Paper - Disturbances in mammalian development produced by radium emanation

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Bagg HJ. Disturbances in mammalian development produced by radium emanation. (1922) Amer. J Anat. 30(1): 134-161.

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Disturbances In Mammalian Development Produced By Radium Emanation

Halsey J. Bagg

Huntington Fund for Cancer Research, Memorial Hospital and the Department of Anatomy, Cornell University Medical College, New York City

TEN TEXT FIGURES AND THREE PLATES (FIGURES ELEVEN T0 FIFTEEN)

The effects of radium on animal development has been the subject of several researches since the early work of Bohn (1), in 1903, upon the ova and larvae of the sea-urchin. Experiments on developing nematodes, molluscs, amphibians, fishes, and birds are associated with the names of Perthes (2), P. Hertwig (3), Schaper (4), O. Hertwig (5), and G. Hertwig (6). These investigators report developmental retardations following radiation of the ova and developing embryos. They found a particular susceptibility of the nuclei of the cells and a general slowing up in the developmental processes, especially in the case of the central nervous system. The total disturbances, depending upon the period of development when the radiation was applied, resulted in the formation of monstrosities conforming more or less to a general type}

Similar experiments concerning the effects of x—rays on development have been conducted by many investigators. After exposure to x—radiation, Perthes (7) noted abnormal cell division and a retardation in the development of the ova of Ascaris mega 1 In connection with the above statement, and applying to x—ray treatments as well, the question of dosage is an important one. A survey of the literature shows that there was a very wide range in the severity of the close employed, and in several eases the experimental settings were inadequately described (Bohn used ‘some centigrams’ of pure radium bromide for from twenty minutes to two hours). The amount of radium metal used in the investigations that have been mentioned varied from 2 mg. to 35.1 mg., and the time from a few seconds to several hours. The deleterious changes in the animal tissues varied with the amount of radium and the time of exposure.

locephala. Gilman and Baetjer (8), after radiating the ova of Amblystorna, and Baldwin (9), the fertilized ova of frogs, were able to produce a fairly constant type of developmental defect. injurious results have followed in all cases where mammals have been exposed to x-radiation. It has been shown that when any particular part of a young a.nimal is exposed to a sufficient amount of radiation, that part fails to reach its normal size and is unable to exercise a full degree of function.

Arrests in development and the production of abnormal types may be induced not only by radio-activity, but by many physical or chemical agents. Abnormal temperature changes, treatment by many chemicals, lack of oxygen supply, or the overabundance of carbon dioxide, etc., have produced marked changes in the developing embryo.

The present experiments are mainly concerned with disturbances in mammalian development, before and after birth, as a result of exposing the embryos of rats, at various times during the prenatal period, to irradiation from radium emanation. The effect on the embryos following radiation of the mother at varying intervals before mating was also determined. These experiments were designed not only to study the factors underlying the production of abnormal types, but through an examination of the abnormal to gain a clearer insight into the nature of normal development and differentiation?

I acknowledge with pleasure my indebtedness to Dr. James Ewing for his aid in the interpretation of the pathological results.

METHODS AND APPARATUS

Two methods were used for applying the radium emanation. In the first method an ‘active deposit’ was obtained by exposing a definite quantity of common salt to a comparatively large amount of radium emanation, about 500 millicuries were used,

2 Dr. J. F. Gudernatsch was a co-investigator with the writer during the year 1919. A preliminary report of the Work done with him at that time is given in the Proceedings of the Society for Experimental Biology and Medicine, 1920, vol’. 17, p. 183. RADIUM IRRADIATION AND DEVELOPMENT 135

or the amount of radium emanation initially equivalent to onehalf a gram of radium metal. To the radio-active salt thus produced suflicient water was added to make a physiological solution. The pregnant rats were injected subcutaneously in the shoulder region and intravenously through the caudal vein ; 3 to 4 minims constituted the usual dose. Because of the rapid loss of radio-activity of these solutions, the injections were made immediately after the preparation. The details involved in preparing and measuring the doses, as well as the methods for protecting the experimenter, are described elsewhere (10 and 11). The activated solution exhibited all the known phenomena of radium metal itself; alpha, beta, and gamma rays were present, but the greatest physiological effects were probably due to alpharay activity. After long experimentation, a dose of 5 millicuries was found to be the maximum applicable to the aims of this experiment. In the second method gamma-ray radiation was applied through the ventral body Wall of pregnant rats at nearly full term. A large amount of radium emanation was used, an amount equivalent to 1% grams of radium metal, filtered by 2 mm. of lead and % mm. of silver. The source of emanation was 1 cm. away from the animal. The applicator, called a ‘lead tray’ in clinical usage, was 6 cm. in diameter and 1.5 cm. high. This was placed in the bottom of a small Wire cage, 10 by 13 cm. in diameter and 10 cm. high, and was covered by a thin sheet of cardboard. The animal was placed on this paper immediately. above the applicator.

