest stages the dilated bulbous end of the glomerulus contains no lumen, but consists of a solid mass of spherical cells, which in appearance resemble epithelial cells. By a rearrangement of these cells there is a separation which results in the formation of a lumen within this cell mass, which leaves the epithelial cells on one side and the mass of epithelial and endothelial cells on the other.

The epithelial cells forming the peripheral wall of the tubule, however, become quite flat and very much elongated, approxinuiting somewhat the endothelial type of cells and thus may have been mistaken for endothelial cells by other observers. According to previous writers the spherical end of the convoluted tubule increases steadily in size until the invaginating cup reaches its maximum, whereas in my observations the spherical tuft reached its maximum size before the completion of the lumen. Immediately upon completion of the lumen, the visceral and parietal layers of the anlage of Bowman's capsule are formed in situ within the tubule fi'om undifferentiated spherical cells. In fixed sections, these layers of Bowman's capsule show up very clearly and it is easily seen how other investigators, considering these walls to be the boundaries of the tubule, would obtain the impression of an invaginating cup. In the fixed sections, however, interrupted stages only can be observed, whereas, in the living, the entire process of lumen development can be followed as a whole.

Concomitant with the formation of the lumen and differentiation of the cells adjacent thereto, there is difl"erentiation of the cells within the spherical tuft, resulting in the development of endothelial cells in the most intimate association with the epithelial cells. These endothelial cells later form channel-like tracts which are covered by a single layer of epithelial cells. Hematopoiesis in the spherical tuft occurred in the cultures before any demonstrable connection with the sinuses coubl be seen, and it must be borne in mind that there was no circulation of blood elements whatever within the explant. At a later stage the connection of endothelial channels with the surrounding sinus was always observed to take place through the small neck of tissue connecting the spherical tuft of cells with the wall of the tubule, a communication with the ingi-owing branches from the aorta being established still later.

The idea that endothelium and blood develop in situ was first suggested as a speculation by Herring. To him it seemed possible that tlie capillaries might develop in situ and not as ingrowths from the dorsal aorta during the formation of the glomerulus. Schreiner and Huber, on the other hand, claimed that the glomerular tuft is formed entirely by an ingrowth of pre-existing capillaries.



The later work of Sabin in blood-vessel formation, in which she states that not only the endothelium, but also the blood of the early vascular system arises in situ, suggests the possibility that, in other regions where primitive tissue exists, the vascular arrangement may also develop in situ. Certainly the observations on the development of the glomerulus in tissue cultures, where the mass of primitive mesenchyme from which this organ develops is entirely isolated from any circulation, show that not only epithelial cells, but also endothelial cells and bloodcells, differentiate in situ from the mass of undifferen tiated spherical cells within the glomerular end of the convoluted tubule which goes to form the glomerulus. The intimate relation of the glomerular capillaries and the epithelial covering is thus explained, for it is quite difficult to figtire out how this epithelial covering of eacli capillary could be acquired by the mere ingrowth of the capillaries into an epithelial sac. In addition to this, the sinuses alsoi develop in- situ, as could be seen not only from a study of spreads and tissue cultures but also in fixed sections. This development of the blood spaces in situ might have been surn^ised by Colberg (1863) from injections. He injected the blood-vessels of the kidney and found that some of the glomeruli were injected while others were not. The latter he called pseudo-glomeriili. These were undoubtedly glomeruli at an early stage of development before connection with the peripheral circulation was established. The same opinion was expressed by Herring in 1!)00. In 1911 Jeidell studied three injected pig embryos and found that a rich capillary network was formed in the mesencliyme surrounding the renal anlage from the inferior mesenteric and middle sacral arteries, the drainage occurring into the post-cardinal vein, in ferior mesenteric veins, and capillaries of the Wolffian body. None of these injections, however, so far as I can make out from her paper and figures, penetrated farther than the primitive capsule. She concludes that the renal anlage is supplied by this means but does not state how the blood enters from the surrounding mesenchyme. In a study of human embryos of different ages, Kelly and Burnam (1911) claimed from injections that the development of the permanent blood supply of the kidney occurs when that organ reaches its definitive position and that, previous to this time, the circulation is maintained by a communication of the capillaries of the kidney with capillaries of the surrounding mesenchyme. Thus, in the ascent of the kidney, the capillaries which are left behind atrophy, while new ones fonn about the superior pole. At the end of seven weeks a connection with the aorta is established. Before this, no injection of tlie kidney could be made. Somewhat the same phenomenon occurred in the chick. Previous to complete development of the secreting tubule, I was unable to reach the sinuses or glomeruli of the metanephros by way of the peripheral circulation by injections of India ink into the heart, whereas the liver and mesonephros became so heavily injected as to appear entirely black. I think it can be concluded, therefore, that in the incompletely developed metanei>hros there is no continuity of or direct connection with the peripheral circulation ; that there is a sinusoidal circulation, which by continued growth and constant sprouting is diminished and transformed into a capillary circulation, has been shown above. The presence of blood elements and a fluid medium inside these sinuses insures a vehicle to carry oxygen or nutritives to the developing renal body. The nourishment of the kidney in its early stages of development is therefore somewhat analogous to the nourishment of a foetus by way of the placenta.

Sabin (1920), from studies of the blood-vascular system of living chick embryos up to four days, concludes that the various white blood-cells have different origins. The lymphocytes and monocytes arise from the endothelium lining the capillaries, while the granulocytes arise from the mesenchyme cells and migrate into the lumen of the blood-vessels. A particular study was not made of the exact origin of each type of blood-cell, but it was seen that not only the capillaries, but also the sinuses, often were filled with Ijniphocytes which had arisen from the endothelial walls, and in many instances, granulocytes were observed scattered among the mesenchyme cells, as though they had differentiated in that region.

Conclusions

1. In the above study of the development and growth of the metanephros of the chick embrj-o it is found that a continuous sheet of endothelial cells entirely surrounds the collecting and convoluted tubules.

2. The convoluted tubules differentiate in situ from a comma-shaped mass of undifferentiated nephrogenic tissue. The S-shaped tubule is developed out of the commashaped mass by different degrees of growth in different areas of the mass, and not by elongation of a renal vesicle.

3. The glomerulus, including its capillaries and blood elements, differentiates in. situ from an undifferentiated cellular mass which completely fills the distal end of the convoluted tubule. There is no cup formation with subsequent ingrowth of capillaries.

•1. The blood system surrounding the metanephric tubules is at first principally sinusoidal, as in the pronephros and mesonephros, being later almost entirely replaced by a capillary system. These sinuses are not at first in direct continuity with the capillaries of the peripheral circulation, but circulation takes place by diffusion between them and the capillaries.

5. The convoluted tubules grow directly Into the intact collecting tubules.

6. In tissue cultures of the metanephros there does not occur a dedifferentiation of tissue but an active growth and differentiation takes place not only in the explant but also, within certain limits, in the marginal outgrowth.



BIBLIOGRAPHY

Atterbury, R. R., 1920. Potentialities of the different parts in the l^idney anlage. Anat. Rec, vol. 18-19, p. 219.

Carrel and Burrows, 1910. Cultivation of adult tissues and organs outside the body. Jour. Amer. Med. Assn., vol. 5.

Champy, C, 191314. Quelques resultats de la methode de culture des tissues; generalit^s. (Note pr^liminaire.) Arch, de zool. exper. et gen., t. 3, pp. 42-51.

Idem, 1914. Quelques resultats de la methode des cultures de tissus; le rein. Arch, de zool. exper. 'et gen., t. 4, pp. 307-38G.

Colberg, A., 1863. Zur Anatomic der Niere. Centralbl. med. Wissensch., Bd. 1.

Disse, J., 1902. Harn und Geschlectsorgane. Von Bardelebens Handbuch der Anatomie des Menschen, Bd. 7, Jena.

Felix, W., 1912. The development of the urinogenital organs. Human Embryology, Keibel and Mall, vol. 2.

Herring, P. T., 1900. The development of the Malpighian bodies of the kidney and its relation to pathological changes which occur in them. J. Path, and Bact., vol. (J.

Huber, C. G., 1905. On the development and shape of urlniferous tubules of certain of the higher mammals. Amer. J. Anat., vol. 4.

Jeidell, H., 1911. A note on the source and the character of the early blood vessels of the kidney. Anat. Rec, vol. 5.

Kelly, H. A., and C. Burnani, 1914. Diseases of Kidney Ureters and Bladder: Development of the renal vascularization.

Lillie, F. R., 1919. The Development of the Chick.

Minot, C. S., 1910. A Laboratory Text-book of Embryology.

Minoura, T., 1921. A study of testis and ovary grafts on the hen's egg and their effects on the embryo. J. Exper. Zool., vol. 33.

Ribbert, H., 1899. Ueber die Bntwicklung der bleibenden Niere und uber die Enstehung der Cystenniere. Verhandl. der deutsch. path. Gesellsch., p. 18.

Sabin, P. R., 1920. Studies on the origin of the blood-vessels and of the red blood-corpuscles as seen in the living blastoderm of chicks during the second day of incubation. Contributions to Embryology, vol. 9; Carnegie Inst. Wash., Pub. 272.

Schreiner, K. E., 1902. Ueber die Entwicklung der Amniotenniere. Zeitschr. f. wiss. Zool., Bd. 71.

Stoerk, O., 1904. Beitrag zur Kenntnis des Aufbaus der menschlichen Niere. Anat. Hefte, Bd. 1, 23.

EXPLANATION OF FIGURES Plate 1.

Fig. 1. — 16 mm. lens. Seven days' growth of metanephros of 8-day chick embryo. Extensive growth of the collecting-tubule tree not only within the explant but also out into the marginal growth. The collecting tubules have grown into the mairgin as intact tubules, branching in the characteristic dichotomous manner, the line of growth being a relatively straight one. In this figure the branches are all intralobar, the main stem to the left of the figure being a quarternary branch. The large clear areas between the basic branches are the sinuses; smaller ones are seen toward the periphery. Throughout the culture there are several displaced collections of undifferentiated nephrogenous tissue. The margin of the growth appears in the lower right hand corner of the figure.

Fig. 2. — 16 mm. lens. Seven days' growth of the metamephros of a 10-day chick embryo. Entire excretory unit completely differentiated inside the explant. Union between the convoluted and collecting tubules has occurred in most instances. The main stem of the collecting tubule here is made up of two tertiary tubules. In the upper portion of the figure there are some convoluted tubules which have grown more nearly in a straight line than usual. All of the convolutions show typicad dilation of


the glomerular ends. The glomeruli here are quite small. Numerous macrophages can be seen throughout the culture clearing up the remaining undifferentiated tissue by phagocytosis.

Fig. 3. — 16 mm. lens. Seven days' growth of metanephros of 8-day chick embryo. Marginal outgrowth of collecting tubules on the right. The tubules grew out intact within the thicker region but became flattened out into a sheet-like growth in the more attenuated portion of the margin. At different points in the marginal outgrowth can be seen endothelium composed of cells larger and flatter than the mesenchyme cells, which make up the greater part of the outgrowth. Owing to the heavy staining, the structures inside the explant cannot be made out. Formed sinuses in the explant at the left are well shown about the base of the collecting-tubule outgrowth.

Plate 2.

Fig. 4. — 16 mm. lens. Six days' growth of metanephros of 8-day embryo. Extensive outgrowth of formed collecting tubule into margin, flattening in the attenuated edge of the culture.

Fig. 5. — 16 mm. lens. Seven days' growth of metanephros of 8-day embryo. Extensive outgrowth of renal epithelium from collecting tubule in the explant. There is a lumen in this for a short distance in the marginal growth. The endothelium can be seen a/bout the tubule and is quite characteristic in this mode of outgrowth. This endothelial growth origmates from the endothelial covering about the tubules and presents the same type of outgrowth that occurs from endothelium elsewhere.

Fig. 6. — 4 mm. lens. Four days' growth of metanephros of 9day chick embryo. Iodine vapor. Comparatively straight collecting tubule into which the convoluted tubule has grown. The point of union is shown in the lower left hand corner of the figure, where the convoluted tubule appears to be constricted. The tubules are sharply defined by the characteristic etchedlike endothelium. The lumen formation in the glomerulus has been completed and the capsule of Bowman is well shown. The tuft of epithelial-like cells, among which endothelial are intermingled, shows as yet no evidence of vascularization. This figure demonstrates clearly the ease with which the terminal walls of the convoluted tubule cam be seen and also how these intratubular changes can be distinguished from those taking place outside the tubule. About the tubules many white blood-cells and phagocytes are evident. Vacuolization of cells, exactly as occurred in the formation of the sinuses, is well shown in the lower right quadrant of the figure.

Fig. 7. — Seven days' growth of metanephros of 8-day chick embryo, illustrating the same points as figiire 5.

Plate 3.

Pig. 8. — Five days' growth of metanephros of 9-day chick embryo; 16 mm. lens. Collecting tubules have grown straight out into the marginal growth, carrying with them the undifferentiated nephrogenic tissue which went on to complete differentiation in their final position. They have grown out as intact tubules. The characteristic type of growth for the collecting and convoluted tubules is well shown, the former growing in a straight line and the latter being coiled. At the uppermost point in the figure there is a very small glomerulus in which red bloodcells can be seen. Union of the convoluted and collecting tubules has occurred.

Fig. 9. — Higher power picture of Figure 8; 4 mm. lens, showing extreme upper pole of collecting tubule, convoluted tubule, and glomerulus. The characteristic convolutions of the secreting tubule are well shown, despite the fact that the growth is in the margin where the physical factors attending it were entirely different from those obtaining in the embryo. The glomerulus appears as a small sphere in which red blood-cells are seen as black dots. The defining endothelial cells surrounding the tubules show plainly.

Fig. 10. — 4 mm. lens. Three days' growth of metanephros of 9-day chick embryo. Small explant containing collecting tubule, which presents a very interesting growth. The forward or normal growth is accompanied by the usual dichotomous type of branching, while the posterior growth is in a straight line, with no tendency to branch. On either side of the collecting tubule, in the angles, there are twoS-shaped tubules which differentiated in their characteristic position as regards the collecting tubule. These S-shaped tubules differentiated in the culture. The right side is at a more advanced stage than the left side. Certainly the tendency of the convoluted tubules to be coiled in this culture may be attributed to an inherent tendency of the cells. The collecting tubule, for the same reason, has grown in a straight line.

Fig. 11. — 4 mm. lens. Four days' growth of metanephros of 9-day chick embryo. Immediately at the beginning of the marginal outgrowth there are two convoluted tubules with their glomeruli in the course of development. The one to the right side is not as far advanced as the one to the left. Lumen formation has occurred in the tubule to the left. The tubule to the right is a solid core of cells with a dilated bulbous end in which the formation of a lumen has not as yet occurred. Tbe characteristic pinched-off appearance of the neck of the glomerulus, which results from the relatively slow growth of the cells composing this region, is shown.

Figs. 12 and 13. — Five days' growth of metanephros of 9-day chick embryo. Sinus formation well shown about the convoluted tubules. The white blood-cells show up as dark spots, more pronounced in Figure 12.

Fig. 14. — Same as Figures 12 and 13, except that the sinuses are located more in the denser region about the convoluted tubules.

Plate 4.

Fig. 15. — Metanephros, 8-day embryo. Section 5 micra. x 750. Collecting tubule formation with inverted comma-shaped mass of nephrogenic tissue about end. The cells making up the body and tail of comma are spherical in type with their axes perpendicular to the central core. In the head of the comma there is marked proliferation evidenced by mitotic figures. About the periphery of the comma-shaped mass the endothelial cells can be seen with their axes parallel to the central core. These endothelial cells are just in a stage of transformation from the round spherical type to the long slender endothelial type. The head of the comma Is turned somewhat towards the observer and at the inner angle about the middle of the mass (at X) is an endothelium-lined space which approximates the sphere and, when several sections are studied, proves to communicate with an endothelium-lined sinus, which is somewhat poorly showoi in this figure, just beneath the head of the comma. This is the portion of the sinus surrounding the comma^shaped mass that will later be included in the first curve of the S-shaped tubule. TTils figure shows the comma just prior to the development of the lumen and demonstrates very clearly that the tail and body of the comma, as well as the head, are utilized in the formation of the 2-shaped tubule.

Fig. 16. — Metanephros, 9-day embryo. Section 5 micra. x 750. A "Y"-shaped collecting tubule with two comma-shaped bodies along its inner sides and about its ends. Between the commas there is an endothelium-lined sinus which surrounds the entire anlage. The endothelial walls of the sinus are in opposition along the lateral side of each of the collecting tubules, but the connection with the large sinus at the lower portion of the


figure can be traced even in the photograph. The tubule is here shown in a later stage of development than in Fig. 1. The endothelial covering is evident, as well as the endothelium lining the sinus, the two layers being in apposition. Lumen formation has just begun and is somewhat more advanced on the right side than on the left. The characteristic polarity of the endothelial and epithelial cells, as well as the more rapid proliferation of the spherical head pole of the tubule, is well shown.

Fig. 17. — Metanephros, 9-day embryo. Section 5 micra. x 1500. Cellular tuft in glomerular end of convoluted tubule shown immediately below a loop of the tubule. The lower loop of tubule belongs to another unit. The capsule of Bowman is partially formed about the spherical tuft of cells and the tuft is bounded by a row of radially arranged epithelial-like cells. The flattened epithelial cells making up the parietal layer of Bowman's capsule are evident. The point of junction of the glomerular end with the convoluted tubule occurs just to the right in the figure. A double line of flattened endothelial cells running across the superior surface of the glomerular tuft is evident. In the distal end of the tuft a black spot is seen which is a newly formed red blood-cell. On each side of this cell there are endothelial cells which form a channel in the middle of the epithelial cells making up the major portion of the tuft. This is better shown in Plate 5. A mitotic figure can be seen towards the proximal end of the tuft. The clear areas in the figure, in which red blood-cells appear as black spots, are the sinuses in the immediate neighborhood of the glomerulus.

Plate 5.

Fig. 18. — Collecting tubule covered by cap of metanephrogenlc tissue. The collecting tubule contains granules and is lined with columnar epithelium. Three layers of endothelium can be seen about the cap and one layer surrounding the collecting tubule. The spherical epithelial cells composing the cap are radially arranged about a central core corresponding to the single line shown. Blood cells are shown in the sinus existing between the two outermost endothelial layers.

Fig. 19. — Collecting tubule buds increased in length. The metanephrogenlc cap has become transformed into an inverted comma-shaped mass. The cells making up the tail of the comma show the same arrangement as in Fig. 18. The head of the comma is the seat of most marked proliferation causing a dilatation of the head pole.

Fig. 20. — Collecting tubule only partly shown. Growth of bud extending peripheralward carrying small portion of metanephrogenlc tissue. The comma-shajped mass has approximated its definitive position in regard to the collecting tubule. At points 15 and IG the most rapid proliferation of cells takes place.

Fig. 21. — Transition of comma-shaped mass into that of an ©shaped tubule has occurred, angulation having been brought about by proliferation and more rapid gi-owth of cells at points 17 and 19, point 17 being the first curve of the ©-shaped tubule formed. A sac-like protrusion of the sinus (No. 11 in Fig. 24) has been included between the superior surface of the bulbous glomerular end (19) and the inferior surface of the first curve (17). Blood cells are shown in this sac. Metanephrogenlc cap shown at 14.

Fig. 22. — Collecting tubule and metanephrogenlc cap remain about the same as in Fig. 21. ©-shaped tubule more angulated. Second curve No. 18. First curve increased in size. Glomerular end a dilated bulb of spherical epithelial cells.

Fig. 23. — ©-shaped tubule growing in the collecting tubule at point 13. Lumen formation occurring in second curve with abutting cells becoming cuboidal in shape. Heavy black lines in tuft of epithelial cells composing glomerular end are the endothelial cells. No. 8 in Fig. 24.


Fig. 24. — S-shaped tubule completely joined with collecting tubule. Lumen complete with the formation of Bowman's capsule (5). Note that this formation is entirely inside the endothelial covering layers 1. 2 and 3, 4 constituting the parietal layer of Bowman's capsule and 6 the visceral; 7, spherical epithelial cells composing the tuft surrounded by Bowman's capsule. Intertwining endothelial cells and channels (8) among the epithelial


cells. Blood elements are also shown forming in situ. The connection of the endothelium inside the tuft with the surrounding sinus is shown at 10, and the included endothelial sac or sinus at 11; 9, a connection between the surrounding endothelial sinus and sinuses in other parts of the metanephros. The lumen of the collecting tubule is shown at 12.

NOTES ON NEW BOOKS

A Textbook of Oynwcological Surgery. By Berkjxet and Bonnet. 829 pp. 2nd edition. Cloth, ?11.00 (N. Y., Paul B. Eoeber, 1920.)

It is a pleasure to turn over the pages of a book that is as well prepared as this one. The subject matter is for the most part presented in an up-to-date and clean-cut manner. The illustrations, although not elaborate, on the whole are clear and supplement the text well. To a large extent one of the commonest faults of recent gynecological text-books has been avoided — the tendency to distort the sense of balance and proportion by laying undue stress on minor pet procedures in which the authors are particularly interested. The sections on post-operative complications and care and on ultimate results add materially to the value of the work.

