Anatomical Record 10 (1915-16): Difference between revisions

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W. B. KiRKHAM AND H. W. Haggard. The anatomy of a three-legged kitten. Three figures 537
W. B. KiRKHAM AND H. W. Haggard. The anatomy of a three-legged kitten. Three figures 537


{{Ref-Scammon1916}}
{{Ref-Scammon1916}} Ten figures 543
 
Richard E. Scammon. On the development of the biliary system in animals lacking a gall-bladder in post-natal life. Ten figures 543


===No. 9. JULY===
===No. 9. JULY===
Line 14,446: Line 14,444:
ScHAEFFER, J. P. AND Nachamoffsky, L. H. 1914 Some observations on the anatomy of the upper extremities of an infant with complete bilateral absence of the radius. Anat. Rec, vol. 8, no. 1.
ScHAEFFER, J. P. AND Nachamoffsky, L. H. 1914 Some observations on the anatomy of the upper extremities of an infant with complete bilateral absence of the radius. Anat. Rec, vol. 8, no. 1.


ON THE DEVELOPMENT OF THE BILIARY SYSTEM IN ANIMALS LACKING A GALL-BLADDER IN POSTNATAL LIFE


RICHARD E. SCAMMON Institute of Anatomy, University of Minnesota^
===On The Development Of The Biliary System In Animals Lacking A Gall-Bladder In Postnatal Life===
 
Richard E. Scammon Institute Of Anatomy, University of Minnesota


TEN FIGURES
Ten Figures


It is well kjiown that the gall-bladder is always absent in some species of vertebrates. There seems to be no general rule governing the distribution of this peculiarity. Thus in the cyclostomes a gall-bladder is found in the myxinoids but is absent in the fully developed Petromyzon. It is present as a rule in fishes although absent in a few species of sharks. A gallbladder is said to be present always in amphibians and reptiles; it is wanting in a number of birds including the pigeons. Among mamnaals no gall-bladder is found in a number of rodents and in noany ruminants, pachyderms and some other ungulates. It is absent in the Cetacea. In other forms the gall-bladder may or ntiay not be present. This is the case in several species of birds and in a few mammals. With the latter may be included man where congenital absence of the gall-bladder is an unusual anonaaly. In man the gall-bladder alone ntiay be lacking, or more rarely both the gall-bladder and the larger biliary ducts.^
It is well known that the gall-bladder is always absent in some species of vertebrates. There seems to be no general rule governing the distribution of this peculiarity. Thus in the cyclostomes a gall-bladder is found in the myxinoids but is absent in the fully developed Petromyzon. It is present as a rule in fishes although absent in a few species of sharks. A gallbladder is said to be present always in amphibians and reptiles; it is wanting in a number of birds including the pigeons. Among mamnaals no gall-bladder is found in a number of rodents and in noany ruminants, pachyderms and some other ungulates. It is absent in the Cetacea. In other forms the gall-bladder may or ntiay not be present. This is the case in several species of birds and in a few mammals. With the latter may be included man where congenital absence of the gall-bladder is an unusual anonaaly. In man the gall-bladder alone ntiay be lacking, or more rarely both the gall-bladder and the larger biliary ducts.^


Only three of the forms consistently lacking a gall-bladder are commonly available for embryological study. These are the
Only three of the forms consistently lacking a gall-bladder are commonly available for embryological study. These are the
Line 14,658: Line 14,657:
chez Petromyzon. M^m. Soc. de la Zool. de France, T. 26.
chez Petromyzon. M^m. Soc. de la Zool. de France, T. 26.


REGARDING THE LENGTH AND EXTENT OF THE HUMAN MEDULLA SPINALIS
===Regarding The Length And Extent Of The Human Medulla Spinalis===


ROLLO E. McCOTTER From the Department of Anatomy ^ Univereity of Michigan
Rollo E. Mccotter From the Department of Anatomy ^ Univereity of Michigan


Most of our modern text-books on human anatomy state that the spmal medulla is variable in length and in extent within the vertebral canal and that there is no definite relation between the length of the spinal medulla on the one hand and the length of the vertebral colmnn and the height of the individual on the other. It was with these well known facts in mind that the study of this series of 234 cases was undertaken.
Most of our modern text-books on human anatomy state that the spmal medulla is variable in length and in extent within the vertebral canal and that there is no definite relation between the length of the spinal medulla on the one hand and the length of the vertebral colmnn and the height of the individual on the other. It was with these well known facts in mind that the study of this series of 234 cases was undertaken.

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THE ANATOMICAL RECORD

EDITORIAL BOARD

Irving Hardestt Wabrbn H. Lewis

Tulane University Johns Hopkins University

Clarence M. Jackson Charles F. W. McClurb

University of Minnesota Princeton University

Thomas G. Lee Wiluam S. Miller

University of Minnesota University of Wisconsin

Frederic T. Lewis Florence R. Sarin

Harvard University Johns Hopkins University

George L. Streeter

University of Michigan

G. Carl Huber, Managing Editor

1330 HlU Street. Ann Arbor. Michigan

VOLUME 10 NOVEMBER, 1915-JULY, 1916

PHILADELPHIA THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY COMPOSED AND PRINTED AT THE WAVERLY PRESS

Br THB Williams & Wilkins Company

Baltimore, Md., U. 8. A.

Contents

No. 1. NOVEMBER

John Lewis Bremer. The mesonephric corpuscles of the sheep, cow and deer. Three figures 1

Ingalls NW. Truncus arteriosus communis persistens. (1915) Anat. Rec. 10(1): 9-14 One figure

Reeves TB. A double umbilicus. (1915) Anat. Rec. 10(1): 15-18. Three figures

Atwell WJ. The relation of the chorda dorsalis to the entodermal component of the hypophysis. (1915) Anat. Rec. 10(1): 19-42. Twelve figures.

Wayne J. Atwell. On the conversion of a photograph into a line drawing. One figure 39 Edmond Souchon. Preservation of anatomic dissections with permanent color of muscles, vessels and organs by newer methods 43

No. 2. DECEMBER

Henry R. Muller. Notes on the bursting strength of the alimentary tract of the cat. Four figures 53

C. J. Bartlett. Direct union between adrenals and kidneys (subcapsular location of adrenals). Five figures 67

Franklin P. Reagan and J. Monroe Thorinoton. The vascularization of the embryonic body of hybrid teleosts without circulation. Fifteen figures 79

Franklin P. Reagan. A further study of the origin of blood vascular tissues in chemically treated teleost embryos, with especial reference to haematopoesis in the anterior mesenchyme and in the heart. Twenty-six figures 99

No. 3. JANUARY

Frontispiece. Portrait of Charles S. Minot 132

Lewis FT. Charles Sedgwick Minot. An Address. (1915) Anat. Rec. 10(3): 133-164.

Proceedings of the American Association of Anatomists. Thirty-second session 165

Proceedings of the American Association of Anatomists. Abstracts 171

Proceedings of the American Association of Anatomists. Demonstrations 265

List of officers and members 271

No. 4. FEBRUARY

M. R. Lewis. Sea water as a medium for tissue cultures. Four figures 287

William Cogswell Clarke. Experimental mesothelium. Eleven figures 301

George B. Jenkins. A study of the morphology of the inferior olive. Six figures 317

John Locke Worcester. Hernia of the small intestine into a persistent great omental cavity 335

P. G. Shipley. The development of erythrocytes from hcmoglo!)in-free cells and the differentiation of heart muscle fibers in tissue cultivated in plasma. Two figures 347

C. H. Danforth. The use of early developmental stages in the mouse for class work in embryology 355

C. M. Jackson. Book Review. Rat Book 359

No. 5. MARCH

Bexjamin B. Lipshutz. Studies on the blood vascular tree. I. A composite study of the femoral artery. Five figures 361

RoLLo E. McCoTTER. Three cases of the persistence of the left superior vena cava. Three figures 37 1

Charles H. Spurgeon and Ralph J. Brooks. The implantation and early segmentation of the ovum of Didelphis virginiana. Fifteen figures 385

Danchakoff V. The origin of blood cells. (1916) Anat. Rec. 10(5): 397-414.

Vera Danchakoff. Concerning the conception of potentialities in the embryonic cells. . 415

H. E. Jordan. Evidence of hemogenic capacity of endothelium 417

No. 6. APRIL

H. E. Jordan. Richard Henry Whitehead, Portrait 421

Theodore L. SguiER. On the development of the pulmonary circulation in the chick. Three text figures and two plates 425

P. G. Shipley. The vital staining of mitochondria in Trypanosoma lewisi with janus ; green. Eight figures 439

S. S. Schoc^het. a suggestion as to the process of ovulation and ovarian cyst formation. . 447

Marcus Ward Lyon, Jr. A porcupine skull with a pair of supernumerary well developed incisors in the upper jaw. One figure 459

Morris Kush. Ernest Philip Boas. The carrying angle. One figure 463

No. 7. MAY

Weed LH. The formation of the cranial subarachnoid spaces. (1916) Anat. Rec. 10(7): 475 - 481.

Vera Danchakoff. The wandering cells in the loose connective tissue of the bird and their origin 483

H. E. Jordan. The microscopic structure of the leg muscle of the sea-spider, Anoplodactylus lentus. Seven figures 493

Frederick S. Hammett. The correlation of certain chemical findings with histological structure 509

J. R. Callahan. Collateral root canals and multiple foramina in human teeth. Three figures 513

W. M. Smallwood. a short spinal cord in the toad. Two figures 515

No. 8. JUNE

Raphael Isaacs. Properties of colloids in relation to tissue structure 517

Robert T. Hance. A simple paraffin ribbon winder. Two figures 523

Simon H. Gage and Benjamin F. Kingsbury. Some apparatus for the microscopical laboratory. Fifteen figures 527

W. B. KiRKHAM AND H. W. Haggard. The anatomy of a three-legged kitten. Three figures 537

Scammon RE. On the development of the biliary system in animals lacking a gall-bladder in post-natal life. (1916) Anat. Rec. 10(8): 543 Ten figures 543

No. 9. JULY

RoLLo E. McCoTTER. Regarding the length and extent of the human medulla spinalis. . 559

Ezra Allen. Studies on cell division in the albino rat (Mus Norvegicus, var. alba). one text figure and two plates 565

H. D. Senior. Reference models of the abdominal viscera. Three figures 591

P. G. Shipley and C. C. Macklin. The demonstration of centers of osteoblastic activity by use of vital dyes of the benzidene series 597


The meeonephric corpuscles of the sheep, cow and deer

John Lewis Bremer

Department of Anatomy, Harvard Medical School

THREE FIGURES

In this paper I have employed the name corpuscle to indicate the entire blind end of an excretory tubule, the term glomerulus to indicate the knot of capillaries, covered by the inner wall, projecting into the expansion of this end. A corpuscle (Malpighian) then includes ordinarily a glomerulus, the slit-like crescentic cavity continuous with that of the tubule, and the outer layer or Bowman's capsule.

The renal and mesonephric corpuscles of mammals have been so long known and their development so fully studied that a new type, not yet described to my knowledge, seems to me worthy of a short note. Most of the investigations on this subject have been carried out in man, rabbit, and pig, and the similarity of the corpuscles in these forms, in shape and in developmental history, to those of birds and reptiles probably discouraged wider research in other classes of mammals. The peculiar corpuscles to be described are found in the sheep, cow, and deer, and thus may be typical of the ruminants. They are not mentioned in the Normentafel of the deer by Sakurai.^

The excretory corpuscles and tubules develop, as is well known, from the nephrogenic portion of the nephrotome. The most detailed work on the changes in this tissue which lead to the formation of the corpuscles is that of Stoerk,^ who studied the renal corpuscles in man, and of Felix' who described the meso 1 KeibePs Normentafel, Reh. 1906.

2 Anat. Hefte, Abt. 1, Bd. 23, 1903.

» Keibel and MalPs Human Emb., vol. 2, p. 796 et seq., 1912.

1


THE ANATOWCAL BSCORD, VOL. 10, NO. 1 NOVEMBER, 1915

2 JOHN LEWIS BREMER

nephric corpuscles in man and rabbit. According to both these authors the nephrogenic tisue first is divided or gathered into separate solid masses or balls, which then become hollow rounded vesicles. These elongate to a tubular form, and the cavity of one end becomes T-shaped, by means of an increased thickness of the epithelimn along the tube, thus narrowing its lumen (the upright bar of the T), and a lateral expansion of the end of the vesicle with a flattening of the end wall (the transverse bar of the T). Later one side of the transverse bar is exaggerated at the expense of the other, and this together with a curve in the opposite direction at the other end of the upright, which opens into the Wolffian duct, gives to the tubular vesicle the shape of the letter S. The blind expanded end becomes flattened, and in the hollow develops a knot of blood-vessels, which with further growth is enveloped in a double-walled cup. The glomerulus then appears suspended in a rounded cavity by a slender neck, through which its vessels pass. The inner wall of the cup closely invests the capillaries, which are arranged in lobules, and its epitheUiun is modified so as to present thin plates in contact with the endothelimn of the blood-vessels. The outer wall of the cup. Bowman's capsule, remains relatively imdifferentiated as a single layer of flat cells resting on connective tissue. The sUt-like, crescentic cavity of the cup is continuous with the lumen of the tubule, and at the junction the flat epitheUiun of Bowman's capsule changes to the cuboidal epitheUiun of the tubule wall.