Preliminary tests showed that a dose of about 1300 millicurie hours was sufficient to produce developmental arrests in the embryos without killing the pregnant animals. Doses as high as 2900 me. hrs., however, were successfully used in some cases. The embryos were killed by ether, and histological material procured at various periods after the treatment. The tissues were fixed in Bouin’s solution, cut in serial section, and stained with haematoxylin and eosin. 136 HALSEY J. BAGG

EXPERIMENTAL RESULTS

Series A. Injections of radio-active solutions

I. Subcutaneous injections after mating. Sixty-five full-grown, normal, pregnant rats were treated in this series. They were divided into four groups, each treated at different periods after mating. Ten pregnant females were injected 7 days after mating; twenty-four, 10 to 14 days after; twenty-one, 15 to 17 days after; and ten, 18 to 21 days after mating. Many of the animals were killed at weekly intervals after treatment, although some were allowed to reach full term.

Various degrees of developmental disturbances were noted, as shown in the following groups:

1. There was a large number of cases where no embryos developed, in others many began development, but were absorbed or aborted at an early time. The females in which no embryos were found, although they were definitely considered pregnant before treatment, occurred among cases treated soon after mating and in those instances where females were autopsied a considerable time after treatment. Figure 1 shows the remnants of maternal and embryonic structures; from the size of the placentae one can see that the foetuses had reached a fair degree of development before the radiation retarded the normal physiological processes. In one case (fig. 2) a small ovoid sac was found attached to the uterine wall by a thin stalk. This apparently represented the remnants of a former embryo and placenta. Extravasated blood and cell detritus were found in this sac and a great many large cells of an epithelioid nature that probably belonged to the former embryonic syncytium. The wall of this cyst was formed by fibrous connective tissue.

2. Embryos were killed by the treatment, but were removed from the mother and preserved before they were absorbed. These showed various extravasations from the vessels of the subcutaneous connective tissue, within the meningeal sinuses, and mainly along the dorsal midline of the body. Figure 3 shows a typical example of such a lesion which was situated in the middorsal line. The mother of this embryo, no. 1167, was mated on RADIUM IRRADIATION AND DEVELOPMENT 137

April 22, 1919, injected with 4.9 millicuries on May 7th, and was killed two days later. When the embryo was cut in serial section, it showed that the haematoma in the dorsal subcutaneous tissues had exerted sufficient pressure upon the spinal column to produce at one place a complete dislocation. Microscopical examination of the viscera showed no pathological changes. Not all the foetuses of a litter were affected in the same degree. In one case seven foetuses were found, three showing haemorrhagic lesions, two beginning to macerate, and two in the process of absorption. This variation in resistance was due either to the higher or lower vitality of the embryos themselves or to the amount of radioactivity which passed the placentae. In another case the foetuses, although injured, were carried to full term, and among a litter of six young, two were apparently normal and four showed haemorrhagic spots on the head, face, and along the dorsal midline of the body.

3. Several young of a single litter showed areas of extravasa— tion and were born alive. Their mother died, however, and foster mothers refused to nurse them.

4. Eight litters gave normal living young. This number is low, because, as previously stated, many pregnant rats were killed by the experimenter at various intervals after treatment. The average number of young per litter was 4.8, as compared with 6.5 per litter for the control rats, but the probable errors indicate that this difference is, very likely, not significant. Only one litter, containing four young, survived a treatment given seven days after mating. Several of the rats of this group, which had apparently escaped the full radium exposure during the uterine period or perhaps they were more resistant to it, when mated inter se produced litters of apparently normal young of normal fertility. The offspring of these animals, about twenty in number, were observed for two generations, but no abnormalities were noted.

II. Subcutaneous injections before mating. Seventy—seven females were treated in this group, eleven died as a result of the injection before they were mated, while several were killed at Weekly intervals after mating, and some were allowed to continue 138 HALSEY J. BAGG

Fig. 1 Two we11—deve]oped placent-ae are shown at the left attached to a uterus which has been partly opened. The remnants of embryonic tissue are superimposed on the placentae. At the right is aplacenta which has been dissected from the uterus, and shows more clearly the remains of embryonic material, here RADIUM IRRADIATION AND DEVELOPMENT 139

to full term. Thirty-four animals were injected between 5 and 7 days before mating; seventeen, 10 to 14 days before, and fifteen, 20 days before mating.

Only three litters in this group showed abnormal young. The most interesting was a litter of seven, in which case the female was treated with 4.2 me., 22 days previous to fertilization, and the foetuses, approximately 16 days old, showed very pronounced areas of extravasation, which in one case (fig. 4) covered a large area on one side of the head and a few small scattered areas on the other side. These areas were not only along the dorsal midline, but also on the lateral surfaces of the body as well (fig. 5). The lesions were much more widely distributed and more variable in size than in the cases recorded under section I. Although the conditions that produced these results were repeated many times, the above is the only case where positive data were obtained. Usually the female had either been rendered sterile or the young were killed and absorbed during early stages. There were two other cases, however, where young were found with haemorrhagic areas, and these occurred in a group of females that were treated seven days before mating. Female 85 was given a dose of 6.6 me. on November 7, 1919. It was mated on November 14th, and

represented as a lighter area in the upper portion of the drawing. Female mated April 22, 1919, injected May 7th, killed May 16th. Dose = 4.6 mc. (subcutaneous).