As the authors state, this book presents the opinions and practices of the school of gynecology to which they belong. In general, we feel that these views are sound and trustworthy, although quite naturally ours differ from theirs in some particulars. Thus, in our opinion they have underestimated the advantages that may at times be gained by draining the pelvis through the cul-de-sac of Douglas rather than through the abdomen, and they have also failed to appreciate the value of posterior colpotomy as a diagnostic or therapeutic procedure in selected cases. We would criticize their treatment of perineal plastic surgery as being incomplete. It seems unusual that more space should be devoted to the directions for dilating


the cervix (fourteen pages) than to the treatment of vesicovaginal fistula (twelve pages). Little or no attention is paid to the subvesical fascia in the treatment of cystocele, vesicovaginal fistula or prolapsus; this may be the cause of some of the failures that they seem to have had. The treatment of complete laceration of the perineum is too cursory to be sufficient; not enough attention is paid to the rectal sphincter or the obtaining of continence, which is usually the only indication for performing the operation. In the treatment of carcinoma of cervix, the authors are far more radical than are most gynecologists of the present day: it is an open question whether the ultra-radical operation involving the wide dissection of the pelvic and iliac glands, the parametrium and vagina is worth while. Their method of taking care of the vagina both before and after perineal operations is rather elaborate; in our experience perineal infections have been much less common since we have simplified our technique. We would question the wisdom of devoting space to operative procedures that are definitely out of date, such as Le Fort's operation for uterine prolapse or the making of a common cloaca out of the vagina and bladder in some cases of vesico-vaginal fistula. Their indications for performing hysterotomy and utriculoplasty also seem questionable. In their treatment of carcinoma of the rectum, they recommend that the combined abdominal and perineal operation be performed in one stage; in some cases, at any rate, it is undoubtedly safer to carry this out in two stages.

From what we have said, we would not wish the reader to get the idea that this is not a good book. On the contrary, it strikes us as being one of the best recent text-books on operative gynecology. With the above reservations, we can recommend it both to the expert gynecologist and to the beginner, for both will find in its pages much that is interesting and instructive.

L. R. W.

The Diseases of Children. By Sik James F. Goodhart and Georce Frederick Still. 11th Edition. Cloth, $10. (New York, Paul B. Hoeber, 1921.)

This is the sort of book which is and was meant to be a text-book of pediatrics for students of medicine and should be judged as such. How well it has served its purpose in the past is indicated by the fact that this is the eleventh edition since it first made its appearance in 1885. In this last revision of the book Dr. Still has studied to retain as much of the text left by Sir James Goodhart as possible and still to bring it abreast of the present day.

The general consideration of the diet and hygiene of the healthy child is very well done and the author offers a reasonably simple, if somiewhat dogmatic, system for the modification of milk for the hand-fed infant. The book is untouched by post-war fads which have grown up out of plausible empiricism masquerading as science. Their omission may stamp the book as obsolete to the beginner in whose novitiate they had their rise and before whose heyday they will pass into the limbo of the forgotten. After all, the question which matters in feeding a baby is not what may be given but what is the safest food for the average child. To teach this is all that can be attempted in a text-book. Experience only can teach a student the art of dealing with the idiosyncrasy of the individual child so as to secure optimal results. Still's feeding is based on the percentage composition of milk, and his formulae are made up with added cream. An infant fed according to this book would receive a good bit more fat than is usually allowed by American pediatrists nowadays and the interval between the feedings is shorter than that which has found favor in this country. Nevertheless, the book offers the student a tolerable ground on which to base further study.

The pathology, symptomatology and diagnosis of the morbid conditions of childhood have been thoroughly and carefully set forth. Considera,ble less emphasis has been placed by the author on laboratory diagnosis than is found in most American books on medicine. As regards etiology, the book is not equally good throughout. For example, the author has gone into the etiology of poliomyelitis with some care and has reported it at sufficient length. On the other hand, if the text may be used as an index, he has entirely ignored the recent work which has been done to reveal the factors which underlie the occurrence of tetany. The chapters on the exanthemata are excellent.

In the treatment of the disease the author is much more prone to rely on administration of drugs than is customary in this country, possibly because he knows them better. There can be no doubt that in the hands of a competent therapeutist the simples and compounds of the pharmacist contribute much to the safe and happy curing of disease, and the list of prescripts at the end of the book contains many excellent formulae. Drugging is, however, easily overdone and many of the diseases


to which children are subject may be confidently and safely handled by regulating the diet and hygiene of the patients. It is well to remember that it one is to have recourse at all to the pharmacopeia in the practice of pediatrics, one should be qualified by a much greater knowledge of pharmacy, pharmacology and therapeutics than the average physician possesses.

Diluis helleborum, certo compescere jjuncto Nescius examen —

In closing, a protest is in order against the exorbitant prices which are charged in America for English books. Ten dollars is far too high a price for this text which, although it is printed well and on good paper, is not expensively illustrated and is wretchedly bound with the cheapest possible material. In such a dress the book would be dear at half the price now charged for it. Not only should books be cheaper but they should be better made. By far the greater number of those published now will be dust before fifty years are past and the book which is worth publishing at all is worthy of publication with a view to a longer life. It may be that the useful existence of a modern book of medicine is but seven years. Granted that this is so, such a statement only supplies an argument for the preservation against time of the only available records of the period of change and investigation which is responsible for its truth.

P. G. S.


Modern Surgery, General and Operative. By John Chalmers DaCosta. Cloth, $8.00. {Philadelphia and London, W. BSaunders Co., 1919.)

The appearance of a new edition of DaCosta is always most welcome. Little comment is necessary or desirable on this wonderfully concise, accurate and up-to-date volume. For a subject so extensive, it is always a marvel how it can be done so well.

W. E. D.


Submucous Resection of the Nasal Septum. By William MedDAUGH Dunning, M. D. (New York, Surgery Publishing Company, 1922.)

This book contains forty-seven pages of text and twenty-five illustrations. The author discusses briefly the anatomy and physiology of the nose, and considers, in a somewhat cursory manner, the etiology of septal deviation and its relation to such general disorders as hay fever and asthma. He classifies septal deviations as (1) bowed or curved, and (2) angular, and describes the operative procedures he has devised for their correction. The schematic drawings illustrating the various steps of the operation could be improved upon. At the end he presents a few case histories which show the beneficial effects that result when the operation is really indicated and is properly performed.

The facts could have been presented in a much more concise manner, and while there are many interesting and helpful suggestions, there is really little that has been added to what we find in the standard text-books on rhinology.

J. W. B.


VOLUME I. 423 pases, 90 plates.

VOLUME IL 570 pages, with 28 plates and fleiires.

VOLUME III. 766 pages, with 69 plates and flsures.

VOLUME IV. 504 pages, 33 charts and Illustrations.

VOLUME V. 480 pages, with 32 charts and Illustrations.

VOLUME VI. 414 pages, Trith 79 plates and figures.

VOLUME VII. 537 pages with lllnstrations.

VOLUME VIII. 552 pages with Illustrations.

VOLUSIE IX. lOBO pages, 66 plates and 210 other Illustrations. Contributions to the Science of Medicine.

Dedicated by his I'upils to William Hexbv Welch, on the twenty-flfth anniveisary of his Doctorate. This volume contains 38 separate papei-s.

VOLUME X. 516 pages, 12 plates and 25 charts.

VOLUME XI. 555 pages, with 38 charts and Illustrations.

VOLUME XII. 548 pages, 12 plates and other Illustrations.

VOLUME XIII. 605 pages, with 6 plates, 201 figures, and 1 colored chart.

VOLUME XIV. 632 pages, with 97 figures.

VOLUME XV. 542 pages, with 87 Illustrations.

Twelve papers on pneumonia. By Drs. Chatard, Fabtax, Emekson, Marshall, McCrae, Steixek, Howard and Hanes.

A Study of Diarrhoea in Children. J. H. Mason Knox, Jr., M. D., and Edwin H. Schorer, M. D.

Skin Transplantation. By John Staige Davis, M. D.

Epidemic Cerebrospinal Meningitis and Serum Therapy at The Johns Hopkins Hospital. By Frank J. Sladen, M. D.

VOLUME XVI. 670 pages with 161 figures.

Studies in the Experimental Production of Tuberculosis in the Genitourinary Organs. By George Walker, M. D.

The Effect on Breeding of the Removal of the Prostate Gland or of the VesiculiB Seminales. or of Both ; together with Observations on the Condition of the Testes after such Operations on \^ hite Rats. By George Walker, M. D.

Scalping Accidents. By John Staige Davis, M. D.

Obstruction of the Inferior Vena Cava with a Report of Eighteen Cases. By J. Hall Pleasants. M. D.

Physiological and Pharmacological Studies on Cardiac Tonicity in Mammals. By Percival Douglas Cameron, M. D.

VOLUME XVII. 586 pages with 21 plates and 136 flgnres.

Free Thrombi and Ball Thrombi in the Heart. By Joseph H. Hewitt, M. D.

Benzol as a Leucotoxin. By Lawrence Selling, M. D.

Primary Carcinoma of the Liver. By Milton C. Winternitz. M. U.

The Statistical Experience Data of The Johns Hopkins Hospital, Baltimore, Md 1892-1911. Bv Frederick L. Hoffman, LL. D., F. S. S.

The Origin and Development of the Lymphatic System, By Blobence K.

The Nuclei Tuberis Laterales and the So-called Ganglion Opticum Basale. By Edward F. Malone. M. D.

Venous Thrombosis During Myocardial Insufficiency. By Frank J. Sladen, M D., and Milton C. Winternitz, M. I).

Leukasmia of the Fowl ; Spontaneous and Experimental. By Harby C. Sch.meisser, M. D.

VOLUME XVIII. 446 pages with 124 figures. Fasciculus I.

A Study of a Toxic Substance of the Pancreas, By E. W. Goodpasture, M, D., and George Clark, M. D.

Old Age in Relation to Cell-overgrowth and Cancer, By E. W. Goodpasture, M. D., and G. B. Wislocki, M, D.

The Effect of Removal of the Spleen Upon Metabolism in Dogs ; Preliminary Report. By J. H. King. M. D.

The Effect of Removal of the Spleen Upon Blood Transfusion. By J. H. King, M. D. B. M. Bebnheim, M. D., and A. T. Jones, M, D.

Studies on Parathyroid Tetany. By D. ^N'^ught Wilson, M, D„ Thornton Stearns, M D,, J. H Janxev, Jr., M. D., and HUdge DeG. Thdblow, M. D.

Some Observations on the Effect of Feeding Glands of Internal Secretion to Chicks. By M. C. Winternitz, M, D.

Spontaneous and Experimental Leukaemia in the Fowl. By H. C.

SCHMEISSER, M. II.

Studies on the Relation of Fowl Typhoid to Leuksemia of the Fowl. By

M. C. Winternitz, M. D., and H. C. Sciimeisser, M. D. Hyaline Degeneration of the Islands of Langerhans in Pancreatic Diabetes.

By M. C. Winternitz, M. D. Generalized Miliary Tuberculosis Resulting from Extension of a Tubercular

Pericarditis Into the Eight Auricle. By M. C. Winternitz, M. D. Acute Suppurative Hvpophysitis as a Compllcatiou of Purulent Sphenoidal

Sinusitis. By T. R. BoGGS, M. D., and M. C. Winternitz, M. D, A Case of Pulmonary Moniliasis In the United States. By T. R. BOGGS,

M. D , and M. C. Pincoffs. M. D. Gaucher's Disease (A Report of Two Cases in Infancy). By J. H. M.

Knox, M. D., H. R. Wahl, M. D., and H. C. Schmeisser, M. D. A Fatal Case of Multiple Primary Carcinomata By E. D. Plass, M. D. Congenital Obliteration of the Bile-ducts. By James B. Hol.mes, M. D. Multiple Abscesses of the Brain in Infancy, By James B. Holmes, M. i>. Gastric Carcinoma in a Woman of Twenty-six Years, Bv R. G. HossET,

M. D. Subdiaphragmatic Aliscess with Rupture Into the Peritoneal Cavity Fol


lowing Induced Pneumothorax for Pulmonary Haemorrhage. By R. G.

HissEY, M. D. Heart Block Caused by Gumma of the Septum. By E. W. Bridgeman,

M. D., and II. C. Schmeisser, M, D. Analysis of Autopsy Records.

A. The Johns Hopkins Hospital. (Table Showing Percentage ot

Autopsies.)

B. The City Hospitals, Bay View. (Table Showing Percentage of

Autopsies.) "The Monday Conferences."

Clinical Representatives on the Staff of the Department of Pathology. Donation,

Fasciculus II. The Role of the Autopsy in the Medicine of To-day. By M. C, Winternitz, M. D. Experimental Nephropathy in the Dog. Lesions Produced by Injection

of B. bronchisepticus into the Renal Artery. By M. C. Winternitz,

M, D., and William C. Quinbt, M. D. Mesarteritis of the Pulmonary Artery. By M, C. Winternitz, M. D., and

H. C. Schmeisser, M. D. A Clinical and Pathological Study of Two Cases ot Milary Tuberculosis of

the Choroid. By Robert L. Randolph, M. D., and H. C. Schmeisser,

M. D. The Blood-vessels ot the Heart Valves. By Stanhope Bayne-Jones, M. L>. Equilibria in Precipitin Reactions, By Stanhope Bayne-Jones, M. D. Carcinoma of the Pleura with Hypertrophic Osteoarthropathy. Report of

a Case with a Description of the Histology of the Bone Lesion. By

Stanhope Bayne-Jones, M. D. The Interrelation ot the Surviving Heart and Pancreas of the Dog In

Sugar Metabolism. By Admont H. Clark, M. D. Congenital Atresia of the Esophagus with Tracheo-Esophageal Fistula

Associated with Fused Kidney. A Case Report and A Summary of the

Literature on Congenital Anomalies of the Esophagus. By E. D.

Plass, M. D. Ectopia Cordis, with a Report of a Case in a Fifteen-Month-Old Infant.

By JA.MES B. Holmes, M. D. Studies in the Mechanism of Absorption from the Colon, By Samuel

GoLDSCH.MiDT, M. D., and A. B. Dayton, M. D. Report ot Two Fatal Cases Following Percy's Low Heat Treatment of

Carcinoma of the Uterus. By V. N. Leonard, M. D. and A. B. Dayton,

M. D. The Relationship in Tvphoid Between Splenic Infarcts and Peritonitis

Unassociated with Intestinal Perforation. By A. B. Dayton, M. D. Left Duodenal Hernia. By A, B. Dayton, M. D.

Histological as Related to Physiological and Chemical Differences in Certain Muscles of the Cat. By H. Hays Bullard, M. D. A Method ot Clearing Frozen Sections. By H. Hays Bullard, M. D. On the Occurrence and Significance of Fat in the Muscle Fibers ot the

Atrioventricular System. By H. Hays Bullard, M. D. Studies on the Metabolism of Cells in vitro. 1. The Toxicity of o-Amlno Acids for Embryonic Chicken Cells. By Montrose T. Burrows, M. D.,

and Clarence A. Xey.mann, M. D. The Significance of the Lunula of the Nail. Bv Montrose T. Bdbbows,

M. V. The Oxygen Pressure Necessary for Tissue Activity. By Monteose T.

Burrows, M. D. The Functional Relation ot Intercellular Substances in the Body to Certain Structures in the Egg Cell and Unicellular Organisms. By

Montrose T. Burrows, M. D. Studies on the Growth of Cells in vitro. The Cultivation of Bladder and

Prostrate Tumors Outside the Body. By Montrose T. Burrows,

JI. D., J. Edward Burns, M. D . and Yoshio Suzukl, M. D. The Study ot a Small Outbreak of Poliomyelitis in an Apartment House,

Occurring in the Course of an Epidemic in a Large City. By Montrose

T. Burrows, M D., and Edwards A. Park. M. D. Papilloma of the Larynx Report of a. Case Treated with Radium with

Resultant Chronic Diffuse Thyroiditis. By William C. Duffy. M. D. Analysis of Autopsy Records. Autopsy Statistics.

(a) Bay View.

(b) Johns Hopkins Hospital. Report of the Photographic Department. General Improvements.

Donations.

VOLUME XIX. 358 pages with 29 plates.

The Structure of the Normal Fibers of Purkinje in the Adult Human Heart and Their Pathological Alteration in Syphilitic Myocarditis, Bv O. Van Der Stricht and T. Wingate Todd. M. D.

The Operative Story ot Goitre. The Author's Operation. By William S. Halsted, M. D.

Study ot Arteriovenous Fistula with an Analysis ot 447 Cases. By Cuble L. Callander. M. D.

VOLUME XX. 314 pages with 82 plates. „ ^

The Pathology of the Pneumonia in the United States Army Camps During the Winter of 1917-lS. By William G. M.acCallum. M. D.

Pathological .\natomv of Pneumonia Associated with Influenza. By William G. MacCallum, M. D. ,, j , •

Lymphosarcoma. Lymphatic Leukwmia. Leucosarcoma. Hodgkin s disease. Leslie T. Webster. M. D.

CONTENTS

  • The Diagnostic and Therapeutic Use of Uveal Pigment in Injuries of the Uveal Tract and Sympathetic Ophthalmia. By Ai.AN C. Woods, M.D., and Arnold Knapp, M.D.
  • Observations on the Production of Toxic Substances by Pneumocoeci. By Ai.AN M. Chesney, M.D., and Alfred B. Hodges, M.D 425
  • Influenza-like Bacilli. Growth of Influenza-like Bacilli on Media Containing Only an Autoclave-Labile Substance as an Accessory Food Factor. (Illustrated.) By T. M. Rtvers, M.D 429
  • A New Method of Testing Liver Function With Phenoltetrachlorphthalein. Second Communication. By Sanford M. Rosenthal 432
  • A Study of Trichomonas Hominis, Its Cultivation, Its Inoculation Into Animals and Its Staining Reaction to Vital Dyes. By Mary Jane Hogue, Ph.D 437
  • Entero-Urethral Fusion in a Fetus, Simulating Fetal Ascites. (Illustrated.) By John C. Baldwin
  • Report of a Case of Tubal Pregnancy Probably Caused by a Parovarian Cyst. (Illustrated.) By Leo Brady, M.D
  • The Isolation of Nucleic Acid From Tissues. By Walter Jones and Caspar Folkoff.
  • A Case of Congenital Osteosclerosis. (Illustrated.) By Ralph K. Ghormley, M.D
  • Boylston Medical Prizes
  • Index to Volume XXXIII of the Johns Hopkins Hospital Bulletin 447
  • Errata 450


THE DIAGNOSTIC AND THERAPEUTIC USE OF UVEAL PIGMENT IN INJURIES OF THE UVEAL TRACT AND SYMPATHETIC OPHTHALMIA

By Alax C. Woods, M. U. ami Arnold Knapp, M. D.

(From the Departments of Pathology and Ophthalmology of the

Johns Hopkins Hospital and University, and from the

Herman Knapp Memorial Hospital, New York)


In a former paper b^- one of n.s (Woods '), clinieal and experimental studies on the immune reactions following injuries to the uveal tract were presented. These studies showed that: {1) In an intraocular injuiy involvinf> tlie uveal tract, where normal healing took place without the occurrence of a .sympathetic disturbance in the fellow eye, substances developed in the blood serum which gave a positive complement fixation reaction with an antigen made from the pigment of the uveal tract. (2) In the cases of the intraocular injury involving the uveal tract


when normal healing was delayed, and where sympathetic disturbance in the second eye sometimes occurred, this complement fixation reaction to pigment antigen was absent. Furthermore, there was not only the absence of this complement fixation reaction, but in one case of sympathetic disturbance there appeared to be a definite hypersensitiveness to jjigment. Evidence was also presented which had led the author to believe that the occurrence of this complement fixation reaction was evidence of the development of an immunity by the organism to the pigment, and gave definite protection against the outbreak of a sympathetic disturbance in the fellow eye. Likewise, it seemed clear that the failure to develop this complement fixation reaction was evidence of the failure to develop an immunity, and was so attended by a persistance of inflammatory symptoms and the liability to the development of a .sympathetic disturbance in the fellow eye.

The possible clinical use of this phenomenon for the purpose of early information as to the prognosis, in any given case of intraocular injury- involving the uveal tract, is at once evident. Should the.se facts hold time, the development in the blood serum of a positive complement fixation against pigment antigen would warrant the surgeon in giving a favorable prognosis, and allow him to leave the injured eye without fear of the development of a sympathetic disturbance in the fellow eye. On the other hand, the failure to develop a positive reaction would give definite information to the surgeon that .sympathetic ophthalmia was an event to be feared, and would allow removal of the injured eye before the outbreak of the sympathetic disease.

Likewise, the possibility for the therapeutic use of uveal pigment in a sympathetic ophthalmia is manifest. As much as the hypersensitivity predisposes to the outbreak of the disease and an immunity protects against the disea.se, as soon as a hyper.sensitivity is demonstrable, the self-evident course is to de-sensitize the patient, and then, as a therapeutic measure, proceed either to active or passive immunization.

It is the purpose of this paper to report cases of intraocular trauma invoking the uveal tract, in which this serum complement fixation reaction against uveal pigment was used as a diagnostic procedure to determine the status of the case with regards to a possible outbreak of .sympathetic ophthalmia. Further, one case of malignant sympathetic ophthalmia is reported in which uveal pigment was used as a therapeutic procedure.

The Diagnostic Use of the Scrum Complement Fijcation Reaction against Uveal Pigment

The sera from seventeen cases of intraocular injury involving the uveal tract, occurring in the private and dispensary practices of the authors, were examined iu the complement fixation reaction against an antigen of uveal pigment. The technique of the reactions and the pi'eparation of the antigen have been previously reported.^

Grouping these cases according either to the results of the reaction or to the clinical picture and result, they fall into three rather general groups. The first group comprises ten cases which gave a positive complement fixation reaction, and in which healing took place without the occurrence of any sympathetic disturbance. In three of these cases, however, for one or another reason enucleation was performed. The second group comprises three


cases which all showed negative complement fixation reactions and at the same time showed more or less severe signs of a sympathetic disturbance. The third group comprises three cases which showed alarming symptoms in the injured eye, but without any manifestation of sympathetic disturbance in the second eye, and gave negative reactions.