SUght variations from this normal type have been noted. MacCallum* foimd by injections that in the WolflSan body of older pig embryos the efferent glomerular arteries, of which there may be many, usually proceed from the side opposite the entry of the afferent vessels;" this indicates either a very broad neck or base from which the glomerulus projects, or the division of the neck by the encroachment of the cavity, as happens in the perforation of the mesocardium. Fused corpuscles and instances where two or more tubules lead from a

Am. Jour. Anat., vol. 1, no. 3, 1902. MESONEPHRIC CORPUSCLES — SHEEP, COW, DEER 3

single cavity have also been described. In the first case the outer wall, Bowman's capsule, of two or more neighboring corpuscles have fused and then degenerated, leaving two or more glomeruli suspended in a single cavity from which two or more tubules arise; in the second case it is probable that after the fusion of the cavities all but one of the glomeruli have disappeared, making the fused cavities appear like a single one. In petromyzon Wheeler*^ has described the actual fusion of several glomeruli, separate at first, into a single large glomus, with many separate afferent and efferent vessels. But in all these cases a distinct glomerulus, projecting into and nearly filling the cavity, is present, and in all there is a distinct Bowman's capsule.

In the sheep the posterior corpuscles of the fully formed Wolffian body correspond in all essential details with those of birds, reptiles, and the previously studied mammals. In the more anterior portions a different type presents itself. If a tubule here is followed from the Wolffian duct toward its blind end it is foimd to open ultimately into a rather wide cavity, with a nearly circular wall in the transverse plane, but with the horizontal walls flat and nearly parallel. The neighboring cavities are in relation to each other by their flat walls, the top of one making the bottom of the other. (Figure 1. In transverse sections the two types do not appear strikingly different.) Frequently the cavities are wedge-shaped in sagittal section, the dorsal and ventral walls being of imequal length. Into this cavity, which represents the crescentic, sUt-like cavity of the usual mesonephric corpuscle, no large single glomerulus protrudes; instead the top and bottom walls are provided with a close network of capillaries, some projecting slightly in little tufts. The endotheUal cells are covered by the epitheUum lining the cavity, here again taking the form of thin plates, as in the true glomeruU. The vessels in these horizontal walls supply the surface of the cavities above and below, so that in sagittal sections each septum shows an irregular border on each side.

» Zool. Jahrb. Abt. Anat., Bd. 13, 1899.

4 JOHN LEWIS BREMER

In the anterior end of the gland the horizontal walls of these separate cavities are wanting in places, and become more and more irregular. A large cavity with many partially closed compartments results, from which open many tubules. As the incomplete horizontal walls retain the same character as those of the separate cavities, with vascular tufts projecting on each side, they might easily be mistaken, in sagittal sections, for long narrow glomeruli; but the lack of any neck and the presence of a connective tissue core reveal the true condition. This large irregular cavity bears the same relation to the closed cavities below as do the fused corpuscles already mentioned to the more typical ones.

It will be seen that in these compartments Bowman's capsule does not exist as such, and can only be represented by those portions of the walls free from the protruding capillaries. In these places, where the lining epithelium rests on the subjacent connective tissue, the epithelial cells are flat, but without the extremely thin plates, and not in direct relation with the few blood-vessels present. The largest areas of this type are on the perpendicular circular walls, where the capillary tufts are fewest. The flat epithelium changes to cuboidal at the opening of the tubule.

The large fused cavity represents the corpuscles of the anterior fifteen to twenty tubules; the separate, individual cavities, opening each into a single tubule, number about eight or ten. The ordinary corpuscles begin then at the 23rd to the 30th tubule, and extend throughout the remainder of the gland. The point where the anterior tjrpe ceases is marked roughly by the groove on the genital ridge between the anterior and middle thirds, i.e., between the part destined to form the rete and that to form the sex gland proper; and it is with the anterior corpuscles that the rete estabUshes relations. Also at this point, near the ventral edge of the organ, there is an especially prominent cross connection of the mesonephric veins. At the line of demarcation there are two or three corpuscles, apparently always present in the same form, which show, by the enlargement of certain tufts almost to the proportions of true glomeruli, a transition from one type to the other.

MESONEPHRIC CORPUSCLES — SHEEP, COW, DEER 5

The difference between the two types of corpuscles Ues m the fact that m the more usual type one side only of the original cavity comes into close relation with the blood-vessels, the other remaining undifferentiated as Bowman's capsule, while in the anterior corpuscles of the sheep all sides, but especially the cranial and caudal sides of the original cavity are equally in relation to the vessels and equally differentiated. The cause of this seems to me to lie in the prococious development of the corpuscles in the sheep, cow, and deer, at a time when the Wolffian ridge is neither long enough nor broad enough to permit their proper expansion. As can be seen in figure 2 the cavity of the corpuscle is quite large and distended while the tubule is of insignificant length and quite straight. This is the stage at which the transverse lines of the T should be extending craniocaudally in the plane of the figure, according to Felix.^ ^'The transverse bar has a cranio-caudal direction. In the angle between the upright and transverse bars the glomerulus appears, sometimes lying on the cranial and sometimes on the caudal side of the upright; the part of the transverse bar with which the glomerulus is associated becomes much more strongly developed than the other part. The extension of the transverse bars in a cranio-caudal direction seems, in these animals, to be rendered impossible by the close crowding of the vesicles. In the earUest formed, more anterior vesicles the attempt at such an extension leads to the fusion and dissolution of the end walls of the transverse bars, and the confluence of the cavities. (Upper part of figure 2.) With slightly more room the cavities do not join but the transverse bars cannot develop, and each vesicle remains symmetrical. (Lower part of figure 2.) In both cases the capillaries, when they appear, may come into equally close relations with the cranial and caudal walls, and because of the restricted length of the transverse bars a single group of capillaries may be in relation with the cranial wall of one vesicle and the caudal wall of that next anterior.

Such an inhibition of the transverse bars does not occur in the lower part of the Wolffian body, where the vesicles are formed

•Loc. cit., p. 803.

6 JOHN LEWIS BREMER

only after the Woffian ridge has expanded; nor does it occur in the kidney where the nephrogenic tissue is widely scattered. The normal process of glomerulus formation, similar to that described by Stoerk and Felix, can therefore take place, as is shown in the caudal end of the Wolffian body of a deer embryo of 6.4 mm. (fig. 3), where the increased growth of one limb of the transverse bar, and the consequent formation of glomerulus and crescentic cavity can easily be seen.

From the standpoint of physiological function this type of corpuscle, without glomerulus, seems capable of greater efficiency than the more common one, because it lacks to a great extent the supposedly inactive surface of Bowman's capsule. The capillary tufts might become larger and closer together until they practically filled the cavity, and offered an enormous secreting surface in the aggregate. As the corpuscles are f oimd, with a rather large central cavity and small capillary tufts, I doubt if the secreting surface nearly equals that of the true glomeruli with their deep lobulation.

PLATE 1

EXPLANATION OP FIGURES

1 Sheep, 14.6 mm. H.E.C., no. 1109, sect. 162. Sagittal section of the Wolffian body, lateral to the genital ridge, from about the 18th to the 32d tubule. The caudal six or seven corpuscles are of the ordinary type, the cranial ones of the special t3rpe. The fused corpuscles are not shown, (r., transitional corpuscles with large capillary tufts; gL, glomerulus; caps.. Bowman's capsule; con. tub,, convoluted tubule leaving corpuscle, others seen above, and again on dorsal (left) side of drawing; ve., large cross connection of Wolffian veins, marking transition zone. X 96 di.jn.

2 Sheep, 5.0 nmi. H.E.C., no. 1896, sect. 63. Sagittal section of the Wolffian body, from the 13th to the 19th tubule. Tubules 13, 14 and 15 have fused, making a large cavity continuous in another section with the anterior tubules. 17 and 18 are S3munetrically expanded, but not fused, and open into the Wolffian duct at the left of the drawing. 19 is in the solid stage, som.f somite. Numbers refer to corpuscles. Note flat epithelium, comparable to that of Bowman's capsule at end of corpuscles. The growth of the tubules later rotates their jimction with the corpuscles to the ventral side (c/. fig. 1). X 200 diam.

3 Deer, Cervus capreolus, 6.4 mm. H.E.C., no. 1519, sect. 46. Sagittal section of lower part of Wolffian body, to show the asymmetrical extension of one side of the transverse bar, and the formation of the ordinary corouscles. GLf glomerulus; caps., Bowman's capsule. X 200 diam.


Truncus arteriosus communis persistens

N. WILLIAM INGALLS

Anatomical Laboratory y Western Reserve University y Cleveland ^ Ohio

ONE FIGURE

The comparatively uncomplicated defect which this case presents, dating from an early period of embryonic development, and the evidence it affords of certain developmental processes, seem to justify its addition to the already almost endless hterature of cardiac malformations.

The development of the heart in question has been normal except for the complete absence of the septum aorto-pulmonale and the anomalies of the ventricular septum and valves of the common arterial ostium which this defect necessarily entails. In addition, as is common in these cases, there is no ductus arteriosus formed.

The organ, which came from a child of 5 months, is very large, weighing ca. 65 gm. The ventricular portion is broad, the apex not very well defined, the bulging of the ventricles hides the root of the tnmcus, especially on the right side.

The atria are normal, the foramen ovale is closed; two left pulmonary veins open by a short common trunk into the left atrium, the condition of the right veins can not be determined. The Thebesian and Eustachian valves are well formed.

In the ventricles, the walls are of approximately equal thickness, ca. 8 mm. The capacity of the left seems rather greater than that of the right, both ventricles communicate freely with the truncus, the left being perhaps the favored one. The mitral valve shows nothing unusual, the anterior and posterior papillary muscles being well developed. The tricuspid valve possesses three well-marked leaflets but the incisures between the cusps fall some distance short of the attached base of the valve. The papillary muscles and their attachments to the

9

10 N. WILLIAM INGALLS

valves may be considered normal. The anterior papillary receives a large, well-defined moderator band. From the septum below and in front of the ventricular defect arise three strong chordae, the papillary muscle of the conus — of Luschka,


Fig. 1 Heart viewed from the left and in front. In the ventricle the medial cusp and posterior papillary muscle of the mitral valve are shown. The truncus and left pulmonary artery are opened up, the origin of the right pulmonary is also visible. Of the valves of the truncus only the right and posterior are well shown; below the former is the ventricular defect and behind this the intact pars membranacea. Through the defect can be seen some of the chordae of the anterior papillary muscle.

although the muscular part is but faintly indicated. These chordae pass to the adjacent ends of the anterior and medial cusps, and one crossing the medial cusp can be traced directly across the pars membranacea. The noduli Albini are very distinct on both mitral and tricuspid valves.

TRUNCUS ARTERIOSUS COMMUNIS PERSISTENS 11

The large truncus communis arises almost equally from both ventricles, its origin, which is distinctly constricted, is hidden in the deep atrioventricular groove, being over-lapped particularly by the conus portion of the right ventricle. Distal to the semilunar valves it suddenly enlarges, especially toward the right, this wall being strongly convex while the left wall is practically straight. From the left side of the truncus, and rather nearer its origin than the concavity of the arch of the aorta, arises the left pulmonary artery. The right pulmonary artery arises at the same level from the dorsal wall of the truncus on left side, close to the left pulmonary. The right artery turns at once sharply to the right to assume its normal position behind the truncus. Both vessels are of equal calibre and much thinner than the truncus or the aorta. There is no trace of the ductus Botalli. No evidence whatever of the distal portion of the septum aorto-pulmonale is present, i.e., there is here a total persistence, as contrasted with cases of partial persistence, of the tnmcus arteriosus. Indeed the only feature indicating even an intended subdivision is the shifting to the left of the right pulmonary — of the right sixth arch. Beyond the pulmonaries the aorta decreases rapidly in size, its arch giving off the three usual branches. The semilunar valves of the truncus are three in number and perfectly formed. The cusps are so arranged that one is posterior the other two anterior, of these last two the left is more lateral than the right. From the corresponding right and left sinuses arise the coronary arteries, while above the posterior cusp is a slightly marked, transversely elongated depression, limited above by a faint ridge (indistinctly seen in the photograph) resembling an occluded vessel, but nothing can be seen externally.

The ventricular defect appears as an elongated slit between the upper border of the muscular septum and the lower surface of the truncal valves. Its lower and anterior limits are formed by the muscular septum and heart wall, behind it is bounded by the anterior concave margin of the pars membranacea. The pars membranacea — not including the septum atrioventriculare, is a thin, translucent membrane, devoid of muscular fibers

12 N. WILLIAM INGALLS

and triangular in outline. Its posterior rounded apex is situated slightly below the center of the posterior truncal valve, its base or anterior margin is free and sharply concave, running out below onto the muscular septum while above it is lost in the interval between the right and posterior semilunar valves, being confluent with, or giving attachment to, the anterior leaflet of the tricuspid valve. The left side of the membranous septum is easily seen, the right forms the mesial boundary of a small pocket which is limited laterally by the medial, and to a slight extent by the anterior leaflet of the right venous valve. It is on this surface that the above mentioned chorda is found. Between the upper attachment of the free border of the membranous septum and the right semilunar valve, and close to the latter, is a small nodule resembling the noduU Albini. The pars membranacea is much larger than usual and, as far as can be ascertained now, is bent over to the right along its attachment to the muscular septum, so that its left surface looks upward and is brought close to, if not in contact with, the right half of the posterior semilunar valve, particularly during closure of the truncal valves.