Fig. 2 A stalked sac partly dissected from the uterus, showing the remnants of a former embryo and placenta. Female mated April 22nd, injected May 7th, killed May 16th. Dose = 4.8 me. (subcutaneous).

Fig. 3 This is a dorsal view of a rat embryo, showing a characteristic area of extravasation due to the treatment of the mother during pregnancy. Female mated April 22nd, injected May 7th, killed May 9th, at which time seven foetuses were found about fifteen days in development. Two of the litter were macerated and two absorbed. Dose = 4.9 mc. (subcutaneous).

Fig. 4 Areas of extravasation are shown in the two views of this embryo, similar to the condition shown in figure 3, but in this case resulting from treating the mother twenty-two days before fertilization. There are a few small scattered areas over the right side of the head and a large area of extravasation on the left side. Female injected April 22nd, mated May 12th, killed May 30th. Seven foetuses were found, fifteen to sixteen days old. Dose = 4.2 Inc. (subcutaneous).

Fig. 5 These are three views of another foetus, a litter mate of the one shown in figure 4, showing the wide distribution of the extravasated areas over both sides and back of the animal. The experimental conditions are the same as for figure 4. 140 HALSEY J. BAGG

as three young were born December 11th, fertilization took place about fourteen days after the treatment. Two of the young were apparently normal, but one showed a large haemorrhagic area, which involved most of the right side of the snout, the right eye, and a portion of the lower jaw on that side. This area disappeared after three days. Female 99, injected and mated at the same time with female no. 85, received a dose of 5.6 me. Five young, three males and two females, were born on December 13th, making the date of fertilization about sixteen days after treatment. One male and one female showed definite haemorrhagic areas on the face. Consideration of these cases will be deferred until later.

Seventeen females following treatment were killed at varying intervals after mating and showed markedly-haemorrhagic or cystic ovaries and congested uteri. In these cases radium emanation apparently had either so altered the maternal tissues as to prevent fertilization or development when started was soon followed by the death of the embryo and its absorption. N any nodules were found in the uteri in which it was impossible to differentiate between embryonic and maternal structures.

The remaining females (as previously stated, eleven died between the period of treatment and mating) produced either full—term normal young or young apparently normal at autopsy. Several of those living young grew normally and were mated inter se, but produced no abnormal offspring, although observed for two generations.

III. Intravenous injections after Imating. The intravenous injections were primarily planned to act as a check on the series of subcutaneous treatments. The object was to determine the immediate reactions that might occur in the embryo as a result of injecting a comparatively large dose of radio—aotive solution into the circulation of the pregnant female, and whether these reactions would be similar to those already recorded for the subcutaneous series. The toxic reactions were so prompt and fatal that it was not necessary to treat many animals to settle this point. A typical case in that of female no. 128. "i his animal, of about nineteen days’ pregnancy, was treated with 30 me. injected directly into the blood stream through the caudal vein. This was six times greater than the usual dose in the first two series. Three young were born dead twenty—four hours later. They showed Very definite radium changes, typical of those already recorded for the subcutaneous series. Figure 6 shows a foetus still attached to an apparently normal placenta, but a characteristic area of extravasation was found over a considerable portion of the left side of the head. In figure 7A a dorsal View shows another embryo with two comparatively small haemorrhagic areas along the dorsal midline, and the placenta in this case is also normal. The third foetus in this litter was apparently normal, but the placenta (fig. 7B) had acted in the nature of a ‘shock absorber’ in protecting the foetus from exposure to the radio-activity, and it was so swollen and completely filled with blood as a result of its injury, that it had the appearance of a large haemorrhagic sac.

Series B. Results from radiating nearly fall-term pregnant rats with ganma—ray radiation

Ten rats were treated at the end of about nineteen days of pregnancy. It was found that exposure to about 1350 mc.hrs. of radium emanation was sufficient to produce Very decided changes in the embryo and yet leave the pregnant females sufficiently uninjured to be able to nurse their young and care for them until after the weaning period. When the dose was increased to 3378 mc.hrs., the young were severely injured, and were either killed outright or died two or three days after birth.

The following are the conditions that resulted in the first generation of animals treated in utero with a dose of about 1350 mc.hrs.:

1. The young of each litter were born two or three days after the treatment, alive and apparently normal.

2. About ten days after treatment, about half of each litter became markedly anemic, showed symptoms of diffuse edema, and promptly died. There was an easily recognizable slow development of meningeal and spinal-cord haemorrhages, similar to those already described as a result of treatment by radio-active Fig. 6 There is a large and a small area of extravasation on the head of this foetus. The mother was injected intravenously with 30 Inc. of radio-active solution and three young, about full term, were born twenty-four hours later. All were dead. In this case the attached placenta is apparently normal.

Fig. 7 The lower figure (A) shows an embryo with two dorsal head lesions and an apparently normal placenta. This is a litter mate of the animal shown in figure 6. At B is indicated a large haemorrhagic placenta from the third young of this litter, which itself was apparently normal.