Groui' I. — The following reports are of the cases in which the blood serum showed a positive complement fixation reaction against pigment antigen, following wounds of the uveal tract.

No. 1 (A.)— struck in the left eye April 16. 1921 by a piece of steel; the wound of entrance was at the lower corneal margin. The splinter of steel passed back through the iris and was localized by the X-ray, in the region of the lower ciliary processes. Size of splinter, 12x2 mm. Removed by magnet extraction April 17, 1921. Serum reaction against pigment antigen on May 2, 1921 wjas -f + + and on May 21, 1921 -f -}- -f . The injured eye gradually underwent pthisis. In March 1922, the injured eye was small, sightless, but not inflamed. Right eye held 20/15 vision throughout and has remained free from trouble to date.

No. 2. (O.) — Struck in right eye by a flying piece of steel on June 13, 1921. Wound of entrance at corneo-scleral border. The X-ray showed an intra-ocular splinter of steel. 13x3 mm. Successfully removed by magnet extraction on June 14, 1921. Serum reaction against pigment antigen on June 25, 1921, + + +. The injured eye healed quickly and retained 20/40 vision. The left eye held normal vision and has shown no subjective or objective signs of disturbance.

No. 3. (J.) — Struck in right eye on Sept. 14, 1921. Perforating wound of cornea with prolapse of iris and ciliary body. Pi'olapsed tissue excised within three hours after accident, scleral suture and conjunctival flap made. Uneventful healing, but a pthisical eye resulted. Serum reaction on Oct. 1, 1921. + -f +. There was no disturbance of any kind in the left eye, but on Oct. 2G, 1921, the right eye was enucleated for cosmetic reasons.

No. Ji. (B.) — Struck in right eye Dec. 14, 1921; the patient's spectacles were broken. There was a perforating wound of the right eye, involving cornea, iris, lens, and ciliary body. Prolapsed tissue excised after accident. Traumatic cataract developed and was removed.

The eye showed a continual cyclitis of varying intensity, which, finally, starting about the middle of February, gradually subsided with a retention of 20/50 vision, with correction.

The following were the serum reactions in this case: — Dec. 14,

1921, ±; Jan. 9, 1922, + +; Jan. 21, 1922, negative. Feb. 13,

1922, -f -f +; March 27, 1922, + -f +.

Vision in the uninjured eye was 20/15 at the time of the accident. There has at no time been any failure of the visual acuity or field or disturbance of any kind.

This case is of especial interest in that recovery was delayed, and with this delay the patient passed from a positive phase to a negative phase and, finally, to a second positive phase which he has steadily maintained.

No. 5. (B.) — November 1921, while hunting was struck in right eye with spent shot. Treated at home.

Exam. X-ray: Bullet down and in. Iridocyclitis and detached retina below.

Treatment: Enucleation. 12-11-21.

12-20-21. Discharged. Normal recovery.

12-11-21. Serum reaction against uveal pigment -f- +.

No. 6. (McC.)— Nov. 15, 1921. Struck in left eye with a piece of glass.



Exam: x-shaped, penetrating wound in temp, side over region of ciliary body, the wound being 4-G mm. long (Nov. 16, 1921).

Treatment: Scleral wound sutured — conjunctival flap.

Course: Looked very well for first ten days, then eye became red — ciliary congestion — iris showed greenish discoloration — ^Vit, exudate. Tension — .

12- 9-21. Enucleation.

12-16-21. Discharged. Right eye normal.

Dec. 15, 1921. Serum reaction against uveal pigment, ±.

No. 7. (R.) — Dec. 21, 1921, while playing, was struck in eye by a knife.

Exam: Perforating wound of cornea starting a few millimetres above limbus at 12 o'clock and running downwards for about S mm. Iris cut above. Some vitreous in upper part of wound. Lens swollen.

Treatment : 12-28-21. Adm. to hospital. Wound cleaned up and some of cataractous lens removed.

Course: 1-6-22. Serum reaction against uveal pigment negative; no immunity.

2- 4-22. Serum reaction against uveal pigment + + +. 2-17-22. Discharged. Ran through a long drawn out iridocyclitis with remissions and relapses. Finally left eye nearly white. Considerable cortex remaining. Capsule adherent to cornea and iris to capsule. V: R, 20/20; L, H. M. 3-20-22. Iridectomy. 4-15-22. Discharged. Normal recovery.

No. S. (M.) — 1-19-22. Wbile on street was struck by some unknown object in right eye.

Exam,: Linear incision through cornea from 9 to 5 o'clock and extending into ciliary body. Iris prolapsing, also part of ciliary body and some vitreous. V: L. P.

Treatment: Prolapsing iris, ciliary tissue, and vitreous, abscised. Wound closed with scleral and conjunctival flap.

Course: Normal recovery. Discharged 2-7-22. Vision 20/100.

When last seen in clinic V. 20/50 +. Eye was white. No pain, left eye normal. V: 20/30.

Serum reactions against uveal pigment: 2- 1-22. Negative. 2-13-22. Anti-complementary. 2-28-22. +.

No. 9. (P.) — Sept. 1921, struck in right eye with splinter of steel. Three weeks ago noticed right pupil larger than left.

Exam: Corneal scar lower nasal quadrant with laceration of iris and opacity of lens directly behind. Embedded in retina, three disc diameters below disc there is an oblong-shaped foreign body surrounded by degenerated retina and choroid. Numerous small retinal exudates surrounding foreign body. V: 20/30.

Treatment: Giant magnet extraction through zonule and anterior cornea.

Course: 2-11-22. Serum reaction against uveai pigment, -j-.

2-17-22. Vitreous hazy. Some oedema and cloudiness about old choroidal scar.

2-25-22. Discharged. V: 20/70. Eye white. Vit. clearer. Fundus unchanged.

3-16-22. Readmitted. Detachment of retina below. V: R, 10/200; L, 20/20.

Left eye normal.

No. 10. (P.S.)— January 2, 1922, left eye injured by wire in coasting. Prolapsed iris abscised. Eye has remained red. Shrunken. No pain.

Seen on February 6, 1922. Ciliary congestion. Iris discolored. Scar In cornea on temporal periphery. Somewhat indrawn. Beginning phthisis, faulty projection.

Serum reaction February 13, 1922. -f -(- -f .


No. 11. (B.)* — Feb. 2, 1922. Was struck in right eye by nail. He withdrew nail following accident.

Exam: V: H. M. Perforating wound of cornea in lower nasal quadrant. Iris cut and caught in wound. Lens cataractous. P. L., good.

Treatment: 2-6-22. Wound freed of iris.

Course: 2-14-22. Anterior synech. at site of injury, some exudate back of lens. Eye very red.

2-25-22. Serum reaction — negative. No immunity.

3-15-22. Discharged. Eye still very red. Tension normal. V: 10/200; L. proj., N; lens subluxated and cataractous.

4-18-22. Eye still red. No pain. Tension normal. V: H. M, ; L. eye normal.

Group II. — The following reports are of cases in which following injuries of the uveal tract, the blood serum showed negative complement fixation reactions. These cases showed either at the time of serum reactions, or developing later, disturbances in the second eye which were believed to be sympathetic in nature.

No. 12. (H.) — Shot in head Aug. 30, 1921, with a shot-gun. Many shot penetrated skull and one penetrated left eye, passing through eye-ball and lodging near nasal wall of orbit. Craniotomy was performed Sept. 3, 1921 with removal of many shot. Iridectomy was performed on left eye on Sept. 9, 1921, for removal of iris tissue incarcerated in wound of entrance. Serum reaction on Sept. 8, 1921, negative. Serum reaction on Sept. 30, still negative. The eye was still inflamed, and sightless. Enucleation was advised and refused. The child returned to his home in Virginia on Oct. 1, 1921. On Oct. 7, an attack of photophobia with pericorneal flushing in the right eye was reported by the local physician. The child was brought back to Baltimore on Oct. 14, 1921. At that time there was still decided photophobia, but the eye was otherwise objectively negative. Serum reaction on Oct. 14, 1921, was negative. Eiiucleation of left eye was performed on Oct. 16, 1921. Following enucleation, the photophobia of the right eye disappeared, and the eye has remained entirely normal since them.

No. 13. (H.) — Penetrating wound of left eye in 1909. Normal healing with retention of 20/70 vision. In May, 1921, some solder flew up into left eye causing great pain which steadily persisted. Right vision began to fail in July, 1921. On examination, Aug. 23. 1921, the left eye showed a displaced pupil, back of which, bulging the iris forward, a foreign body could be distinctly seen. There was a low grade cyclitis; tension normal, vision 20/70. The right eye showed nothing objectively except marked vascular congestion of the fundus and slight photophobia. Vision was 20/30 — with correction. Visual fields showed normal field in right eye and concentric contraction in left. A complete medical study at Johns Hopkins Hospital showed only negative results. Laryngological and all other special examinations were likewise negative. On Aug. 26, 1921, the serum reaction with pigment antigen was negative. A tentative diagnosis of sympathetic irritation in the right eye was made and enucleation of the left eye advised. The patient returned to his home in North Carolina where enucleation was performed, and he reported that following enucleation all symptoms in right eye cleared.

No. IJf. (H.) — This case of malignant sympathetic ophthalmia was later treated by de-sensitization and immunization with uveal pigment. Full details are reported below. The first serum reaction on Dec. 21, 1921, at the height of the disease, was negative.


• Case incomplete — no serum reaction has been done since negative reaction of Feb. 25th. Case is stiU under observation, and is Included in this series, inasmuch as the report includes all cases tested up to March 15, 1922.



Group III. — The followiug reports are of three cases in which the serum reaction was negative and in which the injured eyes were enucleated as a precautionary measure.

No. 15. (B.)' — Struck in left eye with a bit of steel Dec. 12, 1920; penetrating wound through ciliary body with intraocular retention of piece of steel. Removed by magnet extraction on the same day. 'Eye was sightless, and showed continual low giade cyclitis with periodic violent relapses. All medical examinations were negative. Radiograph showed no further intra-ocular foreign body. Jan. 17, 1921, and Feb. 15, 1921, the serum reaction against pigment antigen was negative. Left eye was enucleated on Feb. 21, 1921. Right eye normal.

A'o. IG. (K.) — In Nov. 1911 was struck by a tree branch and suffered from perforating injury for which he was treated in his home town until Jan. 13, 1922.

Exam: Marked pericorneal congestion. Perforating corneal wound just below center. Iris greenish and adherent to wound. Pupil occluded. P: L., good. V: L., 20/20.

Treatment: Enucleation with fat implantation, 1-14-22.

Course: 1-26-22. Discharged. Normal recovery. V: L., 20/20. Serum reaction against uveal pigment, 1-20-22, negative.

No. n. (P.)— struck in right eye with B.B. shot May 14, 1921. Shot penetrated globe through upper lid through sclera above cornea.

Treatment: Adm. to hospital and discharged as soon as hsemophthalnios had cleared up (6-9-21).

Course: Came to clinic for observation. Had several intraocular haemorrhages. The tension gradually became soft and the eye remained red. Light projection faulty.

1-6-22. Serum reaction against uveal pigment, negative.

1-10-22. Adm. to hospital. Enucleation. Shot found outside globe, but adherent to it near optic nerve.

When last seen (4-18-22) left eye normal; V: 20/20.

Tliere were two other cases in which all the serum reactions were negative, but these cases are of academic interest only. They were both instances of old sympathetic ophthalmia which had finally subsided following enucleation of the exciting eye. One case had followed subconjunctival scleral rupture and showed a sympathetic ophthalmia in the second eye which gradually recovered after enucleation of the injured eye. It is of interest that this patient showed a ])0sitive intradermal tuberculin reaction, confusing the diagnosis. The second case was one of delayed sympathetic ophthalmia in the left eye, following a bayonet wound of the right eye. When seen one year later, the left eye was nearly sightless. The right eye had been enucleated at the time of the sympathetic disturbance in the left eye.

Comment

In these cases above reported, it is frankly admitted that the presence or absence of a positive complement fixation reaction was not the sole index which governed the decision as to whether or not an injured eye should be left or enucleated. In two cases enucleation was performed before the blood was taken for the serum reaction, which in both cases was found positive. In other cases the presence of a positive serum reaction led to the temporarj' postponment of enucleation, which was later


permanently abandoned on account of the favorable outcome of the case. Nevertheless, an analysis of those cases which gave positive results, excluding the two cases in which the picture is confused owing to enucleation having formed prior to the discovery of a positive blood, shows a rather significant fact. Had we relied, in these cases, upon a positive serum reaction as the index of when not to remove an injured eye, our faith would have been justified. In other words, the occurrence of a positive serum reaction, irrespective of the condition of the injured eye at the time the reaction was done, was always followed in time by a subsidence of inflammation without any luanifestation of sympathetic disturbance.

Whether the opposite of this is true — that a negative reaction is sufficient indication for removal of an injured eye for fear of a possible sympathetic ophthalmia — is not so clear. Of the seven patients who showed negative reactions, one is still under observation and three at the time of the serum test already showed, or quickly developed, signs of a sympathetic disturbance. In the remaining three cases, we did not wait for a possible sym])athetic disturbance to develop — the injured eyes were enucleated. But these three cases all showed a long-standing, chronic cyclitis, the eyes clinically appearing dangerous. So in all three enucleation was justified by the clinical sjmiptoms alone.

In the absolute interpretation of negative results, other factors must be considered. In the first place, the blood serum practically never becomes i)ositive before ten days after the injury, and frequently never before four weeks. Furthermore, as is illustrated by case No. 4, Group I, the patient may go from a positive to a negative phase, and then finally to a positive phase. If a single or two negative reactions be taken as a definite indication for the removal of an injured eye, eyes may be needlessly removed, for in a few weeks more it is possible that the patient may go into a permanent positive phase, and be automatically protected against sympathetic ophthalmia. Therefore, if the serum reaction is negative, the time interval after injury must be first considered. However, if in six weeks after injury the patient has failed to develop a positive reaction, it seems unlikely, from the information we now have, that a positive reaction will ultimately develop. In such cases the possibility of sympathetic ophthalmia, and enucleation of the injured eye should be carefully considered.

There is one further point to be considered in the interpretation of positive results. Is the immunity indicated by a positive reaction permanent, or is the immunity likely to fail and the patient later become susceptible to sj-mpathetic ophthalmia? This question is very difficult to answer. We have seen one, and only one, case go from an initial positive phase to a negative phase, and this patient later, within three weeks after entering the negative phase, again became positive, and has so remained.


All immunological fact is known which may be somewhat analogous to this reaction. In an animal experimentally immunized by repeated injections of foreign protein, each fresh injection is followed by u temporary decrease in the antibodj' titer. If the original titer is not very high, this decrease may throw the animal from a positive to a negative phase. This reaction is followed, however, by a further increase over the original titer, so that the ani mal soon again becomes more positive than before.

Every other case examined which showed a positive reaction, so far as our work has gone, has remained posi tive. Yet the following fact is true. In the earlier work on man with this complement fixation reaction, most of the patients tested had a traumatism that was one year or more old; and it must be admitted that positive reactions in these were weaker than in tlie more recent cases. From a review of the work previously reported, it seems clear that immunity, once definitely established, will last for from two to three years, and probably much longer, but graduallj' will become weaker.

Summarizing the results shown by the cases here re ported which showed positive reactions, considered in the light of our previous studies, it seems probably that a positive reaction indicates a definite immunity to the development of sympathetic ophthalmia and that this immunity will last at least several years, if not permanently. From these findings it seems, then, that with the occurrence of a positive serum reaction, unless the clinical condition clearly contra-indicated such a course, the surgeon is warranted in not enucleating injured eyes.

The Thcr(i[>viitU- Use of Uveal Pigment

The basic points tlnis far emphasized in our work have been these: (1) The outbreak of sympathetic ophthalmia seems to be dependent upon an existing hyper-sensitiveness to uveal pigment.^ (2) The occurrence of an immunity against uveal pigment protects against the development of sympathetic ophthalmia. If these two fundamental points hold true, as it appears they do, they point at once to two very definite procedures — preventive and therapeutic — as regards sympathetic ophthalmia: (1) As a preventive or prophylaxis against sympathetic ophthalmia, the obvious procedure is to remove the hyper sensitivity and produce the desired immunity. (2) As a therapeutic measure, to remove the one factor which appears essential to the outbreak of sympathetic ophthalmia, and to substitute for it the one factor that appears to make sympathetic ophthalmia impossible; in other words, again to remove the hy])er-sensitiveness and produce an immunity to uveal pigment.

With the use of uveal pigment as a pr(»])hylactic meas ure we have had no experience. The case reported below is also the only instance in which we have used the pig ment as a therapeutic agent. It may be stated frankly that, inasmuch as this work is scarcely out of the experi


mental stage, we felt considerable reluctance in using uveal pigment in man, knowing that a hypersensitivity to it existed, unless the circumstances surrounding the case were such as to make any procedure, offering the least hope, justifiable. The case below reported moi-e than satisfied the.se conditions. Before any injections were made. Professor Hans Zinsser was good enough to consult with us, and it was only after this final consultation that the procedure was undertaken.

Report of a Case of Sympathetic Ophthwlmia

The patient, a boy aged 8, seen on September 10, 1921, had an unhealed corneal ulcer, with prolapse of the iris in a dense scar, occupying the lower half of the right cornea, the result of a gonorrheal conjunctivitis, which had begun three months before. Vision was reduced to light perception and the tension was increased. The attempt to pre.serve the eye seemed justified ; on September 13th, an iridectomy was done upward, the defect in the cornea was curetted, the prolapsed iris was removed as well as possible and the corneal wound covered with a conjunctival flap. This healed and the patient returned to his home after two weeks with vision of 2/200.

He returned on October 31st, becau.se the other (left I eye had become red three daj's before. The left eye showed slight ciliarj' congestion, posterior corneal deposits, posterior synechiip. V: 20/20. Tension, normal. Interior, normal. The right eye was white. V: 2/200. The area of the corneal defect covered by a conjunctival flap was depres.sed and ocular tension was reduced.

There was no history of indigestion or overeating; the urine was normal ; the stool showed evidence of intestinal fermentation, and the patient was placed on a sugar and starch-free diet and high colon irrigations. The eye was given the usual treatment. November 7th, V: 20/40; vitreous hazy; retinal veins engorged. November 9th, right eye enucleated. Serum reaction against uveal pigment was negative, showing no immunity whatever. A large cyanide subconjunctival injection was given; pilocarpine sweats and mercurial inunctions were begun. November 14th, atropin eczema. I^eft pupil contracting; nodules appeared in iris. Leucocytes, 3000 ; no lymphocytosis. December 8th, V: 20/40. Deep ant. chamber. Iris flat with nodules. Optic nerve blurred, retina hazy. Nonspecific i)rotein therapy was tried ; a course of typhoid vaccines was given by Dr. Boninie. Though these were followed by the usual reactions, the leucocyte count did not increase and the eye continued to grow steadily worse. There was a marked follicular conjunctivitis with pseudomembranes. The general treatment was then changed to salicylate of soda in large doses. The eye showed more and more the characteristic changes of sympathetic ophthalmia with nodules in the iris; peripheric retraction of iris ; complete synechia and capsular opacity in the lens. On Jan. 5, 1922 the injections of uveal pigment were


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commenced. During the period of treatment with the pigment, the progress of the eye disease did not seem to be particularly changed. Anterior chamber shallow; pupil contracting; iris uneven, flat total adhesion; tension increased. The last pigment injection was given Jan. 12.

Jan-tmry 18, 1922. — The eye was better ; cornea and iris were clearer. T. 36. This improvement was only of brief duration and an exacerbation occurred on February 2, 1922, when the eye became more red, harder, and there was more exudation in the anterior chamber and in the iris. On Jan. 20, 1922, eight days after the course of immunization had been completed, the serum reaction had

become -| — | almost complete fixation. Following the

exacerbation of Feb. 2, the eye gradually improved, though the opacity of the lens remained about stationary ; iris retracted with some anterior peripheric adhesions; capsular opacities. On the last examination, the tension was normal and the eye white, V: 20/200. Details in fundus not visible. The blood examination on Feb. 8 gave -| — I — \-, showing complete fixation.

Technique of Teeatjient

Procedure mth uveal pigment. — The suspension of pigment used in the case was made up as before described (2) and preserved with 0.15 per cent tri-cresol. As the pigment is de-naturized by heat, unhealed pigment was given, the only step taken for sterilization being the addition of tri-cresol, which is sufficient to destroy all ordinary non-spore forming pathogenic organisms. Since the critical condition of the patient did not allow sufficient time for animal experiments to exclude the remote possibility of the presence of tetanus spores in the preparation of pigment, it was decided to administer tetanus anti-toxin as a prophylactic measure. A precautionary Intra-dermal test was made with tetanus anti-toxin to determine whether the patient was sensitive to the serum. This was positive, and he was first desensitized with serum in the usual manner, and then given 1500 units of tetanus anti-toxin. An intra-dermal skin test with uveal pigment was then made.

Technique of Intra-dermal test (Jan. 6, 1922). — Three different dilutions of the normal pigment suspension (the pigment of the uveal tract of an eye of a cow to 7.5 c.c. of salt solution is called "normal,") were used. The solutions were 1:100, 1:50, and 1: 10. "normal," were used. The solutions were 1:100, 1:50, and 1:10. 'ihrough a fine hypodermic needle small intra-dermal injections, suflicient to raise a bleb about one half the diameter of a dime, were made with each dilution on the flexor surface of the forearm. A control was made with 0.15 per cent tri-cresol in normal salt solution. At the end of one hour the three blebs where the pigment suspension had been injected were surrounded by a striking, confluent urticarial wheal, whereas the control was practically negative. Within six hours this urticarial reaction had disappeared. Since the patient was strikingly hypersensitive to uveal pigment, we at once proceded with desensitization.