The conus part of the right ventricle is well developed, forming a deep recess above and between the tricuspid orifice and the ventricular defect. Externally this recess is seen as the prominent bulging on the ventral surface of the heart which covers the root of the truncus. The cardiac wall is here semitranslucent, covered with trabeculae which reach almost to the semilunar valves, and is the thinnest portion of the ventricular wall. An undoubted crista supraventricularis cannot be identified, although there is large trabecular mass, forming the right boundary of the thin-walled conus, running up to the right truncal valve, much like the trabeculae which have a similar relation to the left and more especially to the right cusp of the pulmonary valve in the adult.

Viewed from the right ventricle all of the right semilunar valve can be seen, nearly half of the posterior and a small portion of the left. During distension the valves would apparently quite fill the interventricular opening.

TRUNCUS ARTERIOSUS COMMUNIS PERSISTENS 13

The specimen under consideration offers a concrete confirmation of our views of the development in the region of embryonic auricular canal; views which, although by no means new, still do not seem to be universally current. With a complete failure of development of the septum aorto-pulmonale and the subsequent interventricular communication, there is, nevertheless, a well-formed pars membranacea septi, indeed the interventricular portion of the membranous septum is more extensive than usual. The interventricular opening in this case — a defect in the posterior part of the anterior septum according to Rokitansky — has nothing whatever to do with the pars membranacea, but is due to the non-development of the septum aorto-pulmonale, aggravated it may be by some consequent arrest of development in the muscular septum. The great majority of interventricular foramina, regardless of the condition of the septum aorto-pulmonale, do not implicate the membranous septum for the simple reason that the only relation of the two septa is topographical and they are almost as independent in their development as they are in their origin. The entire pars membranacea septi, atrio ventricular as well as interventricular portion, and the major part if not all of the atrio ventricular valves are derivatives of the endothelial cushions of the primitive auricular canal, and the adult relations reproduce essentially those of the embryo. Hence their comihon histological characters in the adult and the intimate relations between the membranous septum and the tricuspid valve. The fused right ends of the anterior and posterior endothelial cushions give rise to the mesial part of the anterior cusp and to the entire medial cusp of the tricuspid valve, while toward the left these same cushions furnish material for the entire pars membranacea although as a rule most of the interventricular portion may be a derivative of the posterior cushion. Evidence for this is provided by the chordae which pass from the membranous septum to the anterior and medial cusps. Although these cusps are as a rule not entirely distinct, cases can be found where the adjacent tips of the leaflets are in no way connected but nm out on the pars membranacea leaving a distinct interval between them. Granted the origin of the valves from the anterior and posterior cushions, one could, in the cases just cited, determine the relative extent of the membranous septum derived from each cushion. In the heart here described the subdivisions of the valve leaflets are not sufficiently marked to determine accurately their relation to the septum. In view of the foregoing one can understand the rarity of pure, uncompUcated defects of the membranous part of the inter-ventricular septum while not denying (Rokitansky) their occurrence.

The development of this heart has been normal except for the septum aorto-pulmonale, even the enlarged conus portion of the right ventricle being present. The blood would leave the right ventricle iq the direction of the pulmonary arteries but would be hopelessly mingled with that issuing from the left side.

The heart, as shown in the photograph, was obtained through the kindness of Dr. V. C. Rowland of this city and was the only material available for study so that nothing is known as to the presence of anomalies elsewhere.

A double umbilicus

T. B. REEVES Anatomical Laboratory, University of Virginia

THREE FIGURES

After reviewing a considerable literature on anomalies and being imable to find such a case reported, it is deemed worth while to put this specimen on record.

The anomaly here described was found in a colored man, estimated to be about 55 years of age, during our regular class work in anatomy. The surface anatomy of the abdominal wall was normal, except for the presence of a small subcutaneous tumor a short distance above the lunbilicus which was supposed to be a Upoma. On removal of the skin a sharply circumscribed knob of fibrous tissue was found in the subcutaneous tissue 4 cm. above the mnbiUcal fovea and 0.5 cm. to the right of the mid Une (fig. 1). This knob of tissue was 2.5 cm. in diameter and 0.7 cm. thick. It was rather soft in consistency and was definitely surrounded by a thin capsule of connective tissue, except posteriorly where it was continuous with the round ligament of the liver. Superficially it was continuous with the subcutaneous tissue though it separated easily, while its deep surface much more dense merged imperceptibly into the obliterated umbilical vein. The latter entered the abdominal cavity through a foramen in the aponeurotic wall of the abdomen. The foramen had a smooth margin unconnected with the wall of the vein; so that the vein could glide easily backwards and forwards through the foramen. On section the knob mentioned above was somewhat loose in texture and strands of tissue nmning in various directions gave it a lobulated appearance.

On opening the abdomen the urachus and obliterated hypogastric arteries were found extending up on the abdominal wall throwing up their usual folds of peritoneum. The urachus which was apparently single became very small in the region of the umbilicus and seemed to terminate in the lower (true) mnbiUcus. The obliterated hypogastric arteries were of about the normal size and in the mnbiUcal region two or three fibrous cords were given off on each side, which terminated in the lower (true) umbilicus while the remainder passed on to the upper (false) umbihcus (fig. 2).


Fig. 1 F.K., Fibrous Knob; R,S., Rectus Sheath; U.F., Umbilical Fovea; iS., Skin.

The obliterated umbiUcal vein was double, one being attached to the lower umbiUcus while the other was somewhat larger and very firmly attached at the upper umbilicus to the fibrous knob previously described as being outside the rectus sheath in the subcutaneous tissue (fig. 2) . After a very short course, however, the two fused forming the round ligament of the hver which extended upward enclosed in the lower margin of the falciform hgament.

Microscopic examination of the fibrous knob mentioned above revealed it to consist of rather dense connective tissue and a good deal of fat.


Regardless of the apparent weakness of the abdominal wall, it should be noted that there was little possibility of an umbilical hernia. In fact the arrangement was so unique that such could not occur (fig. 3), for the reason that whenever the intraabdominal pressure was increased, the pull of the round ligament on the fibrous knob was in the same proportion, thus ensuring the weak area to be closed at all times.



Fig. 2 F.L., Falciform Ligament; L.T., Ligamentum Teres; U.U,^ Upper Umbilicus; L.t/., Lower Umbilicus; 0./f ., Obliterated Hypogastric; C/., Urachus.

Fig. 3 L,T., Ligamentum Teres; F./^., Fibrous Knob; R.S.^ Rectus Sheath; T.C/., True Umbilicus; S., Skin.


To sum up: There entered the abdomen through the upper navel one umbilical vein and the terminal branches of both hypogastric arteries while the urachus, the other umbilical vein and branches of the hypogastric passed through the lower umbilicus.


When one recalls the changes takmg place on the ventral wall of the embryo in very early embryonic life, it seems reasonable to suppose that such a condition as here described would happen much more frequently than it apparently does. An abnormal growth of tissue across the mid line between the allantois and yolk sac, dividing the single foramen into two, would seem to be the most probable cause of the condition. The anomalous fibrous cords on the posterior wall according to this explanation represent anastomoses between the vitelline and umbiUcal vessels. It is possible that the fibrous knob on the end of the umbiUcal vein represents the remnants of the umbilical vesicle. The fact, however, that its structure is directly continuous with the obUterated vein makes it seem more likely that it was merely a locaUzed hypertrophy on the end of that vessel.


The relation of the chorda dorsalis to the entodermal component of the hypophysis

Wayne J. Atwell from the department of anatomy, university of michigan

Twelve figures

The mode of termination of the anterior end of the notochord has been variously interpreted by authors. The older writers thought the chorda to present a causal relation to the head flexure of the embryo; to act mechanically in drawing out the infundibulum of the pituitary from the brain wall; or to form, after a similar manner, the entodermal diverticulum known as SeesseVs pouch. Later writers, while not necessarily arguing for a causal relationship, have noted the existence of contacts between notochord and hypophysis and between notochord and fore-gut. Recently the r61e of the entoderm in the formation of the hypophysis has occupied the attention of a number of observers. It is the object of this communication to call attention to an observed relation of the chorda to the entodermal component of the hypophysis and to attempt the explanation of certain connections of chorda and hypophysis.

An exhaustive review of the literature covering the development of the hypophysis will not be here attempted. For a yery complete bibliography on this subject the interested reader may be referred to Stendell^s monograph in Oppel's Lehrbuch and to the recent contributions of Bruni and Woerdeman (1914). Neither will the much-perturbed question of the origin of the notochord receive attention. For a theoretical study of this question a recent article by Triepel may be consulted. Only the literature which seems especially pertinent will be referred to.

The Entodermal Component of the Hypophysis, Rathke, whose name is used in designating the epithelial pouch from which the main body of the hypophysis arises, thought the structure to be derived from the anterior end of the fore-gut and thus to be entodermal. It was not until in 1873 that the now commonly accepted view of the ectodermal and there is no reason to doubt that the oral membrane had its upper attachment fairly between the two as they now exist. On the ventral side of the angle of Seessel's pouch appears a soUd epithelial bud. The notochord follows the configuration of the entoderm as far forward as SeesseFs pocket, where it dips down and comes into close relation with the soUd bud of epithelium just described. An actual contact does not appear to exist, the two structures being separated by a space of 5m.

Numerous other embryos, closely staged, show the notochord in a similar close relation to a solid bud of epitheUum, always in the same relative position. Gradually SeesseVs pouch is reduced imtil it is represented only by a slight indentation of the roof of the bucco-pharynx. The reduction of the size of the epitheUal bud is not always proportionate, so that it is often relatively prominent. A drawing of a 15-day rabbit embryo is presented in figiu-e 3. The hypophysis shows connection with the epithelium by a solid cord of cells. SeessePs pouch is f oimd in the same relative position as in preceding embryos but is now much flattened. A well-marked epithelial bud extends from the ventral side of the pouch near its apex. This bud, arising from the epithehum by a broad base, tapers rapidly so that its apex is drawn out into a fine point. This point is directed toward the cephalic termination of the notochord, which makes a marked ventral bend and comes in proximity to the end of the epithelial bud. Between the two is a line of cells more densely arranged than the surroimding mesenchyme. The end of the notochord shows signs of degeneration: the boundary of its extremity is not well marked, large spaces occur in it, and the cytoplasm of most of the cells stains heavily with Congo red. The appearance presented warrants the interpretation that this is the last region of contact between notochord and entoderm.

By degeneration of the end of the chorda and of the epithelial bud, and by a rapid increase of connective tissues to form the skull base, the notochord becomes farther removed from any connection with the entoderm. A rabbit embryo of 16 days is shown in figiu-e 4. The chondrocranium is well indicated at this stage and the solid stalk of the hypophysis has lost its although he admits his lack of material to demonstrate their ^'imiige Beziehmig zu einander.

From the scarcity of stages in the rabbit showing any indication of connection between notochord and hypophysis I am led to believe that a union of these two structures, when it does occur, is accidental rather than of normal occurrence. Figiu-e 1 shows that in early stages the end of the notochord lies close to the dorsal wall of the forming hypophyseal pocket. The presence of the notochord is responsible for an indentation of this dorsal wall showing that considerable pressure occurs between the two. It is not inconceivable, then, that a fusion may occur in a certain number of cases.

On the other hand the constancy of relation between the notochord and an epithelial bud from Seessel's pouch is noteworthy. This bud of epithelium appears constantly a short distance ventral to the top of SeesseVs pouch. It is probably to be considered identical with Selenka's 'Gaumentasche' (solid in higher forms), and St. Remy's 'branche descendante' of the notochord.

RELATIONS OF THE CHORDA IN THE CHICK

My observations on chick embryos, beginning with a stage of 17 somites, are in accord .with the generally accepted view that the notochord loses its attachment to the entoderm first caudally, then gradually forward. A mid-sagittal view of a 19 somite chick is shown in figiu-e 6, and a view of the wax reconstruction made of the hypophysis region of this chick is shown in figure 9, A. The hypophysis anlage is indicated by a region of thick^ ened epitheUum. The connection between notochord and entoderm is lost except at the anterior end of the fore-gut. However, the entoderm shows evidences of this recently-severed connection by a considerable region of thickening anteriorly and by several buds just caudal to the present connection. There is no connection between notochord and hypophysis anlage, nor between the latter and the entoderm of the fore-gut.

An older chick (22 somites, figs. 7 and 9, B) shows the hypophysis as a well marked angle with a broad opening into the oral invagination. The notochord shows an attachment to a drawn out portion of the entoderm at the anterior end of the fore-gut and presents evidences of other connections more caudal. The hypophyseal anlage is not connected with either notochord or entoderm.


Fig. 9 Wax models of hypophysis region of chick embryos. X 75. A, chick of 19 somites; J9, chick of 22 somites; C, chick of 29 somites. The embryos from which these models were made are shown in mid-sagittal section in figures 6, 7 and 8, respectively. A and B ahow cephalic end of notochord attached to bud from anterior end of fore-gut. In C this bud has become fused with superior part of dorsal wall of Rathke's pocket. NCj notochord; 5, Seessel's pouch (anterior end of fore-gut); Rj Rathke's pocket; BW^ brain wall.