Fig. 8 Dorsal View of a young rat with the skin dissected to either side. There is a prominent area of meningeal extravasation in the frontal region. This animal was treated in utero with 1350 mc.hrs. of gamma-ray radiation on February 21st and was born apparently normal on February 23rd. It died on March 3rd.

Fig. 9 Dorsal View of a young rat showing areas of frontal and occipital extravasations which were within the meningeal sinuses. There is a smaller lesion in the left dorsal thoracic region. This is a litter mate of the animal shown in figure 8, and the experimental conditions were identical. Death occurred on March 3rd.

Fig. 10 There is an extensive meningeal extravasation over a considerable portion of the hemispheres, and a haemorrhagic lesion is shown on the reflected skin from the dorsal interscapular region. This is a litter mate of the animals shown in the two preceding figures. Death occurred on March 2nd.

142 RADIUM IRRADIATION AND DEVELOPMENT 143

solutions. A series of these lesions is shown in figures 8, 9, and 10. Figure 8 shows a young rat with the dorsal integument partly dissected away, exposing a typical haemorrhagic area in the region of the frontal lobes. The slow development of this lesion could be easily noted through the thin, transparent scalp. This young was one of several treated in utero with 1350 mc.hrs. of gammaray radiation on February 21, 1920. It was born two days later, and died on March 3rd. The young rat shown in figure 9 was a litter mate of the previous animal. It shows the presence of three distinct haemorrhagic areas, a small frontal lesion, a fairly extensive one in the occipital region, and a small lesion in the subcutaneous tissues in the thoracic region, near the middorsal line on the left side of the body. This animal also died on March 3rd. A third animal belonging to the same litter is shown in figure 10. Here is seen a still more acute reaction, as shown by the fact that the animal died a day sooner than in the two cases above. There is an extensive area of meningeal haemor— rhage which covers most of the dorsal portion of the brain, involving the frontal and occipital regions and the medial area between, as well as a considerable portion of the right temporal area. In addition, a distinct, rounded haemorrhagic lesion may be noted on the reflected skin on the left side of the body. This lesion occurred in the midshoulder region of the back.

The heads of several of the young rats showed marked lateral compression. In one case a haemorrhage so affected the spinal cord as to produce complete paraplegia. The tissues of these animals were studied histologically. Save for the mechanical disturbances produced by the presence of the extravasated areas, the most marked pathological conditions were seen in the liver and intestines. In the first case there was a pronounced fatty degeneration of the hepatic cells, and in the second, a desquama— tion of the lining cells of the intestinal mucosa.

3. It is interesting to note that the other half of each litter survived the treatment, grew to a normal size, and some animals have lived for over eighteen months. They showed the

effects of the late uterine treatment by the following arrests in development: 144 HALSEY J. BAGG

a. The first pathological condition noted was that the eyes became smaller, the pupils opaque, and there finally was a complete, or nearly complete, closing of the lids and total blindness. This condition was first observed a short time after the eyes had opened. The photographs in figure 11 show three views of a female rat about one year old with typical eye deformities. The upper view shows the entire animal, which had grown to normal size and Weight for its age. The left eye was nearly completely closed, as is shown more clearly in the lower right—hand View of the head at a higher magnification. Both pupils were opaque, but, as shown in the illustration, the right eyelids were slightly more opened than those of the other side. The animal was one of a litter treated in utero on March 8, 1920, was born six days later, and the photograph was taken on March 1, 1921. The dose in this case was 2920 mc.hrs. of gamma-ray radiation, which was a dose higher than that usually tolerated.

b. Mating tests showed that both the males and females were completely sterile in the first lots, but subsequently a first-generation female, that had been treated with 1350 mc.hrs., mated with a male similarly treated, gave birth to nine apparently normal young.

c. Before these adult offspring of treated animals were killed for histological examination, their neurological reactions were very carefully studied. The animals, being blind, when startled assumed various defensive attitudes, but save for these reactions their behavior was remarkably normal. There was no ataxia in locomotion or in any of the feeding reactions, auditory acuity was normal, and there was no cutaneous hypoesthesia or other sensory disturbances. Except for blindness, there was nothing to suggest abnormal sensory function.

d. When these animals were autopsied, marked developmental disturbances were noted in the condition of the central nervous system. The cerebral hemispheres were greatly reduced in size, and in several cases very little cortical material remained. Those portions of the brain that were ontogenetically older (the archiostriatum and the cerebellum) were apparently normal. The optic tracts were markedly at-rophic. Correlated with this RADIUM IRRADIATION AND DEVELOPMENT 145

disturbance in brain development, the skull was found to be asymmetrical, narrow, thicker than normal, and concave in the frontal region.