Technique of desensitization (Jan. 6, 1922). — Intra-muscular injections In the buttock were made at two-hour intervals. The so-called "normal" suspension was used for the first four doses. The amounts given were 1.0 c.c; 1.5 c.c; 2.0 c.c. and 2.5 c.c. For the fifth injection a suspension of twice normal strength was used, 2.5 c.c. (the equivalent of 5.0 c.c. of the normal) were given. There was no essential change in either the blood pres


sure, pulse, temperature or respiration during this period of desensitization. The only change observed was a generalized urticaria with the resultant unpleasant itching. This was relieved by a 70 per cent alcohol rub, and entirely passed within sixteen hours.

Technique of immunization. — On Jan. 6, during desensitization, a total of 12 c.c. of the normal suspension had been given, the last injection being the equivalent of 5.0 c.c. of the normal. On Jan. 9, the equivalent of 7.5 c.c. of the normal (condensed in a bulk of 2.0 c.c.) were given by intra-muscular injection into the buttock. On Jan. 12, after a second three-day interval, the final injection, the equivalent of 10 c.c. of the normal, was given by intra-muscular injection into the buttock. As for the previous injection, the pigment was condensed in a total volume of 2.0 c.c. No untoward symptoms accompanied either of the latter injections.

Comment

Sympathetic inflammation after perforating ulcers is rare. Peters comments on the fact that sympathetic ophthalmia has been frequently observed after perforation of a corneal ulcer complicating a gonorrheal process and concludes that the conditions in gonorrheal inflammation are more favorable to a development of sympathetic ophthalmia than in other keratitides. As an operation was performed in some of these cases, it may be that the prolapse is not so dangerous as the operation which was undertaken to remove it. The operations performed in these cases (quoted from Peters) were excision of the prolapse, (Schieck), cauterization (Trousseau, Gifford and Fuchs), abscission of corneal staphyloma and conjunctival suture (Fuchs).

In the case just described there seemed to be a definite intestinal autotoxemia. No change in diet or treatment, however, was of any avail. There was no lymphocytosis. Eecent investigations have not shown the occurrence of a lymphocytosis in sympathetic ophthalmia. Treatment I

with typhoid vaccines was without avail. A leucocytosis 1

could not be obtained.

Enucleation after the onset of the sympathetic ophthalmia was, as usual, without influence on the course of the iridocyclitis in the remaining eye.

Microscopic examination of the eye first affected showed a characteristic diffuse infiltration of the iris and the adjoining iris angle, with mononuclear lymphocytes, a moderate infiltration of the ciliary processes and neighboring vitreous body, and only a few scattered foci in the choroid.

In view of the fact that partial recoveries have been observed in sympathetic oiihthalmia, irrespective of the form of treatment, the inflammation having finally exhausted itself, it would be unwise to draw any deductions from a single experience. Yet in this case, after desensitization and immunization, the morbid process, with one slight and short period of exacerbation, came to a standstill; the eye became white and free from irritation and so remained.




Summary

The immune reaction associated with intraocular injuries involving the uveal tract of the eye was made use of in seventeen such cases as a diagnostic procedure. In ten cases the complement fixation reaction was positive. These cases showed a normal healing without the occurrence of anj' sympathetic disturbance. One case is still incomplete. Three cases showed negative reactions, and of these one showed, clinically, a malignant sympathetic ophthalmia, and two showed definite signs of sympathetic irritation. The three remaining cases showed negative reactions and in these cases the injured eyes were enucleated as a precautionary measure. Two other cases of old .sympathetic ophthalmia showed negative reactions.

The one case of malignant sympathetic ophthalmia showed, in addition to a negative blood picture, a positive skin reaction to the intradermal injection of pigment. In this case uveal pigment was used as a therapeutic agent. The patient was first desensitized to pigment and then actively immunized, with apparently beneficial results.


This case of sympathetic ophthalmia occurred, in a boy aged S, after an operation for a perforated corneal ulcer with prolap.se of iris after a gonorrheal ophthalmia. The inflammation pursued a steadily progressive course with all the symptoms of the severe type of sympathetic disease, despite all usual treatment — diet, intestinal irrigations, pilocarpin sweats, mercury inunctions, large doses of sodium salicylate, and non-specific protein therapy. Finally, following desensitization and immunization with uveal pigment, the process came to a stop, the ej'e became white, free from inflammation, the tension fell to normal, and has so remained. The process continued to be active for three months but has now been stationary for two months. Vision: 6/60. Peripheric retraction and flat total adhesion of iris; capsular opacities. Tension, normal.

BIOGRAPHY

1. Woods, A. C: Immune reactions following injuries to the uveal tract. Journ. Am. Med. Assn., 1921, 77, 1317.

2. Idem.: Ocular anaphylaxis, IV. The antigenic properties of uveal tissue as shown by complement fixation. Arch. Ophth., 1917, XLVI, 503.

3. Peters: Graefe-Salraisch Handbuch. Hess. Ill Edition.


OBSERVATIONS ON THE PRODUCTION OF TOXIC SUBSTANCES BY PNEUMOCOCCI

By Alan M. Chesney, M.D. and Alfred B. Hodges, M.D.

(From the Biological Division of the Medical Clinic, Johns Hopkins Medical School)


Introduction

In spite of the advances that have been made in our knowledge of the etiology and epidemiology of pneumococcus lobar pneumonia during the past decade, it must be admitted that but little progress has been effected in our understanding of the pathological physiology of the infection or of the factors that are responsible for the chain of events chai'acterizcd by pneumococcus infection in man and to which we give the name lobar pneumonia. The common explanation for many of the events that take place during the course of lobar pneumonia is the assumption that along with the initiation of an inflammatory reaction in the lungs a substance or several substances are formed which are capable of being ti-ans ported by the lymph or the circulating blood to distant portions of the body, there to bring about alterations in structure or function of vital organs. It is difficult to explain many of the phenomena of pneumonia without resort to this assumption, and it has therefore gained considerable favor with those who have observed the disease in man. That there is a profound alteration in the function of organs at some distance from the primary focus is not to be denied. Furthermore, the time-honored observations that the magnitude of these alterations is


often out of proportion to the extent of the pathological process, that they so often disappear abruptly at an appreciable interval of time before the local pathological process gives evidence on physical examination of beginning to clear up and while viable pneumococci may still be present in the lung tissue,' all support the conception that in pneumonia one has to deal with a toxiemia as well as a local infection and at times a bacten^mia.

The conception of the existence of a toxemia in pneumonia is an attractive one, because it fits in so well with what one observes in the pneumonia patient, and is a relatively simple hypothesis. When, however, one attempts to test the validity of this hypothesis in the light of what is definitely known about pneumococcus infections, one realizes at once how difficult it is to bring forward in support of such a view evidence of an experimental nature which is at all convincing. It is impossible to say at the present time whether or not tlie pneumococcus through its own metabolic activity and independently of the host produces a toxin or toxins, or whether such a substance or substances are the joint product or products of a i"eaction between the parasite on the one hand and the cells of the host on the other. The latter conception has of recent years been gaining ground with some students of the infection.



Historical

Attempts to isolate toxic substances from pncumococci have been manifold. The earlier workers, following the lead given bj' the work on diphtheria to.xin, sought for poisonous substances in the fluid portion of cultures of pneumococcus. The reported results are .somewhat conflicting but in the main negative. Kruse,^ in 1910, and Neufeld and Handel,^ in 1912, after a review of the earlier literature came to the conclusion that active poisons had not been obtained from the pneumococcus which could with any assurance be regarded as being identical with those which are believed to be present in pneumococcus infection in man and to give rise to the symptoms of general intoxication. Wadsworth ^ concluded from his experiments that "dead pneumococcus culture material does not contain the active poisons formed in infection by living pneumococci."

Following Friedberger's work on the production of "bacterial anaphylatoxin" in the test-tube Neufeld and Dold ^ produced a similar substance, using pneumococcus as antigen, and these same workers " showed later that by extracting pneumococci in salt solution containing 0.1% lecithin toxic substances could be obtained, which, upon intravenous injection into guinea-pigs, resulted in death with symptoms of anaphylaxis. About the same time Rosenow,' by extraction of pneumococci in salt solution for forty-eight hours at 37° C, secured solutions which on intravenous injection into guinea-pigs gave similar results. Cole ' Avorked with solutions obtained by dissolving pneumococci in sodium chelate. With such solutions injected intravenously into guinea-pigs he obtained anaphylactic-like symptoms including death with pathological findings similar to those encountered in serum anaphylaxis, and in the test-tube he was successful in producing lysis of red blood cells. Similar results were obtained by him with solutions of pneumococci obtained by freezing and grinding the bacteria. The hemolytic action of solutions of pneumococci could be inhibited by a scrum prepared by injecting the solution into sheep and rabbits, as well as by minute amounts of cholesterin. Cole regarded the substances derived from pneumococci by procedures calculated to disrupt the cell as existing preformed in the bacterial cell and not as being the product of digestion as ordinarily understood by that term. He would class these substances as endotoxins.

S. Solis-Cohen, Weiss and Kolmer " repeating Cole's experiments were "unable to produce uniformly a toxin which was hemolytic and also produced anaphylaxis on primary intravenous injection in guinea-pigs." Weiss,'" however, was able to produce it in sufficient quantities to arrive at the conclusion that it is very labile, tliat it is a true protein, that it inhibits the agglutinating action of antipneumococcal serum but is itself not affected by antipneumococcus serum, and that upon intracutaneous injection into sensitized guinea-pigs it produces an erythema and hemorrhagic edema in tlie suI)cutaneous


tissue overlying the muscle. From our own experience in working with sodium cholate solutions of pneumococci we can testify to the difficulty of securing a toxin from pneumococci by this method with any great degree of regularity. Because of the inconstancy of the results we were forced to abandon our attempts to study this substance.

The search for pneumococcus toxin has not been confined solely to the study of the organism itself. It is but natural that poisonous substances should be sought for in material derived from the pathological process as well. Issaeft" " found that filtered blood of infected animals as well as blood or peritoneal exudate sterilized by heating at 58° C. for two hours was toxic in amounts of one to two per cent of body-weight. The Klemperers '- succeeded in killing young rabbits with the blood serum of infected rabbits in doses of two per cent of the body-weight. F'. Klemperer '^ found that the filtrate of the fresh pleural exudate of a dog was toxic for other dogs. Kruse and Pansini " were not able to demonstrate toxic .substances in the blood of infected animals, nor was Cole.'^ The latter, however, found that the peritoneal exudates obtained from guinea-pigs dying after an intraperitoneal injection of pneumococci contained substances, which on intravenous injection into normal guinea-pigs gave ri.se to immediate .symptoms like those seen in anaphylaxis and killed about one third of the animals with anaphylactic shock. Rosenow and Arkin,'" using extracts of jineumonic lungs and injecting normal dogs intravenously, produced changes which they regarded as similar to those seen in dogs dying after anaphylactic shock. Weiss, Kolmer and Steinfield '" investigated the toxicity of pneumonic lungs, and by subjecting the pathological tissue to pressure in a hydraulic press succeeded in obtaining a juice which, when sterilized by the addition of phenol or by heating to 56° C. for thirty minutes, was two to five times as toxic for mice, guinea-pigs and rabbits as was a juice obtained from normal human lungs treated in the same manner. Extracts of consolidated lungs in which the consolidation was produced by the intra-bronchial injection of aleuronat were in the main more toxic than extracts of normal lung and less toxic than those of pneumonic lungs. These extracts of pneumonic lungs were also found to be lytic for red blood cells. Manipulations, such as heating, drying and filtration through Berkefeld filters, decreased the toxicity of these extracts. It should be pointed out that the differences in toxicity of the extracts of pneumonic lungs and those of normal lungs were not so great as entirely to exclude the experimental error resident in all biological experimentation.

DisorssiON

One may summarize the previous work on this phase of pneumococcus infection as follows: There is general agreement amongst the workers in this field that pneumococci or pathological material the result of pneuniococcus infection can be so manipulated as to yield (somewhat irregularly) substances which upon intravenous injection into laboratorj' animals give rise to symptoms and lesions which are identical with those observed after a second dose of protein-containing material has been administered to an animal previously sensitized to that protein. The correct interpretation of these facts is not so easy ; their relation to pneumococcus infection in man is by no means clear, as Cole and others have pointed out. There is no direct evidence to show that the toxic substances obtained by manipulating the penumococcal cells or exudate produced by them are ever formed in the human body during the course of pneumococcus infection. If it is true that they are formed solely as a result of solution of the bacterial cell, and if they are produced during the course of pneumonia and are responsible for the general toxic symptoms encountei-ed in that disease, we must suppose that many pneumococci are destroyed in the lung during the course of pneumonia and even at the outset of the disease, for it is well known that the toxic symptoms of pneumonia are often quite Severe at that time as well as later. To what extent extracellular destruction of pneumococci takes place in the consolidated lung is likely to remain a matter of conjecture and not susceptible of determination. Whether or not such a process does occur, it is certain that many of the organisms are taken up by the leucocytes and destroyed within the cells, and it is difficult to see how this intracellular process of destruction could result in the setting free of pneumococcus endotoxin unless the leucocytes can excrete such a substance without alteration. The work on agar anaphylatoxin and particularly the studies of Novy and his associates ^^ seem to us to do away with the idea that the animal effects observed after injection of pneumococcus toxin are specific or characteristic, no matter whether the toxin is obtained from the pneumococci themselves or from pathological exudates. In view of their work the query may be raised whether the occurrence of anaphylactic symptoms in guinea-pigs after intravenous injection is a sufficiently safe criterion for the determination of the toxicity of bacterial preparations. Further evidence of toxicity, such as might be elicitated by subcutaneous or intraperitoneal injection, would seem to us desirable before concluding that one was dealing with a toxin capable of being formed in man in amounts sufficient to give symptoms. It should be noted here that Weiss, Kolmer and Steinfield ^^ obtained deaths after intraperitoneal injection of the exudate from pneumonic lungs in amounts considerably less than those needed if cultures were used in the preparation of the toxin. It will be generally admitted that the toxic substances thus far described are of a low potency for the customary laboratory animals. This fact is evidence for the view that such substances play a slight, if any, r61e in the pneumococcus infection in man, although it is possible that a toxin of a low potency operating over a


prolonged interval of time may be just as deleterious as one of high potency acting over a short period.

It has seemed to us much more reasonable to associate the phenomena of intoxication in pneumonia with the growth of pneumococci rather than with their death and dissolution. When one examines the peritoneal exudate of mice dying after an intraperitoneal injection of living pneumococci, one sees evidence of tremendous proliferation of the bacteria and, as judged by the Gram stain, almost all appear to be in a healthy condition. Such mice invariably give a positive heart's blood culture. This same picture, moreover, is seen when mice are injected with sputum containing many other bacteria beside pneumococci. In such instances the pneumococci rapidly outstrip the other bacteria in their growth (frequently the latter are in great part destroyed) and one sees an overwhelming infection with pneumococci as if conditions were ideal for the growth and multiplication of those bacteria and not of the others. One certainly does not get the impression that pneumococci have been destroyed in great numbers in the peritoneal cavity of the mouse, and yet the mouse shows symptoms of intoxication and ultimately succumbs. It is difficult to conceive of rapid growth and rapid extra-cellular death of pneumococci occurring at the same time and in the same place, where conditions are as favorable for growth as they appear to be in the peritoneal cavity of mice.

If the toxic phenomena of pneumonia wei'e associated with the growth and multiplication of pneumococci, it seemed to us conceivable that toxic substances might be present in a culture of pneumococcus while the organisms were growing rapidly, and that the chemical nature of such substances m;ight be altered during the later stages of growth of the culture so that they would no longer be recognizable as such. In some studies on the nature of bacterial lag one of us -" had been impressed by the fact that, when pneumococci are grown in meat infusion broth at 37° C, the maximum number of bacteria is attained within eighteen hours as a rule, sometimes earlier, and that after this period there is often a falling off in the number of living bacteria. The period during which pneumococci multiply at the maximum, rate of growth in favorable fluid media is relatively short, not longer than twelve hours as a rule, and during this time significant changes take place in the H-ion concentration of the medium.-' It seemed to us that this might constitute a desirable time at which to search for pneumococcus toxin, since the more acid condition of the medium later on miight bring about destruction of the relatively unstable substance formed during an earlier phase. Support for this view is offered by the work of De Kruif and Ireland,-who have shown that the hemolysin produced by the streptococcus hemolyticus reaches its maximum at a very early period in the life of the culture, begins to disappear when the period of active growth ceases, and may disappear completely in fourteen hours. Zinsser and his


428


[No. 382


associates ~^ have recently reported findiug substances toxic for rabbits in cultures of a number of different species of bacteria and have found that the potency of these substances, while admittedly low, was in many instances greater in the case of young cultures, and appeared to diminish with age. They failed to find indication of the formation of such substances in the case of pneumococcus, type I, or staphylococcus aureus "in a few isolated experiments."

Because of the importance of the problem and because we have been interested in the question for several years, it seems to us desirable to extend these observations on the pneumococcus, making use not only of the ordinary fluid media but also of substances such as human serum and human blood, in order to approach as closely as possible conditions in the body.

Most workers in bacteriology have in the past dealt with cultures as old as or older than eighteen hours. They have lost sight of the fact that in fluid cultures of actively growing organisms the maximum number of bacteria is usually attained by that time, and that one is no longer dealing with a culture the members of which are multiplying actively. Chemical conditions in a culture of that age are quite different from those obtaining a very short while afterward.^* To test the assumption that there might be found in fluid cultures of pneumococci, during the period of active growth, substances of a toxic nature, a series of experiments were carried out. It may be said in advance that they yielded only negative results. For that reason the results will not be given in detail, but only a general statement of the method and findings will be submitted for purposes of record.

Technique

Two strains of pneumococci type I, isolated from recent cases of lobar pneumonia, were used. The media employed were: (1) beef infusion broth prepared according to the method described by Avery and Cullen;-'^ (2) normal human serum; and (3) defibrinated human blood. The last two media were employed with the idea that conditions existing in the human body would be more closely approximated than in the case of beef infusion broth. Incubation was at 37° C. Specimens were removed at frequent intervals for filtration and plating for counts. Filtration was carried out with previously tested Berkefeld N candles. The clear filtrates were injected intraperitoneally into white mice in am(ounts of one cubic centimeter each. Bacterial counts served to show at what phase in the period of growth the specimens were removed.

Results

In no instance were any toxic effects observed in the mice, no matter whether the specimens were obtained from the culture at the beginning, during the middle


or toward the end of the period of maximal rate of growth. Our experiments, therefore, were entirely negative so far as demonstrating the presence of a soluble toxin in the fluid portion of a rapidly growing culture of pneumococcus in beef infusion broth, normal human serum or defibrinated human blood. They cannot be said to throw any light on the complex problem of the cause of the intoxication in lobar pneumonia.

Summary and Conclusions

1. The relationship of the toxic substances obtained from pneumococci by solution of the bacterial cell or by manipulation of pneumonic exudates to the phenomena of intoxication in pneumonia in man is discussed.

2. Filtrates of actively growing cultures of pneumococci in beef infusion broth, normal human serum and defibrinated normal human blood are not toxic for mice when injected intraperitoneally.

LITERATURE

1. Thomas, H. M., Jr., and Parker, F., Jr.: Arch. Int. Med., 1920, XXVI, 125.

2. Kruse, W.: AUg. Microhiologie, Leipzig, 1910, 958.

3. Neufeld, F., and Handel, L. : Kolle und Wassermann, Handbuch der Pathogenen Mikroorganismen. Jena, 1912.

4. Wadsworth, A. B.: Jour. Exper. Med., 1912, XVI, 54.

5. Neufeld, F., and Dold, H.: Berl. klin. Wchnschr., 1911, XLVIII, 55.

6. Neufeld, F., and Dold, H.: Berl. klin. Wchnschr., 1911, XLVIII, 1069.

7. Rosenow, E. C: Jour. Inf. Dis., 1911, IX, 190.

8. Cole, R.: Jour. Exper. Med., 1912, XVI, 644.

9. Solis-Cohen, S., Weiss, C, and Kolmer, J. A.: Jour. Inf. Dis., 1918, XXII, 476.

10. Weiss, C: Jour. Med. Research, 1918-19, XXXIX, 103.

11. IssaefE: Ann. de I'inst. Pasteur, 1893.

12. Klemperer, G. and F.: Berl. kiln. Wchnschr., 1891.

13. Klemperer, F.: Cited by Neufeld and Handel in Kolle und Wassermann, Handbuch der Path. Mikroorganismen.

14. Kruse, and Pausini: Ztschr. f. Hyg., 1892, XI, 279.

15. Cole, R.: loc. cit.

16. Rosenow, E. C, and Arkin, A.: Jour. Inf. Dis., 1912, XI, 480.

17. Weiss, C, Kolmer, J. A., and Steinfield. F.: Jour. Inf. Dis., 1918, XXII, 469.

18. Novy, F. G., de Kruif, P. H. et ah: Jour. Inf. Dis., 1917, XX, 498-535.

19. loc. cit.