An embryo with the oral membrane in process of rupture (29 somites, figs. 8 and 9, C) shows a marked increase in the size of the fore-brain vesicle which has resulted in a compression of the oral invagination and a flattening of the hypophyseal pocket. But the most noteworthy result of this compression has been the bringing together of Rathke's pocket and the epitheUal bud from the entoderm which is apparently further drawn out by its persisting connection with the notochord. The bud, proceeding from the entodermal epithelium at a point sUghtly ventral to the cephalic end of the fore-gut, lies in connection with the ectodermal epithelium of the dorsal wall of Rathke's pocket for a considerable region near its blind end. The connection is a true fusion of entoderm and ectoderm with no demarcation between the two. The notochord makes a bend to maintain its connection with the entodermal bud. It will now be noted that the end of the notochord is nearer to the wall of Rathke's pocket than to the fore-gut (later Seessel's pouch). Here notochord, ectoderm and entoderm are in close relation. However, the notochord does not touch Rathke^s pocket directly but only by means of the epitheUal bud through which it maintains its connection with entoderm. At the anterior end of the fore-gut is another entodermal bud directed toward the notochord. This is to be considered as one of the remains of the earUer, more general union between notochord and entoderm.^

A strikingly similar condition is presented by another embryo of about the same age (latter half of the third day of incubation). The hypophysis region is shown in figure 10. The oral membrane has not broken but is very thin. A bud from the entoderm Ues fused with the superior part of the dorsal wall of Rathke's pocket for some distance. The result of this fusion is to more than double the thickness of the hypophyseal wall in this region. The notochord shows an unquestionable contact with the end of the entodermal bud by a dense line of cells, which is considered to be the anterior end of the notochord in process of disintegration.

Three other embryos, with the oral membrane in various stages of rupture, show distinctly this fusion of entoderm and

Such appearances are not uncommon, some embryos presenting two or even more such structures, which may persist later than the rupture of the oral membrane.


ectoderm with the remams of the anterior end of the notochord in relation to the place of fusion. These five chicks cover quite Veil the latter part of the third day of incubation, yet in Fig. 10 Reconstructed outline drawing, in mid-plane sagittal section, of hypophysis region of chick embryo in latter half of third day of incubation. X 55. Brain floor shaded. NC notochord; F(j, fore-gut (later Seessel's pouch) ; Rj Rathke's pocket; OM, oral membrane. Shows same relations as figure 8.

Fig. 11 Reconstructed outline drawing, in mid-plane sagittal section, of hypophysis region, chick embryo, 96 hours of incubation. X 55. Brain wall shaded. iVC, notochord; 5, remains of SeessePs pouch; R, Rathke's pocket; X, transition from ectoderm to entoderm. Fusion between Seessel's and Rathke's pockets now lost. A line of cells connects cephalic end of notochord to a bud from dorsal wall of Rathke's pocket.

Fig. 12 Reconstructed outline drawing, in mid-plane sagittal section, of hjrpophysis region, chick embryo, 120 hours of incubation. X 55. Brain wall shaded. NC^ notochord; 5, remains of Seessel's pouch; 72, Rathke's pocket; X, transition from ectoderm to entoderm. Large blood vessel shown between Seessel's and Rathke's pockets. Notochord shows traces of an attachment to a bud from dorsal wall of Rathke's pocket. . :


none of them could be found the fine communication between the cavities of Rathke's and Seessel's pouches as described by St. Remy.

In older stages the fused entodermal bud loses its connection with the fore-gut but remains attached to the ectoderm of Rathke's pocket and causes a thickening of a small portion of its dorsal wall. With this thickening the notochord shows a connection which gradually becomes less definite as its cells are drawn out and separated (St. Remy maintains that they are changed into connective tissue). The hypophysis grows most rapidly at its blind end and thus the point of entodermal fusion (with the degenerated end of the notochord in relation) comes to lie relatively nearer to the mouth of the hypophyseal pocket. This has been noted by Woerdeman, who finds (pig) that in young embryos a fusion of notochord and the dorsal wall of Rathke's pocket is found near the apex of the pocket, while in an older stage the insertion of the notochord is into the middle of the dorsal wall.

A chick of 96 hours incubation (fig. 11) shows Seessel's and Rathke's pockets in commimication in the mid-plane. On the dorsal wall are found two epithelial thickenings. From the more cephalic of the two a line of cells leads to the end of the notochord. This would seem to indicate that this thickening is the location of the former ento-ectodermal fusion. The thickening is bud-like and may be what Bruni calls the 'diverticolo medio.' It is worthy of note that while the outermost part of this thickening is entodermal (remains of the bud that fused with the hypophyseal wall), yet none of this entoderm need be supposed to Ue next to the lumen of the hypophyseal sac. At this stage it is rather diflScult, from mid-sagittal sections, to determine the boundary between Rathke's and SeesseFs pouches. An examination of more lateral sections indicates that this is at the point marked X (fig. 11). The epithelial thickening caudal to this point is the tip of Seessel's pocket. Bruni calls this the

' gemma della tasca di Seessel." A chick of 120 hours incubation (fig. 12) shows much the same relations but SeessePs pouch has again assiraied a more definite demarcation from Rathke's pocket. This is due to growth of mesoderm dorsal to Rathke's pocket and especially to the development of a prominent blood vessel growing between the two. This vessel has been spoken of by several authors as a connecting branch between the int^pial carotids, common in bird embryos. The notochord still shows indications of a connection with a bud (which sometimes shows a lumen) from the inferior part of the dorsal wall of Rathke's pocket. This must be Brum's 'diverticolo medio.' Small blood vessels occur in this location. If the interpretation of the point X as the transition between ectoderm and entoderm is correct, then the hypophysis proper contains no entodermal component except the relatively few calls fused with the dorsal wall at about the time of the rupture of the oral membrane. It is hoped that the fate of SeesseFs pouch may serve as the subject of a future study.

COMPARISON OF RELATIONS IN RABBIT AND CHICK

In comparing conditions found in the rabbit with those in the chick it is seen that in both the anterior end of the notochord tends to maintain a connection with the fore-gut, as St. Remy states. This connection is aided by a bud drawn out from the epithelimn. In the rabbit the bud remains for some time, gradually decreasing as the epitheUum of Seessel's pouch flattens out. In the chick the rapid growth of the fore-brain and the sharpness of the cervical flexure bring this bud into contact with the growing hypophyseal wall and the two become fused. Soon afterwards the bud loses its original connection with the entoderm but remains fused with the wall of Rathke's pocket. Thus a small mass of entodermal cells becomes miited with hypophysis anlage. The notochord shows indications of a connection with this mass of entodermal cells which now form a bud on the dorsal wall of Rathke's pocket. By this means the attachment of the notochord id transferred from Seessel's pouch to Ratlike's pocket.

In the rabbit, on the other hand, the notochord does not usually come into connection with Rathke's pocket. A fusion may occur in a small percentage of cases but must be considered accidental. When such a union occurs a bud of entodennal epithelium may be directed toward the point of union.

These observations may be offered to explain certain connections between notochord and hypophysis as seen by KoeUiker (in the rabbit) and Woerdeman (in the pig). The latter has in two cases, seen a more definite fusion than can be demonstrated in the two rabbit embryos which show evidences of such connection used in this study. These observations on the rabbit and particularly on the chick, may also serve to explain certain entodermal components of the hypophysis as noted by Kupffer (amphibia and mammals), Valenti (amphibia and birds), St. Remy (chick), and Nusbaimi (dog). The findings here recorded, it seems to me, do not substantiate the view, taken by most of these writers, that the entodermal contribution to the hypophysis is of fimdamental importance, especially phylogenetically; nor do they aid in the belief that the hypophysis represents a paleostome.

While the fate of SeessePs pouch and the participation of its epithelium in the composition of the hypophysis may be doubtful in the chick, it cannot be stated that this structure takes part in the formation of the hypophysis of the rabbit. As may be seen from the figures (1-5) Seessel's pouch isat all times sharply defined from Rathke's. This is especially well brought out in numerous wax reconstructions made from embryos closely staged before, during and after the rupture of the oral membrane.

For the rabbit it has not been possible to demonstrate a structure similar to the 'diverticolo medio' which Bruni describes for the rat.

CONCLUSIONS

For the rabbit:

1. In the rabbit the anterior end of the notochord tends to maintain its connection with the entoderm represented by a bud from SeesseUs pouch.

2. The entoderm cannot be said to contribute to the formation of the hypophysis of the rabbit.

3. Usually the notochord does not come into connection with the wall of the hypophysis in the rabbit.

RELATION OF CHORDA DORSALIS TO HYPOPHYSIS 37

For the chick:

4^ In early stages of chick embryos the anterior end of the notochord is attached to a solid bud of epithelium which extends from a point slightly ventral to the cephaUc end of the fore-gut.

5. By the growth of the forebrain and the sharpness of the cervical flexure, this entodermal bud comes into contact with the growing hypophyseal sac and fuses with it. The fusion occurs at about the time the oral membrane ruptures.

6. The bud is soUd; a lumen such as is described by St. Remy could not be demonstrated.

7. The fused bud soon loses its connection with the entoderm. It remains fused with the dorsal wall of Rathke's pocket, however, and contributes a small mass of cells to the hypophysis anlage.

8. The notochord still shows indications of an attachment to the fused bud. Thus the attachment of the notochord (which is gradually becoming less and less a definite attachment) in transferred from Seessel's pouch to Rathke's pocket.

9. The relation of the notochord to this small entodermal increment of the hypophysis, and the fact that no lumen is to be found, tend to disprove the view that the entodermal contribution is anything more than an accidental union of parts.

LITERATURE CITED

Balfour, F. M. 1874 A Preliminary Account of the Development of the Elasmobranch Fishes. Quar. Jour. Micros. Sc. vol. 14, N. S., p. 324.

Bawden, H. H. 1893 Selenka's 'Pharyngeal Sac' in the Duck. Jour. Comp. Neur., vol. 3, p. 46.

Bruni, Anoelo Cesare 1914 SuUo sviluppo del lobo ghiandolare delF ipofisi negli Amnio ti. Intemat. Monatschr. f. Anat. u. Physiol., Bd. 31, Heft 4/6.

Dean, B. 1896 On the larval development of Amia calva. Zool. Jahrb., Bd. 9.

DuRSY, E. 1869 Zur Entwicklungsgeschichte des Kopfes. Tabingen.

Gage, Susanna Phelps 1906 The Notochord in Human Embryos of the Third to the Twelfth»Week, and Comparisons with Other Vertebrates. Science, N, S. vol. 24, p. 295.

Goette, Alexander 1873 Kurze Mittheilungen aus der Entwickelungsgeschichte der Unke. Archiv. f . Mikr. Anat., Bd. 9, p. 396.

Gregory, E. H. 1902 Beitrage zur Entwickelungsgeschichte der Knochenfische. Anat. Hefte, Bd. 20.

His, W. 1868 Untersuchungen liber die erste Anlage des Wirbelthierleibs. Leipzig.

38 WAYNE J. ATWELL

HuBER, G. Carl 1912 On the Relation of the Chorda Dorsalis to the Anlage

of the Pharyngeal Bursa or the Median Fharsmgeal Recess. Anat.

Rec, vol. 6. §

Kkibel, Franz 1889 Zur Entwickelungsgeschichte der Chorda bei Saugem.

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Ende der Chorda dorsalis bei Kaninchen embryonen^ Festschr. s.

Feier d. hundertj&hr. Bestehens d. naturforsch. Gesellsch. in Halle. V. KuPFFER, C. 1892 Entwicklungsgeschichte des Kopfes. Ergebnisse d.

Anat. u. Entw., Bd. 2.

1894 Die Deutung des Hirnanhanges. Sitz. Ber. d. Geselloh. f.

Morph. u. Physiol, zu Manchen, S. 69. Lille, F. R. 1908 The Development of the Chick, p. 80. Henry Holt and

Company. V. MiHALKOvics, Victor 1874 Wirbelsaite und Himanhang. Archiv. f . Mikr.

Anat., Bd. 11. Mt^LLER, W. 1868 Uber Entwicklung und Bau der H3rpophysi8 und des Processus infundibuli. Jenaische Zeitschr. f . Med. u. Naturwiss., Bd. 4,

S.667. NusBAUM, J. 1896 Einige neue Thatsachen zur Entwickelungsgeschichte der

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of Amia Calva. Biol. Bull., vol. 1, no. 2. Rathke, H. 1838 tJber die Entwicklung der Glandula pituitaria. M(Uler's

Arch, f . Anat. Phys. u. wiss. Med. Reichert, K. B. 1840 Das Entwickelungsleben im Wirbelthierreich. Berlin. Reighard, J. 1900 The Development of the Adhesive Organ and Hypophysis

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Bd. 50, Heft 3. Valenti, G. 1895 Sullo svillupo delP ipofisi. Anat. Anz., Bd. 10.

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Woerdeman, Martin W. 1913 Uber einer Zuzammenhang der Chorda dorsalis mit der Hypophysenanlage. Anat. Anz., Bd. 43. 1914 Vergleichenden Ontogenie der Hypophysis. Arch. f. Mikr. Anat., Bd. 86.

On The Conversion Of A Photograph Into A Line Drawing

Wayne J. Atwell From the Department of Anatomy University of Michigan

TWO FIGUBES

The making of suitable drawings for reproduction has always been a problem, especiaUy in laboratories which do not command the services of a competent artist. The modem recourse to a use of photography and photo-micrography with subsequent reproduction in halftone, while greatly reducing the labor of preparation, has not produced pictures uniformly satisfactory in simplicity and definiteness. Wax models do not reacfily lend themselves to photographic methods. Photographs of anatomical dissections do not prove sufficiently clear for easy interpretation after having been subjected to the necessary reduction.