Figure 12 shows a dorsal view of a normal, untreated brain of an adult rat, magnified five diameters. In figures 13 and 14 are dorsal and lateral views of a brain of one of the rats which belonged to the same litter as those of section 2 of this series. This animal was treated with 1350 mc.hrs. on February 21, 1920, was born on February 23rd, and was killed December 31, 1920. This was one of the animals which (except for blindness) showed no abnormal neurological reactions. The magnification in figures 13 and 14 is the same as that for the control brain in figure 12. ‘The dorsal view in figure 13 shows an apparently normal cerebellum and normal olfactory lobes, but the part of the brain which represents the rudiments of the hemispheres shows a great lack of development of cortical substance. In a side View of the brain in figure 14, the cortex may be seen to be very thin; indeed, not completely covering what should normally be the frontal, occipital, and lateral aspects of the brain. The remains of the hemispheres do not sufliciently approach each other in the median line to cover the colliculi beneath. In figure 13 the meninges on the left side of the brain have been removed, but on the other side they have been left in place. It was possible in this specimen to see the lateral ventricles through the transparent membranes. Several other brains have been studied which showed various degrees of developmental arrests resulting from radium treatment. In some cases the hemispheres were markedly reduced in size, were widely divergent in the median line, and yet the pallium was complete over the entire surface. In all these cases there was marked optic atrophy. These brains are now being sectioned, and a study of them in greater detail will be the subject of a separate communication.

e. A histological study of the eye showed that the eyeball was reduced to one-fourth the normal diameter. The retina was missing, but traces of the choroid remained as a few scattered pigment cells. The cornea was three times as thick as normal and covered with four or five layers of opaque squamous epithe146 HALSEY J. BAGG

lium. The optic nerve was extremely small, not more than onethird the normal dimensions.

f. The testes of the radiated animals were decidedly atrophic, and a comparison with the normal is shown in the photograph in figure 15. The diameters of the testicle alone (minus the epididymis) of the experimental animal was 14 mm. for the length and 7 mm. for the width, while the control measurements from normal animals of the same age and weight and with the same method of fixation were 21 mm. for the length and 11.5 mm. for the width. The epididymis of the radiated testis was practically missing. A. small portion of the tail remained, but the head and body of the epididymis had failed to develop. Histological examination shows that there is little evidence of spermatogenesis. Some tubules seem to contain imperfect spermatoblasts and forming spermatozoa, but the great majority of tubules show complete degeneration and loss of epithelial cells, and contain loose granular material, which in places is calcified. Some spermatic tubules are greatly dilated and filled with granular material. Very few interstitial cells are visible.

The ovary of the radiated animals was reduced to one-fourth or one-fifth the normal size. The graffian follicles were entirely missing. Groups of lutein cells persisted in small numbers, but showed marked hydropic degeneration. Some of the large vessels about the ovary were sclerosed.

g. The liver, kidney, lungs, spleen, and the other organs were examined, but showed no pathological disturbance.

CONTROL GROUP

Pregnant rats of the same stock, the same age and weight, were injected subcutaneously and intravenously with equal amounts of solutions that previously had been strongly radioactive, but were allowed to ‘decay,’ until they had lost their radio—activity. These experiments gave absolutely negative results. As a control to the gamma-ray experiments, pregnant rats, sisters of the treated animals, were allowed to breed under exactly the same experimental conditions. No abnormal young were observed. RADIUM IRRADIATION AND DEVELOPMENT

DISCUSSION AND SUMMARY OF RESULTS

It has been shown that when doses of radio—active solutions are injected into an animal marked physiological reactions take place. Large doses produce severe toxemia, resulting in pronounced pathological changes in the various viscera of the white rat (10). A study of metabolic changes in dogs, as determined by urine analysis, showed that, following intravenous injections of such solutions, there were very decided increases in the total nitrogen content of the urine, the urea, creatinine, uric acid, and the total phosphates (12). A prompt reduction occurred in the number of white blood cells of the dog after intravenous injections of these solutions, associated with a marked decrease in the relative percentage of circulating lymphocytes (13). In order to reduce as much as possible the severity of the reaction, very small doses of radio-activity were used in the experiments recorded in this article. But even with comparatively small doses, certain rats treated in utero showed very acute reactions. Many were killed by the treatment and were absorbed or aborted. Others were found showing pronounced areas of subcutaneous extravasations, mainly situated along the middorsal line of the body and within the meningeal sinuses. This condition was probably due to the destructive action of radium on the endothelium of the blood vessels, as well as a possible increase in blood pressure, as was shown to occur in the dog by Burton-Opitz and Meyer (14) after intravenous injections of very small quantities of radium bromide. A similar reaction of the blood vessels to radiation was previously reported by Halkin (15) for the skin of pigs, and by Danysz (16) for radiated mice. This destructive action of radium on the blood vessels is in line with clinical observations on the usual prompt regression of very vascular tumors (the angiomata, in particular) after exposure to irradiation.