20. Chesney, A. M.: Jour. Exp. Med., 1916, XXIV, 387.

21. Cullen, G. E., and Chesney, A. M.: Jour. Exper. Med., 1918, XXVIII, 289.

22. De Kruif, P. H., and Ireland, P. M.: Jour. Inf. Dis., 1920, XXVI, 285.

23. Zinsser, H., Parker, J. T.. and Kuttner. A.: Proc. Soc. Exper. Biol, and Med., 1920-21, XVIII, 49.

24. CuUen, G. E., and Chesney, A. M.: loc. cit.

25. Avery, O. T., and Cullen, G. E.: Jour. Exper. Med., 1920, XXXII, 547.



INFLUENZA-LIKE BACILLI GROWTH OF INFLUENZA-LIKE BACILLI ON MEDIA CONTAINING ONLY AN AUTOCLAVE-LABILE SUBSTANCE AS AN ACCESSORY FOOD FACTOR

By T. M. Rivers, M. D.

(From the Department of Pathology and Bacteriology of The Johns Hopkins University, Baltimore)


For a long time, in spite of uuiuerous studies, very little was definitely known about influenza bacilli. Practically every statement in regard to them has been challenged, leading to numerous controversies, one of which was whether they actually required hemoglobin for their growth. Pfeiffer ' at one time thought that on a purely morphological basis he could distinguish pseudo-influenza bacilli from the true forms. Wolff,^ in 1903, stated that Pfeiffer had decided his original idea of pseudo-influenza bacilli to be incorrect. Later, pertussis bacilli led to more confusion and some workers ^ speak of pertussis-like bacilli without describing them accurately enough for others to recognize these organisms when found. When the pandemic of influenza came in 1918, B. influenza' was characterized as a Gram-negative, non-motile, pleomorphic, aerobic, hemoglobinophilic bacillus. All strains were considered more or less alike serologically, regardless of morphology or virulence.

Since 1918 much profitable work has been done on influenza bacilli. It is known that there are hemolytic and nonhemolytic varieties, that they usually reduce nitrates to nitrites, that some form indole while others do not, that some ferment no sugars while others attack various carbohydrates. The most interesting work, however, has been that dealing with the growth requirements *"' of these organisms. It has been definitely shown that true influenza bacilli require two accessory food substances, one of which resists autoclaving at 1.5 pounds for 30 minutes, while the other does not. Both substances are present in blood. They have been found elsewhere ; the autoclave-stable factor in unfertilized eggs and potatoes, the autoclave-labile one in yeast extract, potatoes, tomatoes, beans and carrots. Respiratory strains differ culturally "-^^ and show a marked serologic heterogeneity.'*. 15 This diflference is not so evident in the group producing meningitis.'** Most of the meningitic strains examined were closely related culturally and serologically. B. hcwoglobinophiliis canis, described by Friedberger '^ and placed in the hemophilic group, has been shown '" to require the addition of only the autoclave-stable substance as an accessory food factor. This bacillus has much in common with influenza bacilli, and several years ago it would not have been easy to differentiate it from them.

Has all this work made it possible to say that any given bacillus does or does not belong to the influenza group? Is there some one thing common to all of them


that no other group possesses? Are there organisms found in throat and lung cultures that are not influenza bacilli, yet which might be mistaken for them? Many of these questions can be answered now. Pertussis bacilli when first isolated are rather difficult to grow. Special media have to be used, but after the strains have been isolated for some time neither of the food accessory factors are required. B. hemoglohuwjjhilus canis requires the addition of only the autoclave-stable substance. Hemolytic and nonhemolytic influenza bacilli, after from 2 to 7 years' cultivation on artificial media, still need both accessory food substances. Certain bacilli to be discussed in this paper, after 2 years' cultivation outside the body, will grow on media to which only the autoclave-labile factor has been added as an accessory food substance. These bacilli might be mistaken easily for influenza or pertussis bacilli and probably have been so mistaken in the past.

Description of Tavo Influenza-like Bacilli

Bacillus Xo. 1 was isolated from a throat culture of a patient with influenza in February, 1920. Bacillus No. 2 was isolated from the right lung of a patient who died of pneumonia following influenza in February, 1920. The throat culture of this patient yielded hemolytic streptococci and hemolytic influenza bacilli. A culture from the trachea showed hemolytic streptococci, hemolytic influenza and nonhemolytic influenza bacilli. From the right lung hemolytic streptococci were recovered in addition to bacillus No. 2. The nonhemolytic influenza bacillus recovered from the trachea was a Gram-negative, nonmotile, pleomorphic bacillus which formed indole, reduced nitrates to nitrites, fermented no sugars, produced little or no change in blood-milk, and required both accessory food factors for growth.

Growth on Blood-Agar. — These influenza-like bacilli grow somewhat more slowly on blood-agar than ordinary influenza bacilli, but the colonies are enough like those of the latter to be mistaken for them. This is especially true of No. 2. Young colonies of No. 1 are regular, round, glassy and transparent. Older colonies have a brownish granular center and occasionally very old colonies have a crinkled surface ^Wth radiating folds (Fig. I, la and lb). The colonies of bacillus No. 2 are flatter, not so glassy, and have a more granular brownish center (Fig. I, 2). The colonies of No. 1 are usually smaller and not so moist as those of No. 2. The difference in size of colonies is not always so marked as in the figures. Colonies vary from 1 to 3 mm. in diameter. Both bacilli grow well under aerobic conditions.

Morphology, Staining, Motility. — The organisms are Gram-negative, non-motile, pleomorphic, non-sporebearing bacilli which cannot be differentiated from influenza bacilli morphologically. As a rule No. 1 (Fig. II) is smaller and more regular than No. 2 (Fig. III).

Hemolysis. — Neither strain hemolyses red blood cells.

Production of Indole. — Neither bacillus forms indole.

Reduction of Nitrates. — Both bacilli reduce nitrates to nitrites.

Production of Amylase. — A starch-splitting ferment is not produced by either strain.

Action on Milk. — No marked change was noticed in the blood-milk tubes after incubation for 3 weeks.

Fermentation of Sugars. — The fluid and solid media for fermentation tests were prepared as described in a previous paper.'" Dextrose, sucrose, maltose and xylose were not fermented in a liquid medium by either bacillus. On a solid medium No. 1 showed a questionable acid formation from dextrose, sucrose and maltose.

Growth Requirements. — The autoclave-stable and the autoclave-labile growth accessory substances were prepared and used as indicated in a previous paper.'* The stable factor was hematin, the labile one was yeast extract sterilized by means of a Handler filter.

The two influenza-like bacilli would not grow for more than a generation or two on meat extract agar, meat infusion agar, 2 per cent peptone agar, 2 per cent peptone agar and hematin, 2 per cent peptone water, 2 per cent peptone water and hematin, or 2 per cent peptone water and ascitic fluid. The results of these experiments are summarized in Table. I.

TABLE I.

Growth Accessory Requirements of Certain So-called

Hemophilic Bacilli.



+ Indicates that successful transplants were made as long as desired. • — Indicates that successful transplants were not obtained, or for one or two generations only.

In meat infusion broth these influenza-like bacilli grow well for one generation ; after that none of the tubes showed any gross turbidity. If, however, amounts of 1 c.c. were inoculated daily from tube to tube, transplants


from the tubes to blood agar showed from 10 to 30 colonies regularly for many generations. Evidently there was a slight multiplication of the bacilli, or at least they did not die for a long time. This could not have been told from the gross appearance of the broth tubes and was discovered by making transplants to blood agar. The meat infusion broth was autoclaved. This discrepancy may be more apparent than real, for it may be possible for the heat-labile factor to withstand a short autoclaving without being entirely destroyed.

In Table II are listed the most impiirtant differential biological reactions -of the hemophilic bacilU so far studied.

TABLE II.

Differential Biological Reactions of Certain So-called

Hemophilic Bacilli



Reduction

of

Nitrates


Indole


Motil

Hemolysis of Red


Fermen of

Sugars


Action Milk


Accessory Food Factors



.^"'.' ity Blood duction g^„^


AutoclaveStable


Auto, clave.

Labile


B. pertussis

not

recently

isolated



-



Strongly Alk.




B. hemoelabino. phitas


+


+




+


Indefinite


+



Influenza.

like

bacilli

Nos. 1 and 2


+





±


Indefinite



+


B. innuen: minutes after the anesthetic was begun, so that the effect of ether upon liver function, as shown by Whipple,-- could be neglected. Paraldehyde anesthesia was used during the experimentation upon dog 6. 15 mgs. of phosphorus in oil were given intramuscularly. Four days later the animal was quite ill ; considerable bile was present in the plasma and urine; on this day a high curve was obtained (Fig. 7). The animal died while under anesthesia. Autop.sy showed the characteristic liver changes of phosphoi'us poisoning.



Dog 14 (Fig. 8) became quite ill by the second day following the injection of 15 mgs. of phosphorus, although little bile was present in either the plasma or urine. A moderately high curve was found at this time; the low readings were rendered only approximately accurate because of hemolysis. The animal died at the end of the experiment.

The normal curve of dog 16 was obscured by lipemia. Forty-eight hours after the injection of 15 mgs. of phosphorus, the phthalein test was again made, and readings fell within the range of early abnormality ; the animal did not appear ill. Five days after the injection, icterus was present, and the dog appeared to be in a toxic condition. A curve characteristic of moderately severe liver damage was found (Fig. 9). The animal was killed because of a septic condition of the neck ; the liver presented typical gross changes of necrosis and fatty degeneration.


December, 1922]


435



J~-> SO 35


11


JO IS 10 25 30 35 +0


Ligation of the Common Duct

In acute obstructive jaundice there is dilatation of the biliary passages behind the obstruction ; the hepatic cells and cells of Kupfer become deeply pigmented, but there is very slight cell degeneration, except after prolonged obstruction.-'- -*- "• ""• -~ The liver cells apparently continue their functions, including the excretion of bile, which is taken up by the blood capillaries and lymphatics.-"' -'• ^^' -'

It therefore seemed of interest to find out how rapidly the dye would be removed from the blood stream following ligation of the common bile duct. After the normal disappearance time had been determined, the common ducts of five dogs were ligated, and after allowing varying intervals of time to elapse, curves were again obtained. The striking fact was brought out, that in these


animals the tetrachlorphthalein was removed from the blood stream in normal, or almost normal time, except that a trace of dye remained present in the blood as long as these experiments were continued.


Minutes


2 3 14

1


s


10


15


30


45


1 Hr.


2Hrs.


3 Hrs.


4 hrs.


i


Day before Ligation


15.




8.


T.


T.


T.







a


1 hour after


13.





T.


T.








Z Q


2 days before Ligation



10.




T.



O.







2 days after



12.5




3.9.


F.T.


F.T.


F.T.


F.T.





O


3 brs. before Ligation




7.3



P.T.


F.T.








2 days after



10.




F.T.


F.T.








d


1 hour before Ligation



7.




3.


V.F.T.








2 days after



10.




5.


L.T.


L.T.


L.T.


L.T.


L.T.


L.T.


L.T.



Per cent of dye in plasma


The high curves in Dog No. 3 are due to poisoning by chloroform administered a week previously.


Only one hour had elapsed after ligation in dog 3, when the second test was made; there was no essential difference from the curve obtained previous to ligation (Fig. 10). Xo bile had appeared in the plasma or urine. Two days after ligation dogs 7, 10 and 21 received their second injections of the dye. The cystic ducts as well as the common ducts were ligated in dogs 10 and 21. All of these animals became icteroid. Curves very slightly higher than the normals for these dogs were reached in the second determinations, but the differences are negligible (Fig. 10).

A curve was obtained in dog 21 three days after ligation of the common duct (Fig. 11). It shows the practically normal rate of disappearance and the persistence of a trace of dye in the plasma is demonstrated, in that, twelve hours after this injection of dye, the "ring test" on the plasma was positive and at this time a large amount of phthalein was present in the urine. Twentyfour hours after the injection, the ring test on the plasma was just barely detectable, and the dye was still present in a catheterized specimen of urine. Following the injection of phthalein in these animals with obstructive jaundice, varying amounts appeared in the urine of all of them except dog 3, who was killed 15 minutes after the injection was made. The appearance of this dye in the urine during experimental obstructive jaundice has been previously studied by Whipple.*


Fig. 11

It is felt that these curves are doubly significant. Firstly, it seems probable that the taking up of the dye by the liver cells, and its excretion in the bile, are independent functions, bearing in mind that chronic obstruction will undoubtedly produce cell damage. The former function seems to be dependent upon the extent and degree of degeneration of the liver cells. The latter function and the time of appearance of the dye in the bile would appear to be influenced greatly by the patency of the biliary passages, and would not necessarily be an index of the condition of the liver cells.

Secondly, while it is felt uiiwise to draw broad conclusions until the test has been given the trial of time and usage, the writer wishes to point out that, in these experiments, a method has been demonstrated that differentiates the early jaundice due to mechanical obstruction in which cell damage is slight, from the jaundice associated with degeneration of the hepatic epithelium. Experiments are being conducted with phenolsulphonphthalein to determine whether an analogous behavior occurs in renal conditions, in an attempt to develop a test for kidney function upon the same principle.

Summary and Conclusions

Phenoltetrachlorphthalein has been intravenously injected and its behavior in the blood stream has been studied in normal animals, in dogs following chloroform poisoning, phosphorus poisoning, and in dogs after ligation of the common and cystic bile-ducts.

The concentrations reached in the plasma of normal dogs, after 5 mgs. per kilo injections, are strikingly constant. Approximately 10 per cent is present in 2 minutes, 8 per cent in 3 minutes, and from a large trace to absence in 15 minutes. The rate of disappearance was found higher in two cats, and more rapid in one rabbit. Variation of the dose has a direct influence upon the curve obtained.

There is a marked increase in these concentrations, with a greatly prolonged rate of disappearance, after injury to the liver parenchyma; the extent to which this occurs depends upon the degree of damage to the liver cells, as evidenced by the condition of the animals, and by autopsy findings. Curves approaching normal are obtained if repair has taken place.


Curves have been obtained in jaundice due to mechanical obstruction that differ widely from those found in the jaundice associated with extensive degeneration of the hepatic cells.

A simplified method is herein presented for quantitative determination of the amount of dye in the plasma. A practical test for its qualitative detection is also described.

A clinical study is in progress, and a series of cases will be reported in the near future.

REFERENCES

1. Rosenthal, S. M.: Jour. Pharm. and Exp. Therap., 1922, XIX, 385.

2. Abel, J. J. and L. G. Rowntree: Jour. Pharm. and Exp. Therap., 1909, I, 231.

3. Rowntree, L. G., A. L. Bloomfield and Hurwitz: J. H. H. Bull., 1913, XXIV, 329.

4. Whipple, G. H., V. R. Mason and T. C. Peightal: J. H. H. Bull., 1913, XXIV, 207.

5. McLester, J. S. and B. Frazier: Jour. A. M. A., 1915, LXV, 383.

6. Kahn, M. and J. R. Johnston: N. Y. Med. Jour., 1915, CII, 848.

7. Sisson, W. R.: Arch. Int. Med., 1914, XIV, 804.

8. Chesney, A. M., E. K. Marshall and L. G. Rowntree: Jour. A. M. A., 1914, LXIII, 1533.

9. Krumbhaar, E. B.: N. Y. Med. Jour., 1914, C, 719.

10. McNeil, H. L.: Jour. Lab. and Clin. Med., 1916, T, 822.

11. Aaron, A. H., E. C. Beck and H. C. Schneider: Jour. A. M. A., 1921 LXXVII, 1631.

12. Whipple, G. H., and Sperry: J. H. H. Bull., 1909, XX, 279.

13. Joannovicks and Pick: Berlin Klin. Woch., 1910, XLVII, 928.

14. Whipple, G. H. and S. H. Hurwitz: Jour. Exp. Med., 1911, XVIII, 136.

15. Bevan, A. D. and H. B. Favill: Jour. A. M. A., 1905, XLV, 691.

16. Fischler and Bardach: Ztschr. f. Physiol. Chem., 1912, LXXVIII, 435.

17. McCallum, W. G.: Textbook of Pathology, 1916. p. 386.

18. Simonds, J. P.: Jour, Exp. Med., 1918, XXVIII, 663; Arch. Int. Med., 1919, XXIII, 362.

19. Hoppe-Seyler, G.: Dis. of Liver. Nothnagels Encyclopedia, 1903, 839.

20. Hedderich, L.: Munch. Med. Wchschr., 1895, XLII, 93.

21. Opie, E. L. and L. B. Alford: Jour. Exp. Med., 1915, XXI, 1.

22. Whipple, D. H. and J. S. Speed: Jour. Exp. Med., 1915, XXI, 203.

23. McCallum, W. G.: Textbook of Pathology, 1916, pp. 122, 397.

24. Geraudel, E.: Jour, de physiol. et de path, gen., 1906, VIIX, 69.

25. Vaughan, H.: Brit. Med. Jour., 1892, II, 397.

26. Beloussow: Arch. f. Exp. Path. u. Pharm., 1881. XIV, 200.

27. McMaster, P. D. and Rous: P. Jour. Exp. Med., 1921, XXXIII, 731.

28. Eppinher: Ergeb. d. Inn. Med. u. Kinderh., 1908. I, 107.

29. Whipple, G. H. and J. H. King: Jour. Exp. Med., 1911, XII!. 115.



LEGENDS

Fig. 1. — Dog No. 5. Chloroform Poisoning. May 5, normal curve (1). May 7, two hours and fifteen minutes chloroform anaesthesia. May 8, curve (2), eighteen hours later. Dog not very ill; chloroform again given for one hour. May 9, curve (3), second day. May 12, curve (4), fifth day. May 18, curve (5), eleventh day.

Fig. 2. — Table I. Preparation of series of standards to which specimens of undiluted plasma are compared directly. 10 mgs. of phenoltetrachlorphtalein to 100 c.c. of water used as 100 per cent solution of dye.

Fig. 3. — Curves of disappearance of phthalein from blood stream. 13 curves on normal dogs; 5 mgs. per kilo dosage.

Fig. 4. — Variation of the dosage of dye in normal dogs. Curve (1), Dog No. 11: 1.7 mgs. of tetrachlorphthalein per kilo. Curve (2) and curve (3), Dog No. 12 and Dog No. 13: 2.5 mgs. per kilo. Curve (4), Dog No. 15: 10 mgs. per kilo. Curve (5), composite curve constructed from Fig. 3, 5 mgs. per kilo.

Fig. 5. — Curves on normal cats and rabbit. Curve (1), Cat No. 2, 5 mgs. per kilo. Curve (2), Cat No. 2, 10 mgs. per kilo. Curve (3), Cat No. 3, 5 mgs. per kilo. Curve (4),


Rabbit No. 1, 5 mgs. per kilo. Curve No. 5, composite curve from Fig. 3.

Fig. 6. — Dog No. 18. Phosphorus poisoning with convalescence. Curve (1). before intramuscular injection of 12 mgs. of phosphorus. Curve (2), three days after injection. No icterus; dog not ill. Curve (3), six days after injection. Bile in urine and plasma; moderately toxic. Curve (4), eleven days after injection. Dog appears normal; small trace of bile in urine.

Fig. 7. — Dog No. 6. 15 mgs. of phosphorus. Curve (1), normal. Curve (2), four days after injection. Animal quite ill; moderate jaundice.

Fig. 8. — Dog No. 14. 15 mgs. of phosphorus. Curve (1), before injection. Curve (2), two days later. Dog very toxic.

Pig. 9. — Dog No. 16. 15 mgs. of phosphorus. Curve (1), (above), two days after phosphorus injection. Curve (2), (below), five days after injection: dog moderately ill, slight jaundice.

Fig. 10. — Table II. Curves before and after ligation of common bile-duct.

Fig. 11. — Ligation of common bile-duct. Dog No. 21. Curve (1), before ligation. Curve (2), three days after ligation; deep jaundice.


STUDY OF TRICHOMONAS HOMINIS, ITS CULTIVATION, ITS INOCULATION INTO ANIMALS AND ITS STAINING REACTION TO VITAL DYES

By Mary Jane Hogue, Ph. D.

(From the Department of Pathology and Bacteriology of The Johns Hopkins University, Baltimore)


In a previous paper the author ' ha.s shown that a pure line of Trichomonas liominis, i.e., a race started from a single individual, can be isolated, and that this pure line can be kept in culture, the medium best suited for this purpose being Locke-egg. In this medium the organism has to be transferred every two or three days, since the cultures reach their greatest development on the second or third day and rarely live more than five or six days.

In the fall of 1921 a vei-y simple method was devised, by which Trichomonas hominis can be easily kept alive in test-tube cultures for thirty-five days and in some cases for over sixty-six days, without being transferred. This greatly facilitates the work of keeping cultures in stock for experimental work.

The stock, from which the pure line used for these experiments was isolated, has been cultured for eight months in this laboratory. The pure line was started when the stock was twp and one half months old, and has been kept under constant observation for the past five and a half months.

Sodium chloride serum water medium. The method used is the following: To a flask containing 100 c.c. of 0.85 per cent sodium chloride solution, which has been sterilized in the autoclave, add 10 to 15 c.c. of sterile sheep serum water. For these animals sheep serum is better than pig serum, though the latter can be used.


The serum water is prepared by diluting one part of sheep serum with three parts of distilled water, and is sterilized in the Arnold sterilizer, being heated at 100° C. for one hour on three successive days. This solution will then have a pH of 7 to 7.4. After thoroughly mixing, pour 15 c.c. of this solution into sterile test tubes and inoculate with Trichomonas hominis by means of a capillary pipette, the transferred material being deposited at the bottom of the test-tube. The surface' of the medium is then covered with sterile paraftin oil to prevent evaporation and the tubes are incubated at 36° C.

The serum in the medium very soon coagulates, owing to the action of the bacteria which are growing in the culture with the Trichomonas. Tliis coagulated material settles to the bottom. It is here that the organisms can be found in largest numbers.

Trichomonas hominis does not divide rapidly in these deep cultures covered with paraftin oil. In counting the organisms in one field (oc. 10, obj. 1(5 mm.) one rarely finds more than eight or ten individuals and usually there are not more than three or four in a field. At this slow rate of division the waste products do not accumulate rapidly and the culture lives for a long time.