The process of Une drawing on the other hand leaves nothing to be desired in clearness, since there needs to be depicted only what seems to be essential. Still the execution of suitable drawings from such subjects as those above mentioned requires considerable artistic ability and no Uttle valuable time. As is well known the cost of reproducii^ photographs and drawings in half-tone is much greater than that of reproducing line drawings in black and white.

With these considerations in mind it has seemed that a means for more generaUy utiUzing the Une method would be welcome, if, at the same time, it might be possible to ehminate some of its demands on time and artistic skill.

At the suggestion of Dr. Ruber a method which has been in use for many years by commercial artists and engravers, and known as the 'silver print', has been adapted to the use of scientific illustrators.

In brief the method is: 1) To produce a passable negative of the model, dissection or other object to be pictured. 2) Print from this negative an enlargement of suitable size on bromide paper.^ 3) Trace outUne of print with waterproof ink and fill in shaded portions by stippUng, or by a few fines judiciously placed. 4) Bleach out aU traces of photograph.

In making the initial negative only the ordinary precautions of careful fighting, proper orientation, etc., are necessary. The concealing of supporting frames and wires is not required, nor will these need to be removed later by opaquing the background of the negative. A large negative is not necessary,4 X 5 or 5 X 7 plates sufficing for most subjects, while for some even 3} X 4| plates will be found large enough.

If the negative is large enough contact prints on cheaper papers, even blue prints, may be used instead of enlargements.


If the laboratory possesses a 'copjdng, enlarging and reducing' camera the printing of all sizes of enlargements up to and including 8 X 10 inches will be an easy matter. The camera is arranged as ordinarily used in making enlargements or reductions. The spring which usually holds the plate firm in the plate-holder should be removed. The bromide paper is then inserted and supported at the back by one or more sheets of stiff cardboard. If the bromide paper is of fair weight no difficulty will be experienced in keeping it flat and during exposure it may be handled precisely as a dry plate. Of course it will be necessary to select a bromide paper with a smooth surface so that it will later take the pen readily.^

When the print has been allowed to dry thoroughly it may be inked in, using of course waterproof India ink. The coarseness of the Unes must be proportionate to the amount of reduction which the drawing is later to undergo. At all events the lines and dots must not be too close together. It is best not to try to reproduce all the detail of the photograph.

After allowing the ink to dry, the print is placed first in a tray of water for a few minutes and then transferred to a tray containing the following two solutions in the proportion of eight parts of No. 1 to one part of No. 2.

1. Hypo (thiosulphate of sodium), 30 g.; water, 480 cc.

2. Potassium ferricyanide, 30 g.; water, 480 cc.

The print should remain in this bath until the last traces of the photograph proper have disappeared, which may require from 20 to 30 minutes. It is best to do this in a dark room. Then after washing for 15 or 20 minutes in running water, the drawing may be dried flat or mounted on card board. Some care is necessary in handUng the drawing while wet so as not to rub the surface for the ink is then soft enough to smear.

TWs method results in the production of a clean drawing, accurate in outline, and with the shade correctly placed so that perspective should be properly brought out.

The accompanying figures show a model of the hypophysis region of a chick embryo reproduced by both half-tone and line etchings, the latter prepared by the method here described.

• This laboratory has successfully used the 'P.M.C bromide paper in 'Smooth' and 'Glossy' surfaces (Nos. 2 and 4) made by the Eastman Kodak Company.

A CORRECTION— R. W. SHUFELDT

At the time I was preparing my article on the "Comparative osteology of certain rails and cranes, and the systematic positions of the super-suborders Gruiformes and Ralliformes/' which appeared in the October issue of The Anatomical Record (Vol. 9, No. 10, pp. 731750, 1915), I had before me two manuscripts, namely the old one, published many years ago, when I considered that the Aramidae was a family belonging among the cranes and their allies (Gruiformes), and the remodeled one, in which my present views were set forth. In assembling the pages, the old page upon which the classification and some of the remarks under 'conclusions' appeared, was accidentally substituted for the new one carrying the new classificatory scheme upon it. In this shape it was handed over to be typewritten. When galley proof came to hand, I was extremely busy with other work, and it was therefore turned over to an expert proofreader and most carefully corrected. This proofreader knew nothing of the classification of birds, however, and so the galleys went forward, with the result now to be found on pages 749, 750. In so far as my present views are concerned, with respect to the position of the Aramidae in the system, they are correctly set forth in The Anatomical Record of August 20, 1915 (Vol. 9, No. 8, pp. 591-606).


Preservation Of Anatomic Dissections With Permanent Color Of Muscles, Vessels And Organs By Newer Methods

Edmond Souchon, M.D. Professor Emeritus of Anatomy ^ Tulane University of Louisiana

The newer methods for the preservation of anatomic dissections are to be commended for retaining the color in the preparation and for the uniformity of results. They are classified as the Chemical Method and the Paint Method.

The subject must be embalmed with the following solution:

A — Arsenious Acid (Saturated Solution) 2i gallons

Potassium Nitrate pulverized 1 pound

Formol (must be white and clear, not greenish) 6 ounces

B — ^Alcohol 16 oimces

Carbolic Acid, liquified (soluble) 6 ounces

Glycerine 16 ounces

Creosote (Beechwood) 2 ounces

Mix A and B; strain through a towel.

One week after the embalming the vessels are injected with hot tallow, if desired. I use English vermilion deep shade for the arteries and ultramarine blue for the veins.

The further procedures vary according to the nature of the dissection, whether emphasis is to be given to muscles, viscera, or white tissues.

CHEMICAL METHOD Preservation of color of musdes

It is not so very important to preserve the actual color of muscles, which varies from one subject to another, but it is essential that there should exist permanently a marked contrast between the fleshy parts of the muscles and the tendons, fascia, bones and other white tissues, which must remain white. A dark brown color of the fleshy parts of the muscles is satisfactory. The more red the brown is the better and prettier.

The selection of the subject is of great importance. None but lean subjects should be selected. Tlie least fat makes an unsatisfactory preparation. Slim subjects with thin muscles do not make good preparations. Before embalming the subject, a cut three inches long and one inch deep must be made in the deltoid or the external vast muscle, and the color of the muscles examined critically. If the muscles are of a very dnrk color they will make good preparations, but if the muscles are of a light pale color, they will seldom turn out satisfactorily. The method preserves the color presented by the muscles of the subject but seldom gives better color when the color is originally deficient. Negroes usually present darker muscles; also laborers. Subjects suitable for the work are not very common. Only about 8 or 10 per cent of the subjects which reach the Anatomical Department of Tulane are proper subjects. Each subject will yield about eight preparations of muscles and organs with satisfactory color.

The day after the injection with tallow the subject is cut up into the parts which are to be dissected. After which each part is placed in a large glass jar containing one per cent of soluble liquefied carbolic acid (C 1).^ The solution must be changed as soon and as often as it becomes cloudy.

Muscles to be dissected, if not covered by skin, such as the muscles of the interior of the abdomen and the sternal region, should not go into C 1, since this solution destroys the color. They should be put up to drain at once, and dissected as soon as sufficiently drained, about 8 or 10 days.

When the solution remains clear or nearly so, the parts are transferred into an empty jar to drain and cure; hands, feet and knees up. The upper extremities must be fastened to a thin paraffined board to prevent their distortion. This stage should extend for about 4 or 6 weeks, during which time the color or contrast develops in the muscles. It is an important stage since the color depends much on it. The parts must be examined every week and if they show signs of hardening thev must be placed in a 1 per cent solution of carbolic acid (C 1 or in F 1).

The parts are then dissected on a dissecting stand in the position

EXPLANATION OF ABBREVIATIONS

'C r — 1 per cent of carbolic acid in filtered water.

G 60 C r — 60 per cent of glycerine with 33 per cent of water and 1 per cent of carbolic acid.

CH. F 75' — a solution of chlorides with 75 per cent of formol and 25 per cent of filtered water.

'CH. F 20'— a solution of chlorides with 20 per cent of formol and 80 per cent of filtered water.

'CH. F. 5' — a solution of chlorides with 5 per cent of formol and 95 per cent of filtered water.

'G 30 C r — 30 per cent of glycerine. 69 per' cent of water and 1 per cent of carbolic acid.

'A 20' — 20 per cent of alcohol in 80 per cent of filtered water.

'A 20 F 5' — 20 per cent of alcohol, 75 per cent of filtered water and 5 per cent formol.


they are to occupy in the Museum. Not more than one layer should be shown in one cUssection. The structures should not be raised from their beds.

The next step is to increase the color, to reinforce it because when later the preparation is put in the permanent solution the color will pale somewhat. Glycerine helps to fix the color, to increase it.

After the completion of the dissection it is placed for 24 hours in the following solution (G 60 C 1), each gallon of which is to contain:

Glycerine 66 ounces

Carbolic Acid, liquified 1 ounce

Filtered Water 33 ounces

This is about 60 per cent of glycerine. It registers 20 degrees with Baume Syrup Hydrometer. In this solution the muscle as also the tendons may assume a red color, but sometimes they become more or less black and shriveled, the preparations appearing as if they were ruined.

At the end of 24 hours, the preparation is placed in an empty jar with a lid. At the end of 30 days, when the glycerine has ceased to drip the preparation is taken out of the jar. The jar is washed and dried and there is placed in the bottom one-half pound of calcium chloride anhydrous (C. C. Mercks). The preparation^ is returned to the jar and the jar is closed with a lid, a rubber and a clamp. Some muscles darken very quickly, in a few days. They have to be examined daily. The tendons and muscles gradually darken and become as black as coal. The preparation again looks as if \t had been ruined but later on everything rights itself.

When the preparation is thoroughly black, it is placed in filtered water with 75 per cent of formol (Ch. F. 75). The preparation remains in this solution for 24 hours, and is then placed permanently in a solution of chlorides of sodium, calcium and potassium, the proportions of which are given on another page. Twenty ounces of formol, clear and white, are added to each gallon of solution to prevent possible bacterial cloudiness. This is the Chemical or Chlorides-Formol Method (Ch. F. 20). It costs about 22 cents per gallon of solution. When immersed in the solution the muscles pale somewhat and that is why the muscles must be so dark before they are immersed.

Dissections presenting muscles with a light bi*own or pale color are unfavorable ones. They will seldom turn out satisfactorily. It is usually time, labor and material lost. This is an important point and should be well borne in mind to avoid disappointments. It is better to paint such preparations.

Preservation of the color of the viscera

Viscera, such as the lungs, heart, liver, spleen, kidneys, should as soon as dissected be placed directly in G 60 with one ounce of liquefied carbolic acid soluble (G 60 C 1). After 15 days in G 60 C 1, they must be transferred into Ch. F. 5.

The membranous viscera, mouth, nose, pharynx, oesophagus, stomach, intestines should be put directly in G 30 with one ounce of liquefied carbolic acid (G 30 C 1). After 15 days in G 30 CI they must be transferred into Ch. F. 5.

Preservation of color of the white tissues A 20 F. 5.

This method is suited only for dissections of white tissues: fresh bones (periosteum and marrow), articulations without muscular attachments, brain and membranes, spinal cord, pelvic organs in situ, larynx (cartilages and articulations), trachea, bronchi with or without lungs, eye-balls and sections, aponeuroses of the extremities and neck; dartoic tissues of penis, scrotum and pubis, testicles.

A gallon of solution contains:

Alcohol 20 ounces

Formol 5 ounces

Filtered Wafer 103 ounces

This solution contains about 15 per cent of alcohol and 4 per cent of formol. It is cheap, about 12 cents per gallon of solution. It is here designated as A 20 F 5.

The formol prevents the ivory-like bleaching action of the Alcohol, and the alcohol prevents the possible snow-white bleaching of the formol.

Special remarks

The proportions of the embalming solution are the result of many experiments. I have tried to increase the potassium, the formol and the carbolic acid, separately or conjointly, but with no satisfactory result.

Phenol is not exactly the same as soluble liquefied carbolic acid and should not be substituted for the latter.

The carbolic acid,, formol and creosote are added to the arsenic to assist in the preservation, which the arsenic alone may not do thoroughly in warm weather.

The tallow as used for injection should be heated in a water bath to 210'^F.

The connection of the syringe with the aorta or artery must be by tight double ligatures of white cord, not of a stiff material. Rubber tube connections must be wrapped with tape, to prevent bursting when injecting. The syringes must be previously tested with tepid water to make sure that the leather packing does not leak. When the piston meets with a firm resistance no more pressure should be applied, lest rupture of vessels will take place and extravasation follow in the • tissues.

Hot tallow injections of arteries is not always satisfactory as it does not always reach the small branches, such as the digitals and the labials. I have warmed the bodies in a tank up to 100® and above, but with not very much better results. I believe that the embalming fluid filling the smaller arteries prevents the tallow from reaching them. I have had no better results with other materials, gelatine, paint, etc.

When the subject is cut up the muscles present all sorts of color, raw, pale brown, dark brown, etc. But in the course of the next stages the muscles assume a more or less uniform color. However, in some subjects the muscles are irregularly colored. Those of a certain region may present a good dark brown color and those of another region a pale brown color. Sometimes the upper part of a muscle is satisfactory and the lower part of it is not as good. It is only when the parts are dissected that it can be positively ascertained what the color of the muscles is. Unless the muscles in the dissection present a very dark brown color, it is not likely that the ultimate color will be satisfactory.

When a number of preparations have been placed in the glycerine, this should be tested with the Baume Syrup Hydrometer. If it does not register B 20, glycerine should be added to bring it up to B 20 or it shoiJd be boiled down to B 20. It must be tested with the hydrometer when cold or about GO^'F. It is a slow process, but quite a saving of glycerine. It is important to keep the glycerine at B 20.