The changes in the rat embryos of this experiment are interesting in so far as they show that a sufficient amount of radioactivity was able to pass the placenta and subsequently affect the developing embryo. This occurred after subcutaneous as well as intravenous injections of the mother. By far the most 148 HALSEY J’. BAGG

interesting observation concerned the presence of lesions similar to those described above in rat embryos whose mother was treated with radio-active solutions a considerable time before mating. The writer has no explanation to account for this phenomenon. It would appear that the treatment of the mother several days previous to conception has lessened the faculty of the later-developing embryo to form proper endothelium of the blood vessels, and the wide distribution of these lesions over the body of the embryo (peculiar to this group of animals) would tend to substantiate this view. One female was injected twentytwo days before fertilization, and since the solutions lose their radio-activity very rapidly (there is about a 50 per cent reduction in the first hour after the preparation) the likelihood of any radio—activity remaining over during this period and affecting the egg at a later critical moment is remote. The amount of radio~ activity remaining after twenty—two days, if present at all, should, as determined from physical computation, bcinfinitesimally small.

The series of intravenous injections again emphasize the specific action of radium emanation in the production of typical areas of subcutaneous emtravasations in the developing young,

‘ and in addition shows that the placenta may act in the nature of

a ‘shock absorber’ and prevent the embryo from receiving the full effect of the radiation.

We now come to a consideration of the cases wherein pregnant females were treated with external applications of comparatively large doses of gamma—ray radiation. At this time a report is given only for embryos treated towards the end of pregnancy. The writer plans to continue this line of investigation and treat at earlier prenatal periods.

The results emphasize the well—known_delayed reaction associated with gamma-ray radiation. There was approximately a ten—day interval following treatment during which no changes were noted in the embryo, and during this period the young animals were born in an apparently normal condition. Acute reactions promptly occurred at the completion of this time, killing half of each litter. The young rats died showing typical radium changes, such as anemia, diffuse edema, and meningeal, RADIUM IRRADIATION AND DEVELOPMENT 149

spinal—cord, and subcutaneous extravasations. These extravasations were markedly similar to those already described for the series of solution treatments. The liver in these animals showed a fatty degeneration of the hepatic cells similar to the condition reported by Mills (17) after exposing a series of mice to gammaray radiation. The only other pathological change noted in these embryos was a desquamation of the lining cells of the intestinal mucosa. This observation is in line with the results emphasized by Hall and Whipple (18) in their experiments on Roentgen-ray intoxication in dogs.

While the animals described above died after showing acute reactions certain of their litter mates (half of the litter) continued to develop apparently normally. This difference in reaction may possibly be due to individual variability or tolerance for the radiation, but it probably can be explained by the fact that certain embryos were slightly farther away from the source of radiation than others, and as the intensity of radiation varies inversely as the square of the distance, even such slight differences in distances that did exist would be sufficient to subject the embryos to a considerable range in intensity of radiation. This is especially important in this case because the source of radiation was only 1 cm. from the body wall of the mother. The quality of radiation, however, remained the same for all the embryos.

It was soon apparent that the animals that lived over the tenday period had not completely escaped the effect of the radiation, as was shown by a suppression-of the full development of the eyes. Eye defects were noted, such as opaqueness of the pupil, atrophy of the lens, and closing of the lids, which resulted in complete blindness. These animals grew to a normal size, successfully competed with their cage mates for food, and showed absolutely no abnormal neurological condition, except those clearly incident to blindness. At autopsy, in some cases over a year after birth, very decided developmental arrests were noted in the structure of the brain. All grades of such maldevelopment were noted in the condition of the neopallium, from merely a decrease in the size of the hemispheres, which permitted the corpora quadrigemina to be clearly visible from above, to a more

nu: AMERICAN JOURNAL or ANATOMY, von. 30, No. 1 150 HALSEY J. BAGG

marked absence of the cerebral cortex until only a very thin lamina of tissue remained to represent that structure, and there were large areas in the frontal, occipital, and temporal region where no cortex existed at all, so that when the meninges were removed the basal ganglia were clearly seen from without.

This correlation between defects in the development of the eye and the brain has been emphasized by Stockard (19) in his recent paper on developmental rate and structural expression. He states as follows: “The periods of arrest necessary to induce the eye and the brain modifications are so close together or so nearly the same, that one generally finds combinations and mixtures of the defects among the same experimental group of embryos.” Again in the same article Stockard has shown that the type of deformity that results from experimental disturbance depends upon the developmental moment at which the interruption occurs. It is significant that the animals of this experiment showed arrests in the development of the neopallial portions of the brain and not in those regions which are ontogenetically older. Apparently the radium emanation, acting towards the end of pregnancy, had affected the development of the brain after the basal ganglia, the cerebellum, and medulla had become fairly well differentiated, and therefore those portions showed no gross changes. But the radium had slowed the developmental rate of the neopallium (which we know is one of the last portions of the brain to differentiate) during its period of active cell proliferation, and that portion of the brain was never able to reestablish its proper rate of development in relation to the other parts of the brain. If the period of treatment had occurred earlier in prenatal existence, other portions of the brain would probably have shown disturbances as well. The writer does not believe that the deformities in the brains of these animals were due to the early production of vascular disturbances later recovered from, but to an actual inhibitory effect of the radiation upon the developing nerve cells. If extravasations had occurred in this group of animals, and were so situated as to affect the development of the cerebral cortex in particular, they probably would have been detected as were even the comparatively small lesions RADIUM IRRADIATION AND DEVELOPMENT 151

which were associated with the acute reactions. Also, if the effect was largely due to vascular disturbances, from the nature of the radiation employed, one would expect more generalized changes throughout the entire brain. However, on the other hand, Craigie (20), in his recent paper on the relative vascularity of various parts of the central nervous system of the albino rat, suggests that “the vascularization of the more recently evolved centers (of the cortex cerebri) is more susceptible than the more ancient regions to sexual, hereditary or environmental influences.”