When large numbers of Trichomonas are needed for experiments, 10 c.c. of the sodium chloride serum water medium are put into sterile tubes and inoculated with a


438


[No. 382


few drops of a culture containing Trichomonas. Tliis is incubated at 36° C. In twenty-four hours there will be a rich growth of Trichomonas and in forty-eight hours the culture will be swarming with them. Over a hundred have been counted in one field (oc. 10, obj. 16 mm.).

In making counts to determine the relative number of individuals present in a culture, it was necessary to determine whether the organisms are evenly distributed throughout the medium. Samples of the culture were taken from the surface by means of a platinum loop and the number of organisms in one field (oc. 10, obj. 16 mm.) were counted. Specimens of the same culture from the bottom of the tube were taken by means of a capillary pipette, and were studied with the same magnification. The count for the organisms on the surface in five fields was: 15, 14, 12, 17, 13; and for those from the bottom of the same tube, 66, 94, 92, 85, 104. Many such counts were made, but the organisms in the bottom of the tube always greatly outnumbered those on the surface. This would seem to suggest that they are dependent on the bacteria which are collected in great masses in the coagulated serum. Further evidence for this was seen when the surface of the tubes occasionally became covered with a growth of bacteria. The Trichomonas would then often be found swarming on the surface. As many as 280 and 300 individuals have been counted in fields taken from such a surface.

Sodium chloride serum water was made up in flasks, and, with phenol red as indicator, its reaction was adjusted by the colorimetric method to pH 8.4, 8.2, 8, 7.8, 7.0, 7.4, 7.2, 7, 6.8. The scale for this indicator does not run below 6.8, but two other flasks were made up which had a pll of about 6.6 and 6. This was only approximately estimated by the color, but the fluids were known to be decidedly acid. The adjustment was made by adding hydrochloric acid and sodium hydroxide.

From these flasks 10 c.c. were put into sterile tubes by means of sterile pipettes and each tube inoculated with 5 c.c. of a culture of a pure line of Trichomonas hominis which had been grown in a flask to insure a suffiicient quantity for inoculating all the tubes from the same


pH.


Numbef of tuies inoculated


Number of fields examined


Average number organisms in each Held of 48-hr9. culture


Average number organisms in eacb Geld of 96 hrs. culture


6. (?)


5


15


5.8



6.6 (?)


5


15


9.1



6.8


4


12


20.4


4.5


7.


4


12


20.


4.3


7.2


2


6


46.


8.2


7.4


2


6


54.5


10.


7.6


2


6


38.8


5.7


7.8


2


6


51.3



8.


2


6


87.


1.2


8.2


2


6


20.1


4.7


8.4


2


6


44.1


.2


culture. Examinations were made after forty-eight and ninety-six hours. The tubes were first shaken to insure an even distribution of the organisms. Then two loops full of the culture were transferred to a slide and the organisms in three fields counted (oc. 10, obj. 16 mm.).

From the accompanying table it will be seen that Trichomonas grows in an acid as well as in an alkaline medium. The most favorable range of reaction for rapid multiplication is pH 7.2 to pH 8.4, with pH 8 as an optimal point. If it is desired to keep the cultures for a longer time, pH 7.2 and pH 7.4 are better. Here the rate of division is slower and consequently the waste products, which are very poisonous to the organisms, do not accumulate rapidly so that the organisms can live longer.

Animal Experimentation. Several authors have reported inoculating animals with Trichomonas. Escomel ^ said he infected the rabbit, guinea-pig, cat and dog with Trichomonas. Lynch ^ claimed to have infected a white rabbit with Trichomonas from a culture tube b,y injecting them three inches up into the rectum. Three days later he said the rabbit was passing soft brown stools containing many Trichomonas, which had not been previously present. Boyd * said he infected a rat by feeding it milk which contained a portion of a culture of Trichomonas and what he called Trichomonas cysts. The Trichomonas was not plentiful and he said: "It remains to be ascertained whether infection by feeding results from the flagellates or from the cysts. By analogj- the latter would seem probable and my single feeding experiment, taking into consideration the paucity of flagellates present, would tend to support this view." At the present time it is generally recognized that cysts of Trichomonas have not been seen and it is very doubtful whether they are ever formed. With this in mind the results of Boyd seem questionable. Recently Pringault ^ fed Trichomonas to a cat, guinea-pig, rabbit, and white rat and injected them into the rectum of a kitten. All his attempts at inoculation gave negative results.

With these discordant results in mind it was decided to try infecting cats and rabbits, paying especial attention to preliminary fecal examinations, as it has been shown that many intestinal protozoa appear in the stools at intervals several days apart, and, unless the infected animals are watched for a period of at least four or five days, the parasites which they carry cannot be accurately determined.

Accordingly three young cats were isolated and daily fecal examinations made for seven days. None of them was infected with flagellates. They were then fed, by means of a pipette, two-day-old cultures full of Trichomonas hominis. Each cat received from 8 to 9 c.c. of the culture. For six succeeding days the stools were examined microscopically, but Trichomonas did not appear and the stools were normal.


December, 1922]


439


Four kittens, which were three weeks old, were next used. The stools were examined for seven days and found not to contain Trichomonas, though all were infected with Giardia, spirochsBtes and Bclascaris. On the seventh day they were fed 10 c.c. of a culture of Trichomonas hominis containing 20-21 organisms in each field (oc. 10, obj. IG mm.). Daily fecal examinations were made for five days but Trichomonas did not appear. Then 10 c.c. of a three-day-old culture were injected into the rectum of each kitten. The rectal tube was passed two and a half inches up into the large intestine. After this, fecal examinations were made for six days with negative results. The kittens were again inoculated 2)cr rectum with 10 c.c. of a culture containing 25 to 35 Trichomonas per field. The succeeding eleven fecal examinations were negative. A third attempt was made to infect them per rectum, but fecal examinations covering five weeks did not reveal any Trichomonas.

Since both Escomel - and Ljaich ^ have reported successful inoculation of rabbits with Trichomonas, these animals were next tried. Two rabbits were examined daily for nine days and found not to be infected with Trichomonas, though one of them was infected with a flagellate closely resembling Trichomonas which appeared at irregular intervals, often three days apart. The flagellate did not have an axostyle nor an undulating membrane. It resembled somewhat a Prowazekia. It was very plastic. Both rabbits were given per rectum 13 c.c. of a rich culture of Trichomonas. On eight succeeding days fecal examinations did not show Trichomonas, though the other flagellate still appeared at irregular intervals. A second inoculation of 17 c.c. of the culture was given per rectum, but the eight succeeding examinations were negative.



The outlines of Figs. 1, 2, 3, and 4 were made with a camera lucida from a prepared slide (x 3000 diameters). The neutral red granules in Figs. 1 and 2 and the mitochondria in Figs. 3 and 4 were put in by free hand, as the animals were moving too rapidly to draw with the camera lucida.

Fig. 1 shows large neutral red vacuoles containing single neutral red granules.


An attempt was made to inoculate two white rabbits, since Lynch ^ especially designated the use of this kind of rabbit. On these animals preliminary stool examinations were made for four days, which were , negative. They were then inoculated with 25 and 30 c.c, respectively, of a rich culture of Trichomonas. The eight succeeding fecal examinations were all negative.

From the above experiments the conclusion has been drawn that cats, kittens, and rabbits cannot become infected with Trichomonas hominis. Organisms closely resembling Trichomonas were found in the stools of rabbits before and after inoculation. The question is opened whether they have not possibly misled some of the former investigators who claim to have inoculated these laboratory animals with Trichomonas.

Vital Stains. These rich cultures of Trichomonas homitiis offered a good opportunity for studying the effect of vital stains on an intestinal protozoan parasite. Neutral red, methylene blue, brilliant cresyl blue and pyrrol blue were all used in varying dilutions in hanging drops of the culture. The coverslips were sealed with vaseline. Neutral red stained granules in vacuoles which were usually situated in the posterior two thirds of the body (Fig. 1). A few neutral red vacuoles were occasionally seen around the nucleus in the anterior part of the body (Fig. 2). The granules in the vacuoles varied in size and in number, as has been shown to be the case with Amoeba. Brilliant cresyl blue stained the granules a purplish blue and the vacuole pink. Methylene blue stained the granules blue. Pyrrol blue did not stain any part of the Trichomonas.

Mitochondria. With janus green the mitochondria stained a blue green. They were short thick rods arranged in groups and singly (Figs. 3, 4). They were not numerous.



Fig. 2 shows the neutral red vacuoles containing neutral red granules of different sizes. The vacuoles are distributed throughout the body.

Figs. 3 and 4 show the short rod-shaped mitochondria which are scattered about in the body of the Trichomonas.


440


[No. 382


Summary

1. Sodium chloride serum water lias proved an excellent medium for the cultivation of Trichomonas hominis. AVhen the tubes are covered with paraffin oil the organisms live from 35 to 66 days without being transferred.

2. Trichomonas multiplies most rapidly on sodium chloride serum water medium with a pH 8. It lives longest on the same medium with a pH 7.2-7.4.

3. Attempts were made to infect cats, kittens and rabbits with Trichomonas hominis but all attempts failed.

4. Trichomonas hominis contains granules which take


the vital stains, neutral red, methylene blue and brilliant cresyl blue.

5. Mitochondria in the shape of short blunt rods are present and stain with janus green.

BIBLIOGRAPHY

1. Hogue, M. J.: Amer. J. Trop. Med., 1921, I, 211.

2. Escomel, E.: Bull. Soc. Path. Exot., 1913. VI, 120.

3. Lynch, K. M.: Amer. J. Trop. Dis., Prev. Med., 1915. II, 627.

4. Boyd, M. F.: J. Parasit., 1919, V, 132.

5. Pringaiilt, E.: Bull. Soc. Path. Exot., 1920, XIII, 800.

6. Hogue. M. J.: J. Exp. Zool., 1922, XXXV, 1.


ENTERO-URETHRAL FUSION IN A FETUS, SIMULATING FETAL ASCITES

By John C. Baldwin

{From the Department of Pediatrics, Johns Hopkins University)


About 80 cases of congenital fusion of the intestinal and urinary tracts have been described in the literature. These can be divided into a variety of types. In some, the intestine communicated with the bladder, in others, with the urethra. In most instances the junction was in the midline; in many, however, the colon emptied into one side of an asymmetrical bladder. The l)la<l(ler was dilated in relatively few of the cases. With rare exceptions, the abnormality has occurred in males, but a closely related malformation in the female, in which the rectum opens into the vagina, is quite common. A survey of the literature dealing with the subject revealed no case as young as the one here described and none in which the prostatic urethra participated in the formation of the bladder cavity. Most of the reported cases were those of infants born alive, on whom the diagnosis was made because of the passage of meconium through the urethra in the absence of an anal orifice. Surgeons have made use of the nature of the mixture of urine and meconium to locate the site of the fusion. If the fluid passed at the beginning of micturition was meconium and later clear urine was passed, it was assumed that the colon emptied into the urethra; whereas, if the fluid was a mixture of urine and meconium, then the point of union was assumed to be in the bladder. In either case, the communication was usually so small as to preclude the possibility of satisfactory defecation and to make early operation imperative.

The fetus described in this paper (male, 8.5 mm. crownrump length, Carnegie Collection No. 2.")47) was presented to the laboratory by Dr. N. W. Ingalls, of Cleveland, as a case of fetal ascites. Examination showed a bizarre creature with an enormously distended abdomen which made the extremities appear small by contrast ( Fig. 1 ) . The external genitalia were so under-developed as to leave the question of sex in some doubt until after


examination of the gonads. The anal orifice was missing and there was no dear indication of the normal site of the anus. .As fetal ascites is characterized by a disproportionately large abdomen, in the absence of peripheral edema this diagnosis was accepted as correct. The existence of an imperforate anus excited no suspicion of the true condition, since one abnormality is frequently associated with others. Furthermore, a dead-born infant with an imperforate anus alone does not, as a rule, show abdominal distention. At autopsy, however, it was found that the abdominal enlargement was due to an abnormally dilated bladder (Fig. 2).

The thoracic viscera showed no gross anomaly. The liver and small intestine were removed and found to be normal. The kidneys were normal in size and position and sections of them showed no dilatation or cyst formation. The sex glands, which lay just below the pelvic brim, proved to be testicles, thus establishing the sex of the fetus. The ureters were not dilated as far as they could be followed beliind the large bladder. A section of the bladder wall showed the musculature to be made up of non-striated fibers of the usual bladder type.

Transver.se sections 100 fj. in thickness were made from a level just below the lower poles of the kidneys downward througli tlie genitalia. These sections were stained with hematoxylin and eosin. The outstanding malformation shown by a study of these sections is an abnormal relation between the descending colon and the urinary tract. The colon opens into tlie prostatic urethra, which in turn is dilated to such an extent that it has become anatomically a part of the bladder. The bladder, as can be seen from figure 2, is enormously dilated. The wall of the fundus shows little if any hypertrophy, while in the region of the trigone the mucous layers appear to be somewhat thickened and infiltrated by cells with deepstaining nuclei. The posterior wall of the bladder is


PLATE LXlll



PLATE LXIV



folded sharply backward upon itself so as to form a deep longitudlual sulcus, at the base of which the urethra leaves the bladder. Running upward along the posterior wall of the sulcus, from the opening of the urethra, is a band of mucosa strikingly different from that covering the rest of the bladder and resembling closely that of the urethra, with which it is continuous. From this point the urethi'a can be followed to the external meatus on the rudimentary penis. Its lumen, although filled with cell debris in places, is clearly patent. From the point where it leaves the bladder to the meatus it is not dilated. Normally, the prostatic urethra at this age is relatively wide, with its lumen continuous with the bladder cavity. In this specimen it has widened still further until it has become anatomically a part of the bladder. This appears to account for the band of urethral mucosa found on the bladder wall and is confirmed by the relation which the deferential ducts and seminal vesicles bear to this band of mucosa. The ducts at this age normally join the prostatic urethra a little below the bladder, as has been shown by Johnson.' In this specimen they are lost in the bladder wall just lateral to the band of iirethral mucosa mentioned above. In other words, that part of the bladder cavity from which the mouth of the urethra opens is not, from a developmental standpoint, bladder at all, but is in reality a dilated prostatic urethra. The membranous portion of the iirethra, instead of its prostatic portion, constitutes the exit from the bladder cavity.

The ureters follow a normal course and join the bladder in the usual locations. The right ureter is slightly dilated as it passes through the bladder wall and at its point of entry the bladder is folded upon itself so as to form a pocket.

The descending colon in its upper part appears normal in all respects. As it approaches the posterior wall of the bladder, however, it narrows rapidly until the lumen is barely distinguisluible. The muscle layers retain their normal thickness throughout, but the mucosa becomes thin and atrophic as the lumen narrows. This abnormal colon penetrates the posterior wall of the prostatic urethra just above the exit of the membranous urethra from the bladder. No further traces of the intestinal canal are found at lower levels. These relations are shown schematically in figure 3, D'.

Discussion

An interpretation of this condition requires answers to several questions, on some of which our knowledge is inadequate.

What happened to interfere with the usual separation of the urinary and alimentary tracts, and when? The normal transition from the early cloacal phase to complete separation of these tracts is shown in figure 3-A.B. CD. It will be seen that a wedge of tissue pushes down


ward, forcing rectum and urethra apart. In our specimen something happened, in the interval between the stages represented by diagrams A and C, to check the descent of this wedge, leaving the colon to empty into the prostatic urethra. In figure 3, D' represents the condition found in this embryo, while B' and C represent an attempt to bridge the gap between D' and normal A. As B shows the relations in an embryo of about five weeks, normal development must have been interfered with at about this time, probably in the first half of the second month.

Why did the bladder dilate? The simple answer that it was distended by increased internal pressure is not a satisfactory one. In the first place, there is no evidence that at this age there is any secretion from kidneys or intestine that might dilate the bladder. In the second place, the ureters, colon, and urethra are all patent and open freely into the vesicourethral cavity. Any increase in the hydrostatic pressure in the bladder, therefore, would have caused fluids to be forced either into the colon and ureters, thus dilating them, or though the urethra, thus relieving the pressure within the bladder. The ureters, however, are not dilated and the colon is actually narrowed. Hence it is clear that the bladder was not distended from increased internal pressure. The only other explanation that presents itself is that the bladder dilated intrinsically and was filled with fluid only in order to avoid a vacuum.

Anders explains dilatation and a.symnietry of the bladder in these cases as being due to an overgrowth of that organ, similar in nature to neoplastic growth. There is, moreover, a certain amount of experimental support for the belief that trophic changes in the embryo may cause dilatation in a hollow viscus. Streeter ^ found in the tadpole that marked dilatation luid developed in ear vesicles that had been transplanted to abnormal sites.

Finally, what caused the death of the fetus? That the abnormality itself was the cause is highly improbable, as there is nothing about it incompatible with fetal or even postnatal life. Two other possibilities present themselves. First, that the ovum itself was originally defective and that this defect caused both the abnormal development and the death of the fetus. Second, that the original germplasm was healthy, but that some process, perhaps a toxemia on the part of the mother, so profoundly influenced the embryo as to cause the abnormalities and later its death. Between these possibilities there is no ground on which to base a decision.

I wish to express my sincere thanks to Dr. G. L. Streeter for his many helpful suggestions in the pursuance of this study and for his kindness in extending to me the facilities of the Carnegie Laboratory of Embryology. I also wish to thank Mr. Charles Miller for the preparation of the serial sections.



Summary

A fetus is described, the external appearance of which provoked a diagnosis of fetal ascites.

The abdominal enlargement is due to an enormously dilated bladder. The prostatic urethra is so dilated as to form part of the bladder cavity.

The descending colon empties into the prostatic urethra.

The dilatation of the bladder is thought to be due to overgrowth of bladder tissue rather than to distention.

REFERENCES

1. Johnson, F. P.: The later development of the urethra in the male. J. of Urology, 1920, IV, 447.

2. Anders, H. E.: Ueber Kloakenmissbildungen. Virchows Arch., 1920, CCXXIX, 531.


3. Streeter, G. L.: Some experiments on the developing earvesicle of the tadpole, etc. J. Exper. Zool., 1906, III, 543.

4. Felix, W. : The development of the urinogenital organs. Keibel and Mall. Human Embryology, Vol. II, Chap. XIX, p. 752.

DESCRIPTION OF FIGURES

Fig. 1. — Embryo erroneously diagnosed foetal ascites, showing large abdomen. Carnegie Collection No. 2547.

Fig. 2. — Same embryo with abdominal wall folded back, and liver and intestines removed to show large bladder.

Fig. 3. — A. Early relations of colon and urinary tract drawn from model of normal foetus of 5.5 mm, greatest length. Carnegie Collection No. 1380. B. Relations in normal foetus of 7 mm. From Felix,' Fig. 577A. C. Relations in normal foetus of 11 mm. Ibid. Fig. 604A. D. Relations in normal foetus of 85 mm. From dissection. B' and C represent stages in the reported case corresponding to B and C. inferred from Normal A, and relations as found in D'. D'. Relations in reported case as established by serial sections.


REPORT OF A CASE OF TUBAL PREGNANCY PROBABLY CAUSED BY A PAROVARIAN CYST

By Leo Brady, M. I).

(From the Department of Gynecology, The Johns Hopkins Hospital and University)


Although it has long been known that tubal pregnancies can be cau.sed by any factor that interferes with the course of the fertilized ovum as it travels inward along the Fallopian tube, this case presents several rather unusual features which seem to justify its publication.

The brief summary of the case is as follows : — F. B. Gyn. No. 154077. Age 26— white— female. Admitted: February 24, 1922.

Family and general past histoi-j' unimportant. Menstrual history normal until the onset of present illness. Marital history : Married one year, one child born September 1021, that is, five months before the present admission to the hospital, when the patient was still nursing it.

Present Illness. — Three months after the birth of her normal child, that is, two months before admission to the hospital the menstrual period returned. Five weeks later a .second period of normal duration occurred. Three weeks later, that is, sixteen days before admission to the hospital, she began to bleed again, although the menstrual I)eriod was not due until a week later. The bleeding was not of greater quantity than usually occurred with the periods, but instead of stopping at the end of four or five days continued for sixteen days until the patient was admitted to the hospital. Two days before adniission to the hospital the patient felt "generally run down" and went to her doctor who prescribed calomel and salts. The next day she was taken suddenly with a drawing pain over the whole lower abdomen which was so severe that it compelled her to go to bed. The pain was especially bad at stool. She says she felt as if she were going to die.


The pains became more severe during that day and the following night. The next day the attending iihysician noticed the facial pallor, localized the abdominal tenderness in the lower abdomen and sent the patient into the hospital with a diagnosis of ruptured tubal pregnancy.

Admission Note. — The patient is a well developed white woman in extreme prostration, but mentally oriented, complaining of intense drj'uess of the month, weakness and abdominal tenderness. Mucous membranes quite pale. Temperature 98.4° ; pulse 140, weak aiul irregular. Respirations 28, white blood count 15,000. Red blood count 2,160,000. Hemoglobin 33%. Blood pressure, 96/74. General [)hysical examination essentially negative. The breasts are swollen and tender and contain milk. General abdominal tenderness is present. Shifting dullness is demonstrable in the flanks. Pelvic examination : Outlet nuirital, mucous membranes very pale. A slight amount of bloody vaginal discharge. Cervix soft. Filling the whole cul-de sac there is a soft mass which to the examiner has the characteristic consistency of blood-clots.

Pre-Operath'c Duif/nosis. — Ruptured extrauterine pregnancy.