When a number of preparations have been placed in tha glycerine, and it has become very dark in color, in spite of filtering, it should be changed for a new solution.

The object of the immersion in glycerine is to lessen the blackening action of the calcium chloride, to which the preparation is exposed later on. If a preparation is exposed to the action of the calcium directly after it is dissected it will become so deeply black that it will retfiain so regardless of any solutions it may be put in.

When glycerine or other solutions mildew on the top or remain cloudy in spite of repeated filtering, there should be added to the solution ten ounces of alcohol to each gallon of solution.

The calcium is recovered by evaporation and when dry crushing it into small pieces. When the calcium Uquefies, new hard calcium must be put in after drying the jar.

Solutionis must be clarified or filtered as soon and as often as they become slightly cloudy or discolored. Keeping solutions clear all the time is the foundation of the preservation of the preparation. If allowed to stay in a cloudy solution the cloudiness will spoil the color of the colored tissues. If allowed to remain in a discolored solution, the white tissues in the preparation will become discolored. A five gallon percolator filter can be used for filtration.

In packing the percolator filter, a layer of three inches of clean white cotton is placed at the bottom, then a layer of four inches of first qual 48 EDMOND SOUCHON

ity filter paper reduced into a pulp and pressed down; then a layer of five inches of granular bone black (3 pounds or half a gallon), the size of a millet seed. Before using the filter it should be thoroughly washed by pouring filtered water through it until the water comes out clear. Do the same each time the filter has been used, before putting in more solution to be filtered. The filtering must not be faster than fast falling drops.

When the filters are used for a different solution, they must be packed fresh, especially when solutions of G 60 and G 30 are filtered.

The bone black removes the color in the solution, but not the cloudiness. The filter paper removes the cloudiness but not the color.

Some of the solutions will remain hazy in spite of repeated filtering. They are clarified by putting in them five or ten grains of aluminium sulphate to the gallon of water. Stir well and filter. Repeat the aluminium, if necessary. Some solutions will not come out clear in spite of repeated filtering and the use of aluminium sulphate. A new solution should be fixed up to take the place. When the JHushing water ceases to come out clear from the percolator, the percolator should be repacked.

The vessels should be painted before the preparation has been in calcium or in a solution. The vessels are wiped with gauze and then painted and the preparation is put in the solution. The same procedure applies to the affixing of the numerals to the structures. I use artists* paints, Chinese VermiUon for the arteries and dark blue for the veins. Water colors must be allowed to dry about 8 hours before the preparation is placed back into an aqueous solution, lest they will dissolve in it. In course of time the painfted vessels may show yellowish spots or patches. They should be wiped off or scraped and the vessels repainted.

If preparations in Ch. F 20 darken too much in course of time, transfer them into A 20 F 5.

Do not use F 5 with G 60 or G 30.

There are now about 125 satisfactory preparations of muscles and organs made after these methods in the Souchon Museum at Tulane.

Some preparations do not turn out as good as they should and should be made over. Sometimes the same preparation has to be made over two or three times before a satisfactory one is reached. This happens almost solely in cases of dissections presenting a medium brown color. Dissections presenting a dark brown color seldom have to be made over.

The angle at which the Ught strikes the preparation has much effect in bringing out the color.

The numerals affixed to the structures are printed on ordinary paper with ordinary printer's ink, of the desired size, cut into circles or squares and stuck in places with tiny pins.

(The sizes of the jars needed for the work are Museum Jars (Whitall Tatum, Phila.) 24 X llj; 24 X 7^; 36 X 7i)

The Chlorides Formol Solution is composed as follows:

PRESERVATION OF ANATOMIC DISSECTIONS 49

For a Jar containing One Gallon of Filtered Water:

Sodium Chloride (Commercial. Pure Table Salt) 3 oimces

Calcium Chloride (Anhydrous, Merck's) 15 grains

Potassium Chloride 1 drachm

Sodium Bicarbonate 45 grains

Formol 20 ounces

Stir and filter. The formol is to prevent the possible formation of bacterial cloudiness and also to preserve color.

It may be that 5 ounces of formol to the gallon will do as well as 20 and cheapen the solution. Experiments must determine that.

The chlorides fix the hemoglobin.

I am indebted to Professor Mann of Tulane for valuable information on chemical points.

THE OIL PAINT METHOD

The great advantage of the paint method is to utiUze a number of preparations which upon the completion of the dissection do not present a satisfactory color and, therefore, are not suitable for the other method and would have to be thrown away. Dark brown or medium brown muscles are not as favorable for the paint method as muscles with a pale color.

Improved technic has yielded better results than on previous occasions. The oil paint method is a simpler and quicker method than the chemical method.

The subject must also be embalmed as described above.

After the completion of the dissection, the preparation is placed in a one per cent solution of Formol (F 1) for 2 or 3 days. It is then placed in an empty jar with a lid until all the water drips from it, about ten days.

The preparation is then mopped thoroughly dry with cheese-cloth and is painted first with gelatine, the next day with oil paint. When the gelatine is dry, in about 3 hours, another coat is given.

The gelatine solution consists of one leaf of white French gelatine to eight ounces of watfer dissolved by boiling. The gelatine is applied with a soft brush to the fleshy parts of the muscles only. The object of the two coatings of gelatine is to secure a smooth surface on which the paint will more easily and uniformly spread. It fills up the small cracks and holes in which the paint may gather and form streaks and spots. When the gelatine is dry, the muscles are painted.

The paint used for the muscles is French Carmine (Devoe), other carmines are apt to harden in the tube; for the arteries, Chinese Vermilion; for the veins, dark blue; for the portal system of veins, dark green; for the biliary ducts, chrome yellow. The mucous membranes (nose, mouth, pharynx, oesophagus, rectum, vagina, uterus) are painted pink (rose dore); the surfaces must be previously gelatined. The oil paints used are the artists' oil paints of Winsor and Newton, of London, or Devoe of New York, purchasable in artists' materials stores. The brushes used are flat sable hair brushes one-quarter of an inch wide, Devoe No. 8 and one-eighth of an inch wide, Devoe No. 4.

The paint is prepared by squeezing out of the tube one-third inch of French Carmine. Use the brush to prevent curling and to straighten out the Uttle column of paint so as to better measure its length. Dissolve in a teaspoonful of rectified turpentine (Devoe). It takes some patience to secure the right color, dark raw beef meat.

Two thin coats or more are better than one single thick coat. • Too thick paint makes unsatisfactory preparations. Too much paint cannot be wiped off after drying and the preparation is ruined. Sometimes one thin coat is enough if the muscles show a dark color.

When nearing tendons and fascias use a small brush with very little paint on the brush and no paint must reach the tendons and fascias.

The preparation must be painted a little darker than it is expected to look in the permanent solution because when immersed in the permanent solution the color becomes lighter. If the whole of the color or parts of it seems too light, the preparation is taken out of the solution and exposed to the air and mopped with cheese-cloth until dry enough for the paint to stick. Then another thin layer of paint is applied to those parts where it is too light. This may have to be done once or twice before the right tint is obtained. Should the carmine show too brightly add one-eighth of an inch of Vandyke Brown to tone it down. After the last coat of paint is applied the preparation must remain exposed to the air to dry, about 6 hours. The paint being an oil paint and being placed later in an aqueous solution need not be thoroughly dry before being immersed in the permanent solution. During the drying of the paint the tendons and the bones may assume a brown color from drying, but later on, in the permanent solution, they resume their white color.

The painted preparation must not be left exposed to the air overnight, lest it will dry too much. It should be placed for the night in an empty jar with a lid and taken out next morning to finish drying.

Before placing the preparation in the permanent solution, the Unes between the various muscles must be marked with a knife to obtain definition.

When the paint is about dry, the preparation is place permanently in a solution composed of ten per cent of alcohol, and eighty-five per cent of filtered water (A 10).

Solutions must be changed or clarified as soon as they become the least cloudy or discolored.

Other good oil paints are house paints ready for use. Stir well before using. For muscles, use Tuscan Red No. 588 of Devoe Co. of New York one teaspoonful, with Maroon of True Tag Co. of Memphis one-half teaspoonfid. Add three teaspoonfuls of rectified turpentine. These proportions are important. Two or three thin coats are better than one thick coat. Expose to the air to dry about six or eight hours before placing in the permanent solution (A 10). They are hardier paints than the artists' Paints. They should be used to repaint artists' paints which unaccountably faded shortly or did not turn out good. I rather prefer them to the others.

When in the solution if the color* shows too bright red, take out the specimen, wipe it, let dry and apply one coat of maroon color made by True Tag Paint Co. of Memphis, Tenn. Add two-thirds turpentine. Repeat the procedure if the color still shows too bright red when replaced in the solution.

THE WATER PAINT METHOD

In using water paints the muscles must also be previously painted with two coats of gelatine.

When the gelatine is dry then the muscles are painted with a first thin coat of water French Carmine as explained for the oil paint. Water Carmine does not come in a tube but in a little cake. This cake should be sawed into little cubes of about one-quarter of an inch, which is the quantity for a teaspoonful of water. The water paint must not be thick. Two thin coats or more are better than a single thick coat.

When the first coat is dry about 8 hours, the second coat of water paint is applied, if necessary. This must alsp be a very thin coat.

After this second coat the specimen is left exposed to the air until thoroughly dry. If placed in the final permanent aqueous solution, before the paint is dry, the water paint will dissolve. The long time required to dry thoroughly is to the disadvantage of the water paint.

During this drying of the paint, the tendons and bones may become more or less brown from drying, but when placed in the permanent solution they resume their white appearance.

When the paint is diy the preparation is placed permanently in a solution composed of 10 per cent of alcohol, five per cent of formol and eighty-five per cent of filtered water (A 10, F 5).

The solution must be changed or clarified as soon as it becomes slightly cloudy or discolored.

CONCLUSIONS

SuflBcient time has not elapsed to state the final result, but my impression is that oil paint preparations kept in *A 10, will ultimately yield better preparations than when other methods are used.

The appearance of painted preparations in the permanent solutions is satisfactory for educational purposes and for esthetic efifect. The artistic skill, judgment and patience of the painter will tell on the final result.

When in course of time the paint shows the least sign of fading or of deterioration, the preparation must be removed from the solution, the old paint wiped off with cheese cloth and the muscles painted again, two thin coats or more. No gelatine is to be applied.

BOOKS RECEIVED

The receipt of publications that may be sort to tay of the five biological Journals published by The Wistar Institute will be acknowle 'cad under this heading. Short reviews of books that are of special interest to a large number of biolocwts will be published in this Journal from time to time.

AN INTRODUCTION TO NEUROLOGY. C. Judson Herrick, Ph.D., Professor of Neurology in the University of Chicago, 335 pages, 137 illustrations. $1.75. 1915. Philadelphia and London: W. B. Saunders Company.

Extracts from Preface. There are two groups of functions performed by the nervous system which are of general interest; these are, first, the physiological adjustment of the body as a whole to its environment and the correlation of the activities of its organs among themselves, and, in the second place, the so-called higher functions of the cerebral cortex related to the conscious life. The second of these groups of functions cannot be studied apart from the first, for the entire conscious experience depends for its materials upon the content of sense, that is, upon the sensory data received by the lower brain centers and transmitted through them to the cerebral cortex. Since the organization of these lower centers is extremely complex, and since even the simplest nervous processes involve the interaction and cooperation of several of these mechanisms, it follows that an understanding of the workings of any part of the nervous system requires the mastery of a large amount of rather intricate anatomical detail. * * *

The problems which at present chiefly occupy the attention of neurologists are of two sorts — first, to discover the regional localization within the nervous system of the nerve-cells and^bers which serve particular types of function or, briefly architecture, and second, to discover the chemical or other changes which take place during the process of nervous function, that is, the metabolism of the nervous tissues. The first of these problems is at present further advanced than the second; the larger part of this work is, therefore, devoted to a description of architectural relations. Without a knowledge of these relations, moreover, the problems of metabolism are, in large measure, meaningless. * * *

This little book has been prepared in the hope that it will help the student to learn to organize his knowledge in definite functional patterns earlier in his work than is often the case, and to appreciate the significance of the nervous system as a working mechanism from the beginning of his study.

The structure and functions of the nervous system are of interest to students in several different fields — medicine, psychology, sociology, education, general zoology, comparative anatomy, and physiology, among others. The viewpoints and special requirements of these various groups are, of course, different; nevertheless the fundamental principles of nervous structure and function are the same, no matter in what field the principles are applied, and the aim here has been to present these principles rather than any detailed application of them. * * *

52

Notes On The Bursting Strength Of The Alimentary Tract Of The Cat

Henry R. Muller

From the Anatomical Laboratory of the Johns Hopkins University

FOUR FIGURES

INTRODUCTION

Comparatively little attention has been paid by anatomists to the questions of the strength of tissues, and to the mechanical factors concerned in such problems, except in the correlation of the architecture of bones with their tensile strength, and in the mechanism of joints, as developed by Fick in Bardeleben's 'Handbuch der Anatomie/ Anatomists have seemingly neglected this aspect of the physiological anatomy of the alimentary canal, and surgeons alone seem to have been interested in it to any great extent. The experiments performed by these investigators have been, however, rather meagre. Ruptures of the hollow viscera by filUng them to various degrees of tension with air or water, and then striking them with a hammer or board have been produced. By this method the blow causes the organ to flatten out both on the side struck and on the side opposite, and so modifies, as will be shown later, the effect of the internal pressure upon the walls. For this procedure increases the tendency to produce the tear along the edge which is made more round by the blow.