From a neurological point of view, it is interesting to consider that the animals with practically no cerebral cortex reacted so normally in their ordinary behavior. Except for blindness, there was no other apparent sensory disturbance, and motor coordination appeared perfect. The physiological functions localized in the cerebral cortex of the rat were in these animals apparently transferred to the basal ganglia and other paleokinetic portions of the brain, showing the remarkable degree of compensation possible in the mammalian brain when the disturbing element acts at an early period in its development. In this connection it is worth mentioning that the radium emanation did not produce a sudden traumatic effect, as is normally the case with the experimental production of brain lesions, and, in fact, the radium changes were probably prolonged over a considerable period. This condition favored the establishment of compensatory reactions, and exists (as shown by the writer in a recent article (21)) even in the case of radium lesions experimentally produced in adult mammalian brains.

The reproductive system completes its development a considerable time after birth, and so it is not at all surprising that these structures should have shown a considerable amount of atrophy due to the developmental arrest during the prenatal period. The ovaries and testes appeared to suffer with equal severity.

An interesting correlative relation was shown by the fact that the other viscera (digestive, excretory, etc.) of the animals that showed marked developmental arrests of the nervous and reproductive systems were apparently normal and the animals grew 152 HALSEY J. BAGG

to an average size. The lungs, liver, kidneys, etc., had differentiated before the physical agent was employed. Further studies with earlier prenatal treatments should throw seine light on establishing the critical growth periods of the various embryonic structures.

As a final point, the results of this investigation may be of interest to the clinicians and the laboratory workers who handle large quantities of radium and utilize x-rays. Although the results of this paper deal only with irradiation of the female, there is no reason to believe that the germ cells of the male are more resistant to these destructive agents than those of the female, and, in fact, there is very good experimental evidence to show that spermatozoa of some animals are especially likely to produce abnormal young after exposure to comparatively small quantities of irradiation. Physicians should guard against the possibility of producing developmental arrests such as shown in this article when treating pregnant women, as Well as the possibility of altering the human germ cells by irradiation previous to conceptionfi

CONCLUSIO‘.\'S

1. The marked selective action of radium emanation on fastgrowing embryonic structures was noted in these experiments.

2. Very decided developmental arrests occurred in the differentiation of the nervous and reproductive systems of mammalian embryos exposed to irradiation towards the end of pregnancy.

3. Experimental animals with greatly reduced, or practically no neopallium, gave apparently normal neurological behavior, except for blindness.

4. Radium emanation, used either in the form of a radio-active solution injected into the adult female, or employed as an external

3 The Writer does not mean to be understood as stating that present-day clinical irradiation treatments produce such effects in the developing young, but it his personal opinion that such changes are biologically possible. It is not possible to obtain desired information by comparing the amount of exposure that a small mammal can stand with the corresponding dose that a man should tolerate. judging by comparative weights. The small mammal can tolerate very much more radiation in proportion to its weight than a man can. RADIUM IRRADIATION AND DEVELOPMENT 153

gamma—ray radiation, produced marked areas of extravasation in the subcutaneous connective tissue of the developing young. This suggests that the action of radium emanation might be selective upon the endothelium of blood vessels.

5. Extravasations occurred in the developing young of females treated with radio—active solutions a considerable time before fer— tilization, and suggest that in some way the faculty of the later developing embryos to form proper blood vascular endothelium had been interfered with.

6. The results so far obtained indicate that gamma-ray radiation is a physical agent admirably adapted to the study of experimentally produced developmental arrests in mammalian embryos.

7. When women are subjected to therapeutic irradiation, especially during the early stages of pregnancy, the clinician should be forewarned concerning the possibility of producing very grave disturbances in the developing child.

LITERATURE CITED

1 BOHN, B. 1903 Action des rayons du radium sur les téguments. Compt. rend. Soc. de bi0l., T. 40, p. 1442.

PERTHES, G. 1904 Versuche fiber den Einfluss der Rontgenstrahlen und Radiumstrahlen auf (lie Zellteilung. Deut. med. Woch., Bd. 30, S. 632.

3 HERTWIG, P. 1911 Durch Radiumbestrahlung hervorgerufene Veréinder ungen in der Kernteilungsfiguren der Eier von Ascaris megalocephala. Archiv. f. lnikros. Anat., Bd. 77, N0. 2, S. 301.

4 SCHAPER, A. 1904 Experiinentelle Untersuchungen fiber die Wirkung des Radiums auf embryonale und regenerative Entwicklungsvorgange. Deut. med. Woch., Bd. 30, S. 1434.

HERTWIG, O. 1911 Die Radiumkrankheit tierischer Keimzellen. Archiv. f. mikros. Anat., Bd. 77, No. ‘2, S. 1.