When the patient was first seen, it was at once evident that she could not in her condition stand an operation and, unless something were done very soon, she would die. The husband's blood fortunately matching with hers, a transfusion of 600 c.c. of citrated blood was given in the operating room. Although the ]iatient had a slight chill and a rise of temperature after it, her general con


THE JOHNS HOPKINS HOSPITAL BULLETIN. DECEMBER. 1922


PLATE LXV



dition improved at once and the blood pressure rose from 96 to 120. One hour after completion of the transfusion the operation was begun. After division of the fascia and separation of the muscles, the blood-clots tilling the abdominal cavity could be seen shining through the peritoneum, and when the peritoneum was opened, there was an outi>ouriug of a large quantity of blood. The entire pelvis was filled with blood, and blood was seen pouring from a rupture which had occurred in the right Fallopian tube at a distance of about 2 cm. from its fimbriated end. There was a small parovarian cyst present which had kinked the tube just proximal to the point where the tubal pregnancy had occurred and which was in all probability the causal factor of its development. On the left side there was also a parovarian cyst, but this was much larger, measuring 12 cm. in diameter. The tube on the left side was drawn over the cyst. The ovaries on both sides were perfectly normal. I removed the Fallopian tube on the right with the extrauterine i)regiu^ncy, both parovarian cysts and as much of the blood-clots from the adbominal cavity as 1 could without prolonging the operation. At the termination of the operation the pulse was IfiO but of good quality.

Convalescence was quite satisfactory and the patient was discharged in good condition twenty-two days after her operation.

Pathological Report (Dr. Warrinner). — Gyn. Path. No. 2 7.") 77.


The right tube measures 8 bj' 2.5 cm. in its distal third. The tube is ruptured 1.5 cm. from its fimbriated end. Just un<ler the [nnut of rui)ture is a parovarian cyst. This cyst is thin-walled and filled with clear fluid. In the center of one of the large blood-clots removed at operation the fetus and membranes are found. The fetus measures 5 cm. in length.

In addition to being one of the many cases we now see of tubal rupture with complete collapse of the patient, in which a transfusion improves the condition sutficiently to allow the performance of a successful operation, this case presents the two following interesting factors :

(1) This patient developed a tubal pregnancy only five months after the termination of a normal pregnancy and while she was still nursing her baby. (2) It is seldom that one can demonstrate so clearly a mechanical factor causing the arrest of the fertilized ovum in the tube as is shown in this instance. For the excellent drawing of the conditions found at operation I am greatly iiulebted to Mr. Max Broedel.

LEG EX D

Posterior view of pelvic conditions as found at operation. On the right is shown the parovarian cyst which had kinked the tube at a point immediately proximal to the site of the rupture. On the left the larger parovarian cyst is shown -with the elongated and thinned out Fallopian tube extending over its top. The small drawing in the upper right hand quadrant indicates the mechanism by which the right tube has been compressed by the parovarian cyst.


THE ISOLATION OF NUCLEIC ACID FROM TISSUES

By Walter Jones and Caspar Folkoff

(From the Laboratory of Physiological Chemistry, Johns Hopkins Medical fichool)


The well known chemical distinction between animal and plant nucleic acid suggests an inquiry into the chemical nature of bacterial nucleic acid to learn whether in this respect bacteria are to be regarded as animals or plants. Attempts to decide this question failed for want of a method of isolating nucleic acid that is applicable to the material which constitutes the bodies of bacteria, until recently, when one of us (Folkotf), in collaboration with Schatl'er and Bayne-Jones,* prepared a considerable amount of bacterial nucleic acid and found the substance somewhat unique. The method of isolation employed by these experimenters had been previously elaborated by us in connection with yeast and is here described for purposes of reference.

Five liters of cold fresh brewer's yeast diluted with eight liters of cold tap-water are treated with 'ZVo liters of 20% sodium hydroxide in small successive portions, and the mixture is stirred continually for ten minutes,

Schaffer, Folkoff and Bayne-Jones: Johns Hopkins Hospital Bull., 1922, XXXIII, 151.


care being taken that the temperature of the material does not rise. At the end of this time most of the alkali has been neutralized with hydrochloric acid. The solution is finally made faintly but distinctly acid to litmus with acetic acid and after staiuling over night in a cold place the muddy opalescent dark brown fluid is decanted from the sharply settled yeast detritus and treated with hydrochloric acid and alcohol in the usual way for the precipitation of the nucleic acid, which is washed by decantation with alcohol of inci-easing strength and finally filtered tightly on a Buchner funnel.

The crude nucleic acid thus obtained is ground in a mortar with five or six parts of water containing an excess of ammonia, and after the solution of the nucleic acid is complete, an equal volume of alcohol is added. The excess of ammonia is then carefully neutralized with acetic acid. This is the crux of the method. The excess of ammonia must not he neutralized until after the alcohol has been added, and the end-point of the neutralization is determined not with an indicator but by the


appearance of a bulky dark brown precipitate leaving an easily filterable interstitial liquid that is as transparent and nearly as colorless as distilled water.

The clear fluid is filtered ofl', treated with an equal volume of alcohol and the precipitated snow-white flakes are washed and dried with alcohol. As this process of purification does not involve an appreciable loss of material, it may be repeated as often as is suggested by the appearance of the final material, and can be applied to the purification of any of the commercial preparations of yeast nucleic acid.

The final product (30 grams of which are obtained from five liters of yeast) consists of a colorless biuret-free


amorphous powder which dissolves easily and completely in water to form a colorless solution. It yields decomposition products which bear the quantitative relation to one another that is required for the ammonium acetate addition product of yeast nucleic acid and contains exactly half of its phosphorus in easily split form, showing that its purine and pyrimidine groups are present in equivalent amounts.

As the method described cannot be applied to the isolation of nucleic acid from animal glands, its successful execution in a particular case would suggest tliat the questionable material under examination is of plant origin.


A CASE OF CONGENITAL OSTEOSCLEROSIS

By Ralph K. Ghormley, M.D.

Assistant in Orthopedic Surgery, Johns Hopkins University

(From the Orthopedic Service of the Surgical Department

of the Johns Hopkins Hospital)


The following case is presented because of its unique characteristics and in the hope that, if others are recognized, they will be brought to the knowledge of the profession.

Case History

J. C. (Surg. No. 55907), male, while, (Vt. 8, was first seen in the Orthopedic Dispensary of the Johns Hopkins Hospital on February 1, 1922.

Complaint: "Pain in left hip and limp."

F. U. The father and mother are living and well. There is one half brother and one half sister by a former husband of the mother. Since her marriage to the father of the patient there has been one child born prematurely, who lived two months ; then there were five miscarriages all before the end of the fourth month. The patient is the last child born. There is no history of either syphilis or gonorrhoea in the father or mother.

P. H. Premature birth at eight months. Weighed four pounds. Breast fed. The child developed quite normally, however, and aside from varicella, at the age of three years, there have been no acute illnesses. Tonsillectomy and adenoidectomy were performed one year ago. The mother states that two years previous to the present illness the child had some "growing pains" in the back for a short time.

P. I. During the first week in December, 1921, the boy complained of pain in the left hip which was more marked at night and toward the close of the day. This pain would also be noticed after resting from his play. The parents noticed a slight limp. There was no history of any injury. There was never any swelling and no night cries. The condition has not progressed to any appreciable extent.


Physical examination. Blood pressure, 100/65; pulse, 80; I'espiration, 18; temperature, 99.2; Height, 4 ft., 3 in.; weight, 58 lbs.

The patient is a fairly well developed and nourished boy; he is bright and active. There is no suggestion of any mental retardation (Fig. 1).

Head : Negative.

Neck : Negative, thyroid not palpable.

Chest : Heart and lungs clear, no retro-mauubrial dullness, no rachitic rosary.

Abdomen: The abdomen is rather prominent. The walls are everywhere soft. There is no tenderness anywhere and no masses are felt. None of the organs are palpable.

Glands : There is some enlargement of the posterior cervical lymph glands which are discrete and not tender. The axillaries and epitrochlears are not felt. The inguinals are moderately enlarged, discrete and not tender.

Skeletal System. Spine: The spine is quite straight, although there is slight deviation toward the right to compensate for the slight tilting of the pelvis toward the left. The movements of the spine are free in every direction and there is no tenderness anywhere. Upper extremities: The muscular development is good. Tliere is no bony tenderness or thickening. The joints are all normal. Lower extremities: The child walks with a moderate limp toward the left. The left foot is held rotated outward when standing. There is slight atrophy of the thigli and calf on the left. When he lies down, there is about half a centimeter of apparent shortening of the left leg. Voluntary as well as passive movements of the left hip reveal slightly less abduction than on the riglit. There is a slight decrease in the hyperextension of the hip. There is no muscle spasm and no tenderness. The measurements are :



Right Left

Apparent shortening 0.5 cm.

Ant. sup. spine — Int. malleolus 64 cm. 63.5

Great trochanter — Ext. malleolus 61.5 61.5

Base of Bryant's triangle 4.5 3.75

Circumference of thigh 33.5 32.5

Circumference of calf 24.5 24.

Blood. Hemoglobin 70% (Sahli) R. B. C. 4.600,000 W. B. C. 7,050 Differential count:

P. M. N 56.3%

P. M. E 33

P. M. B 33

L. Lymph 2.33

S. Lymph 36.3

L. Mono 66

Trans 3.66

There is slight anisocytosis and poikilocytosis. Hemolysis resistance tests are normal. Platelet count, 128,000. Reticulated R. B. C, 0.9%.

Blood Wassermann, negative; ice-box preparation, negative. Blood Chemistry:

N. P. N., 30 mgm. per 100 c.c. CO^, 47.1 vols % Sugar, .119

Phosphorus, 4.S mgm. per 100 c.c. Calcium, 10.2 mgm. per 100 c.c. The blood culture showed no gi-owth. Urine: Sp. Gr. lOlS; acid; albumin and sugar tests negative; microscopic examination negative.

Basal Metabolism, 11.1% above the average for the age and sex.

During the course in the hospital, while under observation, there was an almost daily afternoon rise in temperature to about 100° F. Nothing was found to account for this. Operation was refused by the parents, so no exploration could be carried out. A plaster of Paris spica was applied with the leg as fully abducted as possible without anesthesia. This was worn for about three weeks after which time a radiograph showed little change in the hip. The bo}% however, had no further pain and the limp seems to be less.

Discussion

On seeing the patient in the dispensary our impression was that we were dealing with a beginning epiphyseal separation of the head of the femur on the left. Radiographs of the pelvis taken at that time showed a curious dense appearance of the bones. This was thought at first to be an artefact, but carefully repeated pictures showed the same condition (Fig. 2). Radiographs of all of the bones were then made and showed much the same condition throughout the skeleton. Beside the curious density of the flat bones and the rather hazy outline along all of the bones, the vertebral bodies showed a marked density at either pole (Fig. 3). There is thickening of the cortex of the ribs (Fig. 4), the marrow cavity in places being apparently obliterated. There is definite


thickening of the cortex of all the long bones, more marked in the femora and humeri (Figs. 5 and 6), and the cortical thickening seems to be greater in the proximal portion of these bones than in the distal portion. The skull (Fig. 7) is thicker than normal. In the femoral necks, and especially in the left, there seems to be a breaking down of the bone just below the epiphysis so that the epiphysis is slipped inward and downward (Fig. 2).

Radiographs of the mother and father were then made, which showed the mother's bones to be normal but that practically the same condition existed in the father as in the child (Figs. 8 and 9). The father's history was then reviewed and it was found that both of his parents had lived to be eighty and that he has seven brothers, all of whom are living and in good health. It is hoped that we may be able later to get radiographs of some of these brothers. The father states that when he was a boy he had "growing pains"' in his legs, but had no treatment and entirely overcame them after adolescence. The blood Wassermann of the father, as well as of the mother, was negative. Smears of the father's blood were normal. No dietary discrepancy in either father or son could be discovered.

A search through the literature has failed to locate any cases similar to this in the living person. There are, however, several cases described at autopsy as osteosclerosis, some as congenital and others as associated with disturbance of the blood-forming organs.

Frangenheim ^ has collected three cases from other writers of autopsy reports of congenital osteosclerosis. Two of these were in infants with a normal blood picture but a third, a case reported by Goodall - was in a child who died at the age of ten weeks of what is described as acute myelocythiemia, of three weeks' duration, with bleeding from the mucous membranes, enlargement of the liver and spleen and a blood picture of myeloid leukiemia. Beside the changes in the organs the bones showed osteosclerosis of the ribs, sternum, femora, humeri and tibiie. Another of these cases was originally reported by Sclimidt of a child who died after thirty hours with enlarged thymus, normal blood smears and an apparently normal bone-marrow. The bones showed marked thickening of the cortex of the long bones, skull and sternum ami a marked density in the vertebral bodies. The other case cited by Fragenheim was originally reported by Assinann. A child died, when one day old, showing beside enlargement of the thymus and thyroid a normal blood picture and normal bone-marrow. Tliere was an associated diffuse sclerosis of the long bones as well as of the skull and vertebrje. In all of these cases the cortical thickening was mostly central with partial obliteration of the marrow cavities, there being some cartilaginous rests and the marrow in places being encased in thickwalled bony spaces.

Assmann ' has collected in addition four cases in adults who died at ages ranging from 17 to 68 years.



Two (lied of leukaemias and the others, it was thought, of pi'imaiy aiia>nnas. All of these cases showed similar bone changes at autopsy.

Neuwerck and Moritz ^ report one case with similar bone changes but draw no conclusions except to refute the statement of Schlazenhofer ° that the osteosclerotic process may be due to arsenic or phosphorus used in the medication. Neither of these drugs had been used in their case.

Schwarz" reports a case of lenka-niia with osteosclerosis in a woman. 47 years old, who died after a six months" illness, the autopsy showing, beside enlargement of the liver and spleen with findings characteristic of leukipmia. a thickening of the cortex especially of the femora and humeri. The ribs, vertebne and skull also showed sclerosis. In the long bones of this case it is noted that the proximal portion of the cortex was more markedly thickened than the distal portion. The marrow cavity is described as grayish red to dark brown in color with bony trabeculse scattered through the marrow.

Heuck ' in autopsies on two cases of lenkaMnia in men, 24 and 25 years of age, found marked thickening of the (liapliyses of the femora and humeri, as well as of the vertebnp and skull. He holds that the osteosclerosis is of long standing and that the blood changes are due to the changes wrought in the marrow by the osteosclerotic process, or that the occurrence of the two is a coincidence.

The above case reports are all pathological without any radiographs. However, the anatomical descriptions are so nearly what one would expect to find at autopsy


in the case here reported that we feel we are dealing with the same condition. The majority of the authors take the view that the sclerosis is secondary to an irritation which goes with the morbid process in the blood. Heuck, however, takes an opposite view, as above stated. In our case we seem to be dealing with a definitely inherited condition not related, so far as we know, to any metabolic disturbance which we can demonstrate. There is possibly some increase in the blood phosphorus, the figures given being above the average normal for the age and sex. There is, however, no evidence of either the father or son having been on a high phosphorus diet. It would seem that the condition itself is not incompatible with life and that the secondary blood diseases may come as a coincidence or as a failure of the decreased bloodforming mechanism to respond to the excessive demands placed upon it.

We are greatly indebted to the various laboratories, for aid in the investigations in this case.

BIBLIOGRAPHY

1. Frangenheim: Neue deutsche Chirurgie, Bd. X.

2. Goodall, Alex.: Edinburgh Med. Jour., 1912, 500-507.

3. Assmann. H.: Beitrage zur pathologische Anatomie und Pathologie, I, 1907.

4. Neuwerck, A. u. Moritz, P.: Deutsch. Arch. f. klinische Medizin, LXXXIV, 1905.

5. Schlazenhofer: Ztschr. f. Heilkunde XXIV, 1904.

6. Schwarz, Emil: Ztschr. f. Heilkunde, XXII, 1901.

7. Heuck, G.: Virchows Arch., LXXVIII.


BOYLSTON MEDICAL PRIZES


These prizes, which are open to public competition, are offered for the best dissertation on questions in medical science proposed by the Boylston Medical Committee.

At the annual meeting held in Boston in 1920 a prize of three hundred dollars was awarded to an essay entitled "Acute Inflammation of the Nose. Pharynx and Tonsils" by Mr. Stuart Mudd of St. Louis.

For 1922 there is offered a prize of Ave hundred dollars and the Boylston Prize Medal, for the best dissertation on the results of original research in medicine, the subject to be chosen by the writer. The Boylston Prize Medal will be added to the money prize only in case the winning essay shows special originality in the investigations detailed.

Dissertations entered for this prize must be in the hands of the Secretary, REID HUNT, M.D., Harvard Medical School. Boston, Mass., on or tefore Fetruary J, 1023.

In awarding these prizes, preference will be given to dissertations which exhibit original work, but if no dissertation is considered worthy of a prize, the award may be withheld.

Each dissertation must bear, in place of the author's name, some sentence or device, and must be accompanied by a sealed packet, bearing the same sentence or device, and containing the author's name and residence within.


Any clew by which the authorship of a dissertation is made known to the Committee wilt debar such dissertation from competition.

Dissertations must be printed or typewritten, and their pages must be bound in book form.

All unsuccessful dissertations are deposited with the Secretary, from whom they may be obtained, with the sealed packet unopened, )/ called for within one year after they have been received.

By an order adopted in 1826. the Secretary was directed to publish annually the following votes:

1. That the Board does not consider itself as approving

the doctrines contained in any of the dissertations to which premiums may be adjudged.

2. That, in case of publication of a successful dissertation,

the author be considered as bound to print the above vote in connection therewith.

The address of the Secretary of the Boylston Medical Committee is REID HUNT, M.D., Harvard Medical School. Boston.

Mass.


PLATE LXVI



liowing distortion and deformity, the result of coxa vara.



Pig. 2. — Osteosclerosis of all the pelvic bones with partial

subluxation of the epiphyses of the femora and some new

bone formation around the epiphyseal lines.


PLATE LXVII


THE JOHNS HOPKINS HOSPITAL BULLETIN. DECEMBER, 1922



Fig. 3. — Showing marked osteosclerosis of the poles of the vertebrae.



Fig. 4. — Sliowing thickening of the cortex of the ribs.



PLATE LXVIII




Fig. 5. — Showing a marked thickening of the cortex.


Fig. .5a. — Showing a marked thickening of the cortex.



Fig. G. — Showing a marked thickening of the cortex.


PLATE LXIX


Fil). 7. — Marked osteosclerosis of the bones at the base of the skull.


Fig, S, — Osteosclerosis of the pelvic bones of patient's father. These show no subluxations of the epiphyses.



Fig, :>. — Sliiiie III iialieul's lallior. .^liuuiiii; ihe .-^aiiic u.-^lcosclerotic condition of the poles of the vertebnt.



Index To Volume XXXIII

Acid, nucleic, On the presence of, in bacteria, 151.

Adrenalin, The pharmacological action of, on the sphincter

pylori of the foetus, 159. Ansesthesia. nerve-block. Localization of cutaneous nerves by

electrical stimtilation, applied to, 338. Andrus, VV. D. W. and Heuer, G. J., The alveolar and blood

gas changes following pneumectomy, 130. Anjemia in rats. Occurrence of, on deficient diets, 163. Anemia infantum pseudoleukemica, A study of so-called infantile splenic anemia or. Secondary anemia of infants, 1. Anesthesia, preliminary, Note on the protective effect of. A study of the relation of the adrenal glands to experimentally produced hypotension (shock), 79. Anesthesia, regional. Posterior resection of the rectum and

rectosigmoid under, 13 4. Bacilli,' Influenza-like. Growth of influenza-like bacilli on media containing only an autoclave-labile substance as an accessory food factor, 429. Bacteria, On the presence of nucleic acid in, 151. Bacteria, The dissemination of, in the upper air passages. 145,

252. Baer, W. S., The use of the bone graft in the treatment of

Pott's disease, 140. Bacterial nutrition. Growth of a hemophilic bacillus on media containing only an autoclave-stable substance as an accessory factor, 149. Baldwin. J. C, Entero-urethral fusion in a fetus, simulating

fetal ascites, 440. Bayliss, W. M., Interfacial phenomena. With especial reference

to colloids and enzymes, 307. Bayliss, W. M., The excitatory state, 347. Bayliss, W. M., Vaso-nnotor reactions, 412. Bayne-Jones, S., Schaffer, A. J., and Polkoff, C, On the

presence of nucleic acid in bacteria, 151. Bayne-Jones, S., and Wilson, D. W., Immunological reactions

of Bence-Jones proteins, 37, 119. Becker, J. E.. McCoUum, E. V., Simmonds, N., and Shipley.

P. G., Studies on experimental rickets. XXI, 229. Bigelow, G. H., and Moss, W. L., Yaws. An analysis of 1046

cases in the Dominican Republic, 4 3. Blackfan, K. D., and Shipley, P. G., The pharmacological action

of adrenalin on the sphincter pylori of the foetus, 159. Bloom, W., Histamine as an inflammatory agent. 185. Bloomfield, A. L., Adaptation of bacteria to growth on human mucous membranes with special reference to the throat flora of infants, 61. Bloomfield, A. L., The dissemination of bacteria in the upper air passages. II. The circulation of bacteria in the mouth, 145. Bloomfield, A. L., The significance of the influenza bacilli, 172. Book received: 153; 303. Brady, L., Report of the case of a tubal pregnancy probably

caused by a parovarian cyst, 4 4 2.

Caesarean section, A clinical and anatomical study of fifty-one

cases of repeated, with especial reference to the healing of

the cicatrix and to the occurrence of rupture through it, 93.