These experiments are, therefore, not identical with those in which the rupture is produced by merely increasing the pressure within the organ by a gas or a fluid. Such pure bursting ruptures have been produced experimentally by only a few observers upon the stomach, intestines and oesophagus of man, dog and pig. Key-Aberg,i using 30 human stomachs obtained

^ A. Key-Aberg. Zur Lehre der spontanen Magenruptur. Nord. Medic. Arkiv., Bd. 22, 1890.

53

THE ANATOMICAL RECORD, VOL. 10, NO. 2 DECEMBER, 1915

54 HENRY R. MULLER

from fresh cadavers, found that the stomach would rupture when the internal pressure was equal to a column of water IJ^ to 2 meters high (from 1/7 to 1/5 of an atmosphere); the tear pro- . duced was quite constantly longitudinal, on or near the lesser curvature, beginning first in the mucosa near the cardia. Hertle* gives his results on six loops of dog intestine and three loops of pig intestine. Those of the dog ruptured at a pressure of 300 to 560 nam. Hg., the tear in each case being longitudinal through all the layers, and most commonly situated on the non-mesenteric border. Those of the pig ruptured at 260 to 300 mm. Hg.*, were also longitudinal, but the tear was near the mesenteric border. Brosch^ found the result of over-distension of ten human oesophagi to be a longitudinal tear, beginning in the submucosa. He gives no figures as to the strength of the oesophagus.

METHOD OF PROCEDURE AND MATERIAL USED

In these observations the fresh material from seventeen adult cats, killed by ether or chloroform, was used inunediately after death. In all cases peristalsis of the intestines could still be obtained. Loops of small intestine, about 7 cm. long, were carefully cut from the mesentery close to the intestinal attachment. In addition to these loops, consisting of all the coats of the intestine, preparations were made of loops deprived of the muscle and peritoneal layers, and so consisting of submucosa and mucosa alone. On account of the meagre fibrous connections between the muscles and submucosa (evident in any histological preparation) the muscle layers together with the peritoneum may be easily stripped off, leaving the submucosa and mucosa intact.

In the routine observation the oesophagus was dissected out of the thorax and removed together with the stomach. The stomach was isolated by ligating the oesophagus at the cardia. In one experiment the oesophagus and stomach were left in situ,

' Josef Hertle. Uber stumpfe Verletzungen des Darmes und des Mesenteriums. Beitrage zur klinischen Chirurgie, Bd. 53, 1907.

' A. Brosch. Die spontane Ruptur der Speiserohre auf Grund neuer Untersuchungen. Virchow's Archiv, Bd. 162, 1900.

BURSTING STRENGTH OF ALIMENTARY TRACT 55

the thorax not being opened, and the pylorus tied off. The greater and lesser omenta in all cases were carefully removed from the stomach. The caecum was cut from the colon about 6 cm. from the tip of the caecum, and the fat and connective tissue bands running from the caecum to the ileum were carefully dissected off.

The pressure to produce the rupture was obtained from the water tap, which afforded a pressure as high as 75 lbs. per square inch. From the faucet the water was led through iron pipes (found necessary to prevent bursting) to pressure gauges and to the glass cannula on which the intestines and the other "hollow viscera were tied. The cannula and gauges were all placed on the same horizontal tubing, thus obviating almost completely any hydrostatic errors. It was found that ordinary steam gauges, registering respectively 30 and 150 lbs. to the square inch, were quite satisfactory.* The system was completely closed except for the opening of the cannula. On this, the trial loops were placed with the opposite end strongly closed by a hemostat.

In the case of the oesophagus the water was allowed to flow in from the upper end, the cardiac end being tied. In the observations on the stomach the water entered the pylorus after closure of the cardiac end. When the caecum was used the water entered it through the cut end where it had joined the colon; the junction of ileum with the caecum was tied off.

Preparations of the submucosa of the intestine were made for the purpose of studying the direction of its connective tissue fibers. Loops of intestine were filled to various degrees of distension with Camoy's fixing fluid. The muscle layers and the mucosa were then dissected off, leaving the submucosa. Pieces of this were placed in very dilute phosphomolybdic acid for several hours, washed in distilled water, and then placed over night in dilute aniUne blue solution. The specimens were then

The pressures necessary for rupture were obtained in these experiments in terms of pounds per square inch. These figures have been converted to the corresponding pressures in terms of millimeters of mercury, considering atmospheric pressure equal to 760 mm. Hg., or 14.7 lbs. per square inch. They have been placed in parentheses after the first figures obtained.

56 HENRY R. MULLER

dehydrated in absolute alcohol, cleared in benzol and mounted in damar. Histological sections of small intestine both longitudinal and transverse, stained for connective tissue by Mallory's method, were also prepared.

RESULTS OBTAINED

Using the technic outUned above it was found that the pressure necessary to rupture the oesophagus varied from the lowest 13 lbs. (672 mm. Hg.) in one instance, to the highest 24 lbs. (1240 mm. Hg.) in another case. The average of all fourteen cases was 17.75 lbs. (911 mm. Hg.). The tear was constantly longitudinal, extending through all the coats, with clean-cut edges, ^beginning on the average 5.0 cm. from the cardia and extending upward for 2.0 cm. The oesophagus apparently lengthened but little, whereas it dilated markedly.

The stomach dilated at first very markedly before the pressure within it rose appreciably. Its point of rupture was then, however, soon reached, varying only slightly in the fifteen cases from the average found — 3.73 lbs. (193 mm. Hg.). Nine times the tear was on or near the lesser curvature, and six times, on or near the greater curvature. (Once its position was not noted with reference to the curvatures of the stomach.) In all instances the tear had clean-cut edges, extended through all the coats of the stomach, and ran parallel with either greater or lesser curvatures. Nine times out of fifteen, the tear was nearer to the cardiac end than to the pyloric end. In one additional case, where the stomach and oesophagus had been left in situ, and the thorax not opened (in a living animal), the cannula was inserted into the oesophagus in the neck, and the pylorus tied off. Rupture of the stomach occurred at 2.5 lbs. pressure (129 mm. Hg.) along the greater curvature, 1.0 cm. in front of the omental attachment, and parallel with this border. The tear was 3.0 cm. in length. The oesophagus remained undamaged.

In 32 trials, pieces of jejunum were used having all their coats intact. The lowest pressure to produce rupture was 17.5 lbs. (905 mm.) in one cat, the average of all cases being 28.2 lbs.

BURSTING STRENGTH OF ALIMENTARY TRACT 57

(1458 mm.). In each individual cat, however, the figures were more constant; the greatest difference was in one cat where two trials of intact jejunum gave 32 lbs. (1654 mm.) at one time, and 37 lbs. (1913 mm.) at another. Such duplicate tests were made in twelve other cats; most commonly there was a variation of only 1 or 2 lbs. (52 mm.-104 mm.) in any one individual.

The total average of 18 trials of portions of the ileum with all coats intact was 24.8 lbs. (1282 mm.). The results ranged from 17.5 lbs. (905 nam.), the lowest figure, to 37.5 lbs. (1939 mm.) which was the highest figure obtained. Here also, where duplicate tests were made in one and the same cat, the greatest difference was only 6 lbs. (310 mm.) in a cat where once the ileum ruptured at 21.5 lbs. (1112 mm.), and the second time at 27.5 lbs. (1422 mm.). In three duplicate tests the figures either agreed or varied only a few pounds.

The caecum showed considerable distensibility, and its point of rupture varied from 8.75 lbs. (452 mm.), which was the lowest figure, to 25 lbs. (1292 mm.), the highest figure obtained. Tlhe average of twelve cases was 16.9 lbs. (874 mm.). In six cases the tear was along the edge opposite the ileo-caecal valve, and in six cases along the edge on which the ileo-caecal valve is situated.

The following table (No. 1) gives the results of rupturing the intact oesophagus, stomach, small intestine and caecum of the cat, giving also the average length of the tear, and in the case of the intestines, the distance the tear was situated from the mesenteric border.

In regard to the nature of the tear it was seen that by slowly increasing the pressure in those intestines which ruptured at a relatively low point, for instance at 18 lbs. (931 mm.), the muscle coats, serosa, submucosa and mucosa, iruptured practically simultaneously, so that it was difficult to say which tore first. In those cases, however, where the rupture of the submucosa took place at a greater pressure, in one case for instance at 43 lbs. (2223 mm.), the muscle coats had already ruptured when the pressure had reached 33 lbs. (1706 mm.). The tears in the muscle layers were usually somewhat longer than the tears in

58


HENRY R. MULLER


TABLE 1 Alimentary tract , intact


Oesophacus.,


Stomach


Jejunum...


Ileum.


Caecum


32.


12


ii 13 lbs. (672 mm.)


2.75 lbs. (135 mm.)


17.5 lbs. (905 mm.)


17.5 lbs. (005 mm.)


8.75 lbs. (462 mm.)


Is


^5


24 lbs. (1240 mm.)


5 lbs. (250 mm.)


43 lbs. (2223 mm.)


37.5 lbs. (1030 mm.)


25.0 lbs. (1202 mm.)


■ tt o

"Si


17.75 lbs. (Oil mm.)


3.7 lbs. (103 mm.)


28.2 lbs. (1458 mm.)


24.8 lbs. (1282 mm.)


16.0 lbs. (874 mm.)


o

ss


sis


32 lbs. (1654 mm.) 37 lbs. (1013 mm.)


21.5 lbs. (1112 mm.) 27.5 lbs. (1422 mm.)


s2


2.0 cm.


2.6 cm.


2.6 cm.


2.1cm.


2.0 cm.


^ « s a w .

ApfiOS •< H

H fc. 9 w >o So


3 mm.


4 mm.


the submucosa. Tears in the mucosa of the stomach were seen to take place usually before the submucosa and muscles tore, at about 2 lbs. (104 mm.) pressure.. These tears appeared on the surface of the distended organ as silvery, longitudinal lines. It was also noted that in every case, without exception, the tear was linear, with clean-cut edges, running in the direction of the long axis of the organ; i.e., in the stomach as already noted; in the intestines parallel to the mesenteric attachment, and nearer to it than to the opposite border; and in the oesophagus extending upward in a longitudinal direction.

Pieces of jejunum deprived of muscles and peritoneum, and consisting only of submucosa and mucosa ruptured on the average of 25 trials at 24.25 lbs. (1254 mm.), which is only 14 per cent lower than the average for the entire gut. The lowest figure in this series was 13.5 lbs. (698 mm.), and the highest 49 lbs. (2533 mm.). In seven dupUcate tests carried out in seven cats the greatest individual difference was 1| lbs. (78 mm.),

BURSTING STRENGTH OF ALIMENTARY TRACT


59


the lowest being 13.5 lbs. (698 mm.), and the highest 15 lbs. (776 mm.). Similar tests with ileum deprived of muscles and peritoneum gave 18.6 lbs. (962 mm.) as the average of 16 trials. This is only 25 per cent lower than the pressure necessary to rupture the ileum with all the coats intact. The lowest instance was 9 lbs. (465 mm.), and the highest 27.75 lbs. (1435 mm.). Three duplicate tests were made, in which the greatest was in a cat which gave 24.5 lbs. (1267 mm.) at one time and 27.5 lbs. (1422 mm.) at another.

The following table shows the results obtained by rupturing the jejimum and ileum of the cat deprived of muscle layers and peritoneum. It also gives the length of the tear and the distance of the tear from the mesenteric border.


TABLE 2 Small intestine deprived of muscle coats


/^'vnum without muftcles or peritoneum


Ileum without muscles or peritoneum


25. 13.5 lbs. (698 mm.)


Olbs. (405 mm.)


U


49 lbs. (2533 mm.)


27.75 lbs. (1435 mm.)


O u P


24.25 lbs. (1254 mm.)


18.6 lbs. (962 mm.)


SK se S & U mm


13.5 lbs. (698 mm.) 15.0 lbs. (776 mm.)


24.5 lbs. (1267 mm.) 27.5 lbs. (1422 mm.)


if

M H


s2


3.4 cm.


2.4 cm.


Ea a w

fe«2


1.0mm.


2.2 mm.


In all cases of the small intestine deprived of muscles the tear was a longitudinal one with clean-cut edges, nearer the mesenteric attachment, and parallel with it. In these cases the mucosa could be seen to tear at relatively low figures, usually at 10 lbs. (517 mm.) or lower. These tears shimmered through the intact submucosa as longitudinal silvery lines, usually more than one in number.

Fourteen times the specimen from the jejunum or ileum used contained a Peyer's Patch, and when this was present the tear

60


HENRY R. MULLER


always took place through it at the pressure indicated on the following table. It gives the results of pieces with and without muscles, from the jejunum and ileum respectively.

TABLE 3 Small intestines containing Peyer^s Patches si

z


LOWEST

PBEHSUBE

PRODUCINQ

RUPTUBB


HIOHB0T PRBSSURS PRODUCING

RUPTURS


AVERAOB

PRESSURE

NBCESS.UIT

TO PRODUCE

RUPTURE


LENGTH OF TEAR


DISTANCE

FROM

MESENTERIC

BORDER


Jejunum, with all coats, through Peyer's Patch.


4.


15.5 lbs. (801 mm.)


24.5 lbs. (1267mm.)


18.5 lbs. (949 mm.)


1.1 cm.


9.0mm.


Jejunum^ without muscles, through Fever's Patch.


3.