6 HERTWIG, G. 1911 Radiumbestrahlung unbefruchteter Froscheier und ihre Entwicklung nach Befruchtung mit normalem Samen. Archiv. f. mikros. Anat., Bd. 77, No. 2, S. 165.

PERTHES, G. 1904 Versuche fiber den Einfiuss der Rontgenstrahlen und Radiumstrahlen auf die Zellteilung. Deut. med. Woch., Bd. 30, S. 668.

S GILMAN, P. R, AND BAETJER, F. H. 1904 Some effects of the Réjntgen rays

on the development of embryos. Amer. Jour. Physiol., Vol. 10, p. 222. 9 BALDW1\', W. l\I. 1919 The artificial production of monsters conforming to a definite type by means of x-rays. Anat. Rec., vol. 17, no. 3, p. 135.

ll) BAGG, H. J. 1920 Pathological changes accompanying injections of an active deposit of radium emanation. Jour. Cancer Res, vol. 5, no. 1, p. 1.

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21

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DUANE, W. 1917 Methods of preparing and using radioactive substances in the treatment of malignant diseases and of estimating suitable doses. Boston Med. and Surg. Jour., vol. 177, no. 23, p. 787.

THEIS, R. C., AND BAGG, H. J. 1920 The efiect of intravenous injections of active deposit of radium on metabolism in the dog. Jour. Biol. Chem., vol. 41, no. 4, p. 525.

BAGG, H. J. 1920 The response of the animal organism to repeated injections of an active deposit of radium emanation. Jour. Cancer. Res, vol. 5, no. 4, p. 301.

BURTON—OPITZ, R., AND MEYER, G. M. 1906 Effects of intravenous injections of radium bromide. Jour. Exp. Med, vol. 8, p. 245.

I‘1ALKIN, H. 1903 Uber den Einfluss der Becquerelstrahlen auf die Haut. Archiv. f. Dermat. u. Syphilis, Bd. 65, S. 201.

DANYSZ, J. 1903 De l’aetion pathogene des rayons et des émanations émis par lc radium sur diitérentes tissus et diflérentes organismes. Compt.

_ rend. Soc. de biol., T. 136, p. 461.

MILLS, G. P. 1910 The effect of radium on healthy tissue cells. Lancet, vol. 2, p. 462.

HALL, C. C., AND WH11>1>LE, G. H. 1919 Roentgen-ray intoxication: Disturbances in metabolism produced by deep massive doses of the hard Roentgen rays. Amer. Jour. of the Med. Sciences, vol. 157, no. 4, p. 453.

STOCKARD, C. R. 1921 Developmental rate and structural expression: An experimental study of twins, ‘double monsters’ and single deformities, and the interaction among embryonic organs during their origin and development. Am. Jour. Anat., vol. 28, no. 2, p. 115.

CRAIGIE, E. H. 1920 On the relative vascularity of various parts of the central nervous system of the albino rat. Jour. Comp. Neur., vol. 31, p. 429.

BAGG, H. J. 1921 The effect of radium emanation on the adult mammalian brain. Amer. Jour. Roent., vol. 8, no. 9, p. 536. PLATES

155 PLATE 1 EXPLANATION QF FIGURE

11 The upper photograph shows an adult rat with eye deformities due to g:umi:a—ray irradiation during the prenatal period. The size and weight are normal for its age. The lower figures show the lateral Views of the head at a higher magnification. Both pupils are opaque and the left eyelids are nearly completely closed. Mother treated on March 8, 1920, young were born on March 14th, and photograph was taken on March 1, 1921. Dose = 2920 me.hrs.

156 RADIUM IRRADIATION AND DEVELOPMENT PLATE 1 HALSEY J. BAGG

157 PLATE 2 EX1’LANATION or FIGURES

12 Dorsal View of a normal untreated brain of an adult rat.

13 Dorsal View of the brain of an adult rat showing the marked developmental arrest in the formation of the neopallium. The meninges on the left side of the brain are removed. Mother treated with gamma-ray irradiation on February 21, 1920, young were born on February 23rd. Brain from radiated young removed on December 31, 1920. Dose = 1350 mc.hrs. (For further reference see text.) The magnification is the same as for the drawing of the normal brain.

14 A lateral view of the brain shown in figure 13. This shows the thinness of the remains of the cerebral cortex and its total absence in the frontal and occipital regions. (For further reference see text.)

158 RADIUM IRRADIATION AND DEVELOPMENT PLATE 2 EAISEY J. BAGS

159 PLATE 3

EXPLANATION OF FIGURE

15 At the right is shown a normal untreated testicle of a white rat surmounted by a well-developed epididymis, which is partly obscured by fat. At the left is a radiated testis showing considerable atrophy. The single small lobe at the very bottom of the photograph represents the remains of the tail of the epididymis, the head and body of that part being completely missing. The animal was treated in utero on February 21, 1920, was born February 23rd, and was killed December 31, 1920. Dose = 1350 Inc.hrs. of gamma-ray irradiation.

160 RADIUM IRRADIATION AND DEVELOPMENT PLATE 3

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