Caesarean sections, A study of the results obtained in sixty-four,

terminated by supravaginal hysterectomy, 318. Calcium, concentration of. Factors which determine the, and of inorganic phosphorus in the blood serum of rats, 313.


Calcium deposition. An experimental demonstration of the existence of a vitamin which promotes. Studies on experimental rickets. XXI, 229. Campos, E. S., and Plrket, J., Generalized megalocaryocytic

reaction to saponin poisoning, 271. Capillaries of the bone marrow of the adult pigeon, 222. Carcinoma of the cervix uteri. A very early case, 221. Cardiac complications of gonorrhoea. On the, 361. Cells, The survival of, after the death of the organism, 284. Cervix uteri, Carcinoma of the. A very early case, 221. Chesney, A. M., and Hodges, A. B., Observations on the production of toxic substances by pneumococci, 425. ChromaflSn body, abdominal, in dogs. Note on the, 377. Chromaffin reaction. Note on a modification of the, with observations on the occurrence of abdominal chromaffin bodies in mammals, 359. Churchman, J. W., The effect of slight increase of temperature

on the bacteriostatic power of gentian violet, 227. Clark, H, M., Zinck, R. H., and Evans, F. A., The protective power of serum in pernicious anemia and other conditions against hemolysis by saponin and by sodium oleate. 16. Collateral circulation. The establishment of, following ligation

of the thoracic duct, 21. Corner, G. W., The morphological theory of monochorionic twins as illustrated by a series of supposed early twin embryos of the pig, 389. Cross, H. B., A suggested modification of the Wright opsonic technique based upon the differential white blood count, 142. Crowe, S. J., and Wislocki, G. B., Note on the abdominal

chromaffin body in dogs, 377. Cullen. T. S., A normal pregnancy following insertion of the

outer half of a Fallopian tube into the uterine cornu, 344. Cunningham, R. S., The changes in the omentum of the rabbit during mild irritations; with especial reference to the specificity of the mesothelium, 257. Cyst, parovarian. Report of a case of tubal pregnancy probably

caused by a, 4 4 2. Cysts, Dermoid, of the ovary. A report of four cases. 66. Dandy. W. E., An operation for the total extirpation of tumors in the cerebello-pontine angle. A preliminary report, 344. Dandy, W. E., Treatment of non-encapsulated brain tumors

by extensive resection of contiguous brain tissue, 188. Diabetic diets, A graphic method for the calculation of, in

the proper ketogenic-antiketogenic ratio, 128. Differential white blood count, A suggested modification of the

Wright opsonic technique based upon the. 14 2. Diphtheria bacillus carriers. Results of re-examination of apparently negative cultures, 110. Doan, C. A., The capillaries of the bone marrow of the adult

pigeon, 222. Embolism, Air, following various diagnostic or therapeutic

procedures in diseases of the pleura and the lung, 321. Entero-urethral fusion in a fetus, simulating fetal ascites, 4 4 0. Evans, F. A., and Happ, W. M., Secondary anemia of infants. A study of so-called infantile splenic anemia or anemia infantum pseudoleukemica, 1. Evans, F. A., Zinck, R. H., and Clark, H. M. The protective power of serum in pernicious anemia and other conditions against hemolysis by saponin and by sodium oleate, 16. Excitatory state, 347.


Fallopian tube. A normal pregnancy following insertion of the outer half of a, into the uterine cornu, 344.

Felton. L. D., and Lewis. M. R.. The hydrogen-ion concentration of tissue growth in vitro, 112.

Fetal ascites. Entero-urethral fusion in a fetus, simulating, 440.

Finesilver, E. M., and Macht. D. I., The effect of saline purgatives on the absorption of other drugs, 330.

Firket, J., and Campos. E. S., Generalized megalocaryocytic reaction to saponin poisoning. 271.

Foetus, The pharmacological action of adrenalin on the sphincter pylori of the, 159.

Folkoff, C, Schaffer, A. J., and Bayne-Jones, S., On the presence of nucleic acid in bacteria, 151.

Gamble, T. O., A clinical and anatomical study of fifty-one cases of repeated Caesarean section with especial reference to the healing of the cicatrix and to the occurrence of rupture through it, 93.

Gentian violet, The effect of slight increase of temperature on the bacteriostatic power of, 227.

Glands, adrenal, A study of the relation of the, to experimentally produced hypotension (shock); with a note on the protective effect of preliminary anesthesia, 7 9.

Glands, para-ocular, The changes in the, which follow the administration of diets low in fat-soluble A; with notes of the effect of the same diets on the salivary glands and the mucosa of the larynx and trachea, 357.

Gonorrhoea, On the cardiac complications of, 361.

Grafts, Reverdin. large. The use of, in the healing of chronic osteomyelitis, 386.

Grieves, C. J., McCollum, E. V., Simmonds, N., and Kinney, E. M., The relation of nutrition to tooth development and tooth preservation, 202.

Guthrie, C. G., and Marshall. B. C, Diphtheria bacillus carriers. Results of re-examination of apparently negative cultures, 110.

Hannon, R. R., and McCann, W. S., A graphic method for the calculation of diabetic diets in the proper ketogenic-antiketogenic ratio, 128.

Happ, \V. M., and Evans, F. A.. Secondary anemia of infants. A study of so-called infantile splenic anemia or anemia infantum pseudoleukemica, 1.

Happ, W. M., Occurrence of ansemia in rats on deficient diets, 163.

Harris, J. W., A study of the results obtained in sixty-four Cssarean sections terminated by supravaginal hysterectomy, 318.

Harris, J. W., Pregnancy and labor in young primiparae, 12.

Hemophilic bacillus. Growth of a. on media containing only an autoclave-stable substance as an accessory factor, 149.

Heuer, G. J., and Andrus, W. D. W., The alveolar and blood gas changes following pneumectomy, 130.

H-ion concentration to specific precipitation. The relation of, 116.

Histamine as an inflammatory agent, 185.

Hitchcock, C. H., and Loveland, R., A study of the AustinStillman-Van Slyke index of urea excretion, 294.

Hodges, A. B., and Chesney, A. M., Observations on the production of toxic substances by pneumococci, 425.

Hogue, M. J., A study of trichomonas hominis, its cultivation, its inoculation into animals and its staining reaction to vital dyes, 437.

Hohman, L. B., Post-encephalitic behavior disorders in children, 372.

Hormone theory, The evolution of human races in the light of the, 155, 195.


Howland, J., and Kramer, B., Factors which determine the

concentration of calcium and of inorganic phosphorus in

the blood serum of rats, 313. Hughson, W., Localization of cutaneous nerves by electrical

stimulation, applied to nerve-block anaesthesia, 338. Hydrogen-ion concentration of tissue growth in vitro, 112. Hysterectomy, supravaginal, A study of the results obtained

in sixty-four Caesarean sections terminated by, 318. Influenza bacilli. The significance of the, 172. Influenza-like bacilli. Growth of influenza-like bacilli on media

containing only an autoclave-labile substance as an accessory

food factor, 429. Intercolumnar tubercle, An undescribed area in the anterior

wall of the third ventricle, 181. Johnston, R. A., and Morgan, H. J., Acute lobar pneumonia

and hematogenous puerperal infection. A case report, 106. Keith, A., The evolution of human races in the light of the

hormone theory, 155, 195. Keith, A. The evolution of human races in the light of the

hormone theory. Racial status and form of body, 195. Kidney, permanent, in chick embryos. Development and growth

of the metanephros or. 392. Kinney, E. M., McCollum. E. V., Simmonds, N., and Grieves,

C. J., The relation of nutrition to tooth development and

tooth preservation, 202. Kinney, E. M., Shipley, P. G., Park, E. A., McCollum, E. V.,

and Simmonds, N., Studies on experimental rickets. XX, 216. Knapp. A., and Woods, A. C, The diagnostic and therapeutic

use of uveal pigment in injuries of the uveal tract and

sympathetic ophthalmia, 419. Kramer, B., and Howland, J., Factors which determine the

concentration of calcium and of inorganic phosphorus in

the blood serum of rats, 313. Labat, G. L., Posterior resection of the rectum and rectosigmoid (Kraske or Modified) under regional anesthesia. 134. Lee, F. C, The establishment of collateral circulation following ligation of the thoracic duct, 21. Lewis, M. R., and Felton, L. D., The hydrogen-ion concentration

of tissue growth in vitro, 112. Lewis, W. H., and McCoy, C. C, The survival of cells after

the death of the organism, 284. Liver function. A new method of testing, with phenoltetra chlorphthalein, 432. Long, E. R., The biochemistry of tuberculosis, 246. Loveland, R., and Hitchcock, C. H., A study of the Austin Stillman-Van Slyke index of urea excretion, 294. McCann, W. S., and Hannon, R. R., A graphic method for the

calculation of diabetic diets in the proper ketogenic-anti ketogenic ratio, 128. McCollum, E. v.. Simmonds, N., Shipley, P. G., and Park, E. A.,

Studies on Experimiental rickets. XV. The effect of starvation on the healing of rickets, 31. McCollum, E. v.. Powers, G. F., Park, E. A., Shipley, P. G.,

Simmonds, N., Study on experimental rickets. XIX, 125. McCollum, E. v., Shipley, P. G., Park, E. A., Simmonds, N., and

Kinney, E. M., Studies on experimental rickets, XX, 216. McCollum, E. v., Simmonds, N., Becker, J. E., and Shipley,

P. G., Studies on experimental rickets, XXI, 229. McCollum, E. v., Simmonds, N., Shipley, P. G., and Park.

E. A., Studies on experimental rickets. XXII, 296. McCollum, E. v., Simmonds, N., Kinney, E. M., Grieves, C. J.,

The relation of nutrition to tooth development and tooth

preservation, 202. McCoy, C. C, and Lewis, W. H., The survival of cells after the

death of the organism, 284.


Macht, D. I., and Finesilver, E. M., The effect of saline purgatives on the absorption of other drugs, 3 3 0. Major, R. H., Studies on a case of chromic acid nephritis, 56. Marshall, B. C, and Guthrie, C. G., Diphtheria bacillus carriers.

Results of re-examination of apparently negative cultures,

110. Martzloff, K. H., Carcinoma of the cervix uteri. A very early

case, 221. Martzloff, K. H., Dermoid cysts of the ovary. A report of four

cases, 66. Mason, V. R., The relation of H-ion concentration to specific

precipitation, 116. Mesenchyme cells. Tolerance and acquired tolerance of the,

in tissue cultures for copper sulphate and sodium arsenite,

375. Monochorionic twins, The morphological theory of, as illustrated by a series of supposed early twin embryos of the

pig, 3 89. Moore, J. E., Studies in asymptomatic neurosyphilis. II. The

classification, treatment and prognosis of early asymptomatic

neurosyphilis, 231. Morgan, H. J., and Johnston, R. A., Acute lobar pneumonia

and haematogenous puerperal infection. A case report, 106. Mori, S., The changes in the para-ocular glands which follow

the administration of diets low in fat-soluble A; with notes

on the effect of the same diets on the salivary glands and

the mucosa of the larynx and trachea, 3 5 7. Moss, W. L., and Bigelow, G. H., Yaws. An analysis of 1046

cases in the Dominican Republic, 43. Myoma, uterine, A study of frozen sections through a cadaver

showing the anatomical relations of a large, 350. Nephritis, chromic acid. Studies on a case of, 56. Nerves, cutaneous. Localization of, by electrical stimulation,

applied to nerve-block ansesthesia, 338. Neurosyphilis, asymptomatic. Studies in. II. The classification,

treatment and prognosis of early asymptomatic neurosyphilis, 231. Notes and news, 3 3. Notes on new books, 35, 117, 153, 267, 268, 269, 302, 303, 345,

346, 379, 380, 381, 416. 417. Novak, E., Pseudomyxoma peritonei, 182. Nowrey, J. E., The effect of sodium germanate upon the total

hemoglobin of the albino rat, 340. Nutrition, The relation of, to tooth development and tooth

preservation, 202. Ophthalmia, sympathetic. The diagnostic and therapeutic use

of uveal pigment in injuries of the uveal tract and, 419. Osteomyelitis, chronic. The use of large Reverdin grafts in the

healing of, 386. Park, E. A., McCollum, E. V., Simmonds, N., Shipley, P. G.,

Studies on experimental rickets. XV. The effect of starvation

on the healing of rickets, 31. Park, E. A., Powers, G. F., Shipley, P. G., McCollum, E. V.,

Simmonds, N., Study on experimental rickets, XIX, 125. Park, B. A., Shipley, P. G., McCollum, E. V., Simmonds, N.,

and Kinney, E. M., Studies on experimental rickets. XX, 216. Park, E. A., McCollum, E. V., Simmonds, N., and Shipley, P. G.,

Studies on experimental rickets. XXII, 296. Pathology, Biometrical studies in. 11, 406. Pearl, R., Biometrical studies in pathology. II. Pathometric

injdex numbers: Preliminary account of a quantitative

method of evaluating pathological phenomena, 406. Pelvis, kyphotic, funnel. Spontaneous labor occurring through

an obliquely contracted, 190. Pernicious anemia, The protective power of serum in, and other conditions against hemolysis by saponin and by sodium oleate, 16.


Phenoltetrachlorphthalein, A new method of testing liver function with, 432.

Phosphorus, inorganic, in the blood serum of rats. Factors which determine the concentration of calcium and of, 313.

Pleura, diseases of the. Air embolism following various diagnostic or therapeutic procedures in, 321.

Pneumectomy, The alveolar and blood gas changes following, 130.

Pneumococci, Observations on the production of toxic substances by, 4 2 5.

Pneumonia, Acute lobar, and heematogenous puerperal infection. A case report, 106.

Poisoning, saponin. Generalized megalocaryocytic reaction to, 271.

Pott's disease. The use of the bone graft in the treatment of, 140.

Powers, G. F., Park, E. A., Shipley, P. G., McCollum, E. V., and Simmonds, N., Study on experimental rickets. XIX, 125.

Pregnancy and labor in young primiparae, 12.

Pregnancy, A normal, following insertion of the outer half of a Fallopian tube into the uterine cornu. 344.

Pregnancy, tubal, Report of the case of, probably caused by a parovarian cyst, 442.

Proteins, Bence-Jones, Immunological reactions of, 37, 119.

Pseudomyxoma peritonei, 182.

Psychopathological conditions. The role of situation in, (Abstract), 152.

Puerperal infection, haematogenous, Acute lobar pneumonia and, 106.

Putnam, T. J., The intercolumnar tubercle, an undescribed area in the anterior wall of the third ventricle, 181.

Reid, Mont R., The use of large Reverdin grafts in the healing of chronic osteomyelitis, 386.

Resection, posterior, of the rectum and rectosigmoid (Kraske or Modified) under regional anesthesia, 134.

Rich, A. R., A study of the relation of the adrenal glands to experimentally produced hypotension (shock); with a note on the protective effect of preliminary anesthesia, 79.

Richards, E. L., The elementary school and the individual child, (Abstract), 265.

Richards, E. L., The role of situation in psychopathological conditions, (Abstract), 152.

Rickets, experimental. Studies on. XV. The effect of starvation on the healing of rickets, 31.

Rickets, experimental. Study on. XIX. The prevention of rickets in the rat by means of radiation with the mercury vapor quartz lamp, 125.

Rickets, experimental. Studies on. XX. The effects of strontium administration on the histological structure of the growing bones, 216.

Rickets, experimental. Studies on. XXI. An experimental demonstration of the existence of a vitamin which promotes calcium deposition, 229.

Rickets, experimental. Studies on, XXII. Conditions which must be fulfilled in preparing animals for testing the antirachitic effect of individual foodstuffs, 296.

Rienhoff, W. F., Development and growth of the metanephros or permanent kidney in chick embryos, 392.

Rivers, T. M., Bacterial Nutrition. Growth of a hemophilic bacillus on media containing only an autoclave-stable substance as an accessory factor, 14 9.

Rivers, T. M., Influenza-like bacilli. Growth of influenza-like bacilli on media containing only an autoclave-labile substance as an accessory food factor, 429.

Rosenthal, S. M., A new method of testing liver function with phenoltetrachlorphthalein, 432.


Saline purgatives. The effect of, on tlie absorption of other drugs. 330.

Schaffer, A. J., Folkoff, C and Bayne-Jones, S., On the presence of nucleic acid in bacteria, 151.

Schlaepfer, K., Air embolism following various diagnostic or therapeutic procedures in diseases of the pleura and the lung, 321.

Shipley. P. G., and Blackfan, K. D., The pharmacological action of adrenalin on the sphincter pylori of the fffitus, 159.

Shipley, P. G., McCollum. E. V., Simmonds, N., and Park, E. A., Studies on experimental rickets. XV. The effect of starvation on the healing of rickets, 31.

Shipley, P. G., McCollum, E. V., Simmonds, N., and Park, E. A., Studies on experimental rickets. XXII, 296.

Shipley, P G., McCollum, E. V., Simmonds, N., and Becker, J. E., Studies on experimental rickets. XXI, 229.

ahipley, P. G., Park, E. A.. McCollum, E. V., Simmonds, N., and Kinney, E. M., Studies on experimental rickets. XX, 216.

Shipley, P. G.. Powers, G. F., Park, E. A., McCollum, E. V., Simmonds, N., Studies on experimental rickets. XIX, 125.

Simmonds, N., McCollum, E. V., Becker, J. E., and Shipley, P. G., Studies on experimental rickets. XXI, 229.

Simmonds, N., McCollum, E. V., Shipley, P. G., and Park, E. A., Studies on experimental rickets. XXII, 296.

Simmonds, N., McCollum, E. V., Kinney, E. M., and Grieves, C. J., The relation of nutrition to tooth development and tooth preservation. 202.

Simmonds, N., McCollum, E. V.. Shipley, P. G., and Park, E. A., Studies on experimental rickets. XV. The effect of starvation on the healing of rickets, 31.

Simmonds, N., Powers, G. F., Park, E. A., Shipley, P. G., McCollum, E. v., Study on experimental rickets. XIX, 125.

Smith, D. T., Giant centrospheres in xanthomatous tumors, 342.

Sodium germanate. The effect of, upon the total hemoglobin of the albino rat, 340.

Sphincter pylori of the foetus. The pharmacological action of adrenalin on the, 15 9.

Strontium administration. The effects of, on the histological structure of the growing bones. Studies on experimental rickets. XX, 216.

Syphilis, congenital, The influence of the treatment of syphilitic pregnant women upon the incidence of, 383.

Thayer, W. S., On the cardiac complications of gonorrhoea, 361.

Titles of papers appearing during the year 1921, elsewhere than in the Bulletin, by the present and former member of The Johns Hopkins Hospital and Medical School staff, 69.


Tooth development and tooth preservation. The relation of nutrition to, 202.

Trichomonas hominis, A study of. its cultivation, its inoculation into animals and its staining reaction to vital dyes, 437.

Tuberculosis, The biochemistry of, 2 4 6.

Tumors, brain, Treatment of non-encapsulated, by extensive resection of contiguous brain tissue, 188.

Tumors in the cerebello-pontine angle. An operation for the total extirpation of, 344.

Tumors, xanthomatous. Giant centrospheres in, 342.

Urea excretion, A study of the Austin-Stillman-Van Slyke index of, 294.

Uterine cornu, A normal pregnancy following insertion of the outer half of a Fallopian tube into, 344.

Uveal pigment. The diagnostic and therapeutic use of, in injuries of the uveal tract and sympathetic ophthalmia, 419.

Vaso-motor reactions, 412.

Williams. J. W., A study of frozen sections through a cadaver showing the anatomical relations of a large uterine myoma. 350.

Williams, J. W., Spontaneous labor occurring through an obliquely contracted, kyphotic, funnel pelvis, 190.

Williams, J. W., The influence of the treatment of syphilitic pregnant women upon the incidence of congenital syphilis, 383.

Wilson, D. W., and Bayne-Jones, S., Immunological reactions of Bence-Jones proteins. I. Differences between Bence-Jones proteins and human serum proteins, 37, 119.

Wilson, J. L., Tolerance and acquired tolerance of the mesenchyme cells in tissue cultures for copper sulphate and sodium arsenite, 375.

Wislocki, G. B., and Crowe, S. J., Note on the abdominal chromaffin body in dogs, 377.

Wislocki, G. B., Note on a modification of the chromaffin reaction, with observations on the occurrence of abdominal chromaffin bodies in mammals. 359.

Woods, A. C, and Knapp, A., The diagnostic and therapeutic use of uveal pigment in injuries of the uveal tract and sympathetic ophthalmia, 419.

Wright opsonic technique, A suggested modification of the, based upon the differential white blood count, 142.

Yaws. An analysis of 1046 cases in the Dominican Republic. 43.

Zinck, R. H., Clark. H. M., and Evans, F. A., The protective power of serum in pernicious anemia and other conditions against hemolysis by saponin and by sodium oleate, 16.


ERRATA


Attention is called to two mistakes in the article by Dr. J. Whitridge Williams entitled "The Influence of the Treatment of Syphilitic Pregnant Women upon the Incidence of Congenital Syphilis" which appeared in the November Bulletin.

On page 386, second column, the last paragraph should read as follows:

In conclusion, I am aware that the present paper is based upon too small a material to possess any statistical value, but at the same time the results wliich we have


obtained are highly suggestive and are interesting from two main points of view. First, because our study is based upon the observations made upon a series of women with whose past history we have long been familiar, and secondly, because the results obtained are extraordinarily stimulating from the standpoint of treatment and of the great hope which it offers for the future.

On page 385, Table II, the total for "Deaths after Treatment" should be 9 instead of 3.


Cite this page: Hill, M.A. (2024, March 28) Embryology The Johns Hopkins Medical Journal 33 (1922). Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/The_Johns_Hopkins_Medical_Journal_33_(1922)

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