12 lbs. (620 mm.)


13.75 lbs. (711 mm.)


12.9 lbs. (667 mm.)


0.8 cm.


7.25mm.


Ileum, with all coats, through Peyer's Patch.


3.


16 lbs. (827 mm.)


24.25 lbs. (1254mm.)


19.75 lbs. (1021mm.)


1.2 cm.


8.0 mm.


Ileum, without muscles, through Peyer's Patch.


4.


9.5 lbs. (491 mm.)


12 lbs. (620 mm.)


10.8 lbs. (558 mm.)


1.4 cm.


5.0 mm.


As is seen, the rupture ta^es place at a distance farther away from the mesenteric border than occurs in ordinary specimens, because the Peyer's Patch is situated nearer to the non-mesenteric border. The direction of the tear is again in the direction of the long axis, and, although the tear is linear, its edges are not as clean-cut.

CORRELATION OF ANATOMICAL AND PHYSICAL FINDINGS

The constancy of the longitudinal rupture suggests strongly that the reason for this linear tear in the long axis of the intestine is to be found in some anatomical characteristic of the intestinal architecture. With this idea in view, a study of the fibrous tissues of the gut wall was made in order to ascertain if in the case of the small intestine (the typical hollow cylinder) this contention could be supported.

BURSTING STRENGTH OF ALIMENTARY TRACT 61

The small intestine ruptures longitudinally. The tear through the muscle layers is also longitudinal. When one considers that the circular layer of muscle fibres is considerably thicker and stronger than the longitudinal layer, one would expect, from the anatomical relations alone, a circular tear of the muscle coats at least. The submucosa together with the mucosa withstood pressures, as was shown above, only 14 per cent for the jejunum, and 25 per cent for the ileum, less than when all coats were present, indicating that the submucosa is the part of the intestine chiefly concerned in giving strength to the gut. It was found that the main bulk of connective tissue fibres of the submucosa runs spirally around the intestine in both directions, crossing when the gut is moderately distended, so that the acute angle f onned by their crossing points in the direction of the long axis of the gut (fig. 1).



This agrees with the findings of Mall^ and Clason.** When the intestine is distended nearly to the point of rupture these fibres tend to run more nearly transversely (fig. 2), cross at right angles, and many fibres at angles greater than a right angle.

Hence the fibres which in the gut ordinarily run more nearly longitudinally, in the greatly distended gut assume a more nearly circular direction. This, as will be shown, is an attempt to reenforce the point where the greatest pressure is to be exerted, the reenforcement being taken from the point where the danger from rupture is naturally less according to the laws of physics. Besides this main bulk of coarse connective tissue fibres there is a layer of somewhat finer fibres running longitudinally among the muscle fibres of the longitudinal layer of the muscularis

F. Mall. A Study of the Intestinal Contraction. The Johns Hopkins Hospital Reports, vol. 1, 1896, p. 41. • Clason. Upsal. Lakareforhandl 7. Hofmann-Schwalbe, 1872, p. 182.

62


HENRY R. MULLER


mucosae. The muscularis mucosae in the jejunum of the cat is equal to about i to J of the total thickness of the submucosa. The connective tissue fibres, as can be seen in histological preparations stained for connective tissue, according to Mallory, make up at least one-half of the total bulk of this layer of the muscularis mucosae. The circular layer of muscularis mucosae is much thinner, and contains scarcely any connective tissue fibres.

Naturally, with the anatomic findings favoring a transverse tear, other factors must be considered to account for the constant longitudinal rupture. A consideration of the laws of physics involved in the bursting of hollow cylinders may aid in finding the explanation. The force tending to produce a longitudinal tear as compared with the force tending to produce a transverse tear in a hollow closed cylinder under internal pressure must be considered. Let figure 3 represent diagrammatically such a hollow cylinder, and figure 4 a cross-section of the cyUnder.

If the radius of the circle O, the end of the cylinder, is equal to one (1), then the area of the circle tt R^ = ir.

If p = pressure per unit of area, then p^r = total pressure on the surface of the circle, or the end of the cylinder.

This force is transmitted to the longitudinal fibres, along the circumference of the circle.

The circumference =27rR=27r. Then per unit of circumference we have ^^ = ^; which is equal to the force acting in a longi2'7r 2

tudinal direction on the longitudinal fibres, and tending to produce a circular tear.

BURSTING STRENGTH OF ALIMENTARY TRACT 63

If the circumference of the circle O represents a circular fibre, then the force tending to burst it at A is equal to the force acting on the arc AD, at right angles to AO.

Or, continuing AO as a plane AOFE running through the centre of the cylmder, and OD as a plane ODKF at right angles to the first plane, then the pressure on AOFE is equal to the area of AOFE multiplied by p. If AE and OF = AO, which is equal to 1, the pressiu-e on AOFE = p.

Tliis means that the force transmitted to the circular fibres along AE, a unit of length longitudinally, and tending to produce a longitudinal tear is twice as great as the force p/2, acting on a unit of circumference on the longitudinal fibres and tending to produce a circular tear.

The stomach, being more or less of a cone-shaped organ, will also have its tears longitudinal, as will the oesophagus, a cylindrical organ.

Also, the force tending to produce a longitudinal tear along the side of a cylinder which is flattened tends to become less than the force acting along the side with the curve of the smaller radius; so that those experiments mentioned above, where ruptures had been produced by striking distended organs, are not identical with those in which bursting was allowed to take place by increasing the internal pressure alone.

SUMMARY

It has been pointed out that the mucosa of the stomach and that of the intestines ruptured at pressures below those of rupture for submucosa and muscle layers of the stomach and intestines respectively. This would indicate that the mucosa is of slight importance in giving strength to the organs. That the mucosa of the stomach, which is ordinarily thrown into many folds (rugae), indicating a considerable reserve for dilatation, should reach its elastic limit so soon, and tear, is therefore somewhat remarkable. The observation agrees with that of KeyAberg who said that the tear in the stomach began first in the mucosa. The peritoneum, a very thin and delicate layer of

64 HENRY R. MULLER

tissue covering the organs, is also practically unimportant in giving strength to the viscera. The two chief tissues giving strength to the hollow viscera, are, then, the muscle tissue, and the connective tissue of the submucosa. Of these, the pressures for producing ruptures of the submucosa are only 14 per cent for jejunurp, and 25 per cent for ileum, less than for the intact intestine. Although there was seen to be considerable variation in the strength of the intestines with and without muscles in different cats, still in the same individual the strength was quite uniform for several specimens obtained from the same regions of the small intestine.

Compared with the other parts of the gastro-intestinal tract, the bursting point of the stomach is remarkably low. That it bursts on or near the greater or lesser curvatures would suggest that either these are anatomical weak places, or that the greater and lesser curvatures are curves of smaller radius than the sides of the distended stomach, and that the tear takes place there because of the physical laws.

Similarly, the tear occurring in the intestines on or near the mesenteric border suggests the same two possibiUties. Here, also, no direct observations were made. The figures obtained for the oesophagus were relatively high, and for the small intestine actually much higher than the few cases mentioned in the literature in experiments performed on the dog and the pig. Whether this is due to a difference in the freshness of the tissue used, or to an actual difference in the species of animal, has not yet been determined.

The caecum, another greatly dilatable organ, ruptured at figures lower than those for the small intestine.

Finally, in all the hollow organs tried the tear took place through all the coats, extending in the direction of the long axis of the organ. This agrees with all previous observations. It is explainable, as was seen, not on anatomical grounds, but on the physical phenomenon that in a hollow cyUnder (when other things are equal), the internal force tending to produce longitudinal tears is twice as great as that tending to produce circular tears.

BURSTING STRENGTH OF ALIMENTARY TRACT 65

CONCLUSIONS

1. The following pressures were found necessary to produce bursting ruptures in the following hollow organs of the cat :

a. Oesophagus 17.75 lbs. per sq. inch (911 mm. Hg.)

b. Stomach 3.7 lbs. per sq. inch (193 ram. Hg.)

c. Jejunum 28.2 lbs. per sq. inch (1458 mm. Hg.)

d. Jejunum, without muscle coats 24.25 lbs. per sq. inch (1254 mm. Hg.)

e. Ileum 24.8 lbs. per sq. inch (1282 mm. Hg.)

f. Ileum, without muscle coats 18.6 lbs. per sq. inch (962 mm. Hg.)

g. Caecum 16.9 lbs. per sq. inch (874 mm. Hg.)

2. Although the bursting pressures vary considerably for the small intestines of different cats, the bursting pressures in the same individual are quite uniform.

3. The tear produced by bursting is linear and runs in the long axis of the organ. This phenomenon is explainable, not on anatomical, but on physical grounds.

4. The submucosa is the layer chiefly concerned in giving strength to the alimentary tract. DIRECT UNION BETWEEN ADRENALS AND KIDNEYS (SUBCAPSULAR LOCATION OF ADRENALS)

C. J. BARTLETT

From the Department of Pathology, Yale Medical School, New Haven, Conn,

FIVE FIGURES

There is a rare condition in which the adrenals lie in part or entirely in direct apposition with the kidney parenchyma with no intervening capsule. It is the so-called subcapsular location of the adrenals. For some reason it has been very generally overlooked both by anatomists and pathologists. It is, however, of importance not merely as a developmental error but, from the pathologist's standpoint, because of its bearing upon the origin of the so-called hypernephromata of the kidney, a question still under discussion. No reference to this condition has been found in any of the modem text-books of pathology, such as Aschoflf, Ribbert, Adami and others, nor in any of the works on anatomy which I have consulted. Orth in his Pathologische Anatomie ('93) says Only seldom is the location of the adrenals in gross changed (from normal). One case is known where the right suprarenal was located at the hilum of the kidney above the renal artery." He makes no reference to its subcapsular location. Broman^ in his work on the normal and abnormal development of man does not mention the condition. Also in special works on the kidneys and adrenals this abnormality is commonly overlooked. Thus Kelly and Burnam* state that in children there is a strong attachment between the kidney and adrenal gland which is less marked in the adult. They make no mention of a direct union between the two organs. On the other hand. Dock' in describing the abnormalities of the adrenals says

Broman. Nprmale u. abnorme Entwicklung des Menschen, 1911. Kelly and Bumam. Diseases of the kidneys, ureters and bladder, 1914. Dock. Osier's Modem Medicine, 6, p. 355. 67

68 C. J. BARTLETT

"Pilliet found the right adrenal under the fibrous capsule of the kidney but he gives no references and mentions no other reported cases. Borst* describes the condition briefly as follows, In certain cases the suprarenal lies entirely or in part close upon the kidney, enclosed in the kidney capsule. (Kelly, Grawitz, Klebs, Ulrich.) Frequently the condition is bilateral and the suprarenal is intimately attached to the upper pole of the kidney as a thin plate.

From this it is evident that the subcapsular location of the adrenal body has been recorded occasionally for years. Grawitz^ ill his second paper on the hypernephromata referred to it. He says that an entire adrenal may be found in the capsule of the kidney or in the kidney cortex, as well as smaller or larger portions of the adrenal. In his earlier paper of the year before ('83) on the same subject he makes no mention of the entire organ being thus displaced. Somewhat earlier than this ('76) Klebs* described what is probably a case of this subcapsular location. This was in a well developed man, 20 years old, who died of pneumonia. In place of the adrenals, each kidney was covered over by a thin, yellowish, caplike plate which was beneath the kidney capsule. Microscopically it showed all the elements of the adrenal cortex. At the usual site of the adrenals there was no trace of these. This is the earliest reported case which I have found, although a more careful search might disclose others even earlier. Later Ulrich^ and Kelly ^ and others reported similar cases. In an excellent summary of the work on the Adrenal Cortex, Its Rests and Tumors (with a good bibliography) by Glynn® the only cases of subcapsular location of the adrenal referred to, six in number, are from German authors.

In order to determine the frequency with which the partial or complete subcapsular location of the adrenals has been observed in this country, I communicated some three years ago, after studying the case reported below, with ten other pathologists^® who have had opportunity to study a considerable abundance of autopsy material. I take this opportunity to again thank them for giving me the results of their experience. Seven of the ten repUed that they had never met with the condition. One other could recall clearly only one case of the kind, while two. Professor Le Count of the University of Chicago and Dr. John H. Larkin of New York, had twice found the adrenal in the subcapsular location. The former stated that in both of his cases the condition was bilateral. It is quite evident that this condition is among the rare abnormalities involving the adrenals and kidneys. »


Borst. Die Lehre v. d. Geschwiilsten, p. 789, 1902. Grawitz. Archiv. f. klin. Chirurgie, 1884, 30, p. 325. « Klebs. Handbuch d. path. Anatomie, Bd. 1, abt. 2, p. 567, 1876.

^ Ulrich. Anatomische Untersuchungen uber ganz und partiell verlagerte u. accessorische Nebennierea etc. Zeigler's Beitrage z. path. Anatomie, 1895, 18, p. 589.

Kelly. Zeigler's Beitrage z. path. Anatomie, etc., 1898, 23, p. 293. ® Glynn. Quarterly Journal of Medicine, 1912, v, p. 157.


Aside from being of such rarity as to make it of interest, this abnormal location of the adrenal is of indirect importance in considering the etiology of the so-called hypemephromata of the kidney. As is well known the origin of these tumors from adrenal rests in the kidney, which had apparently been fully determined by Grawitz and confirmed by others, has more recently been seriously questioned. This has been based in part upon the histological differences between the tumors of the kidney and the adenomata found in the adrenal itself, in part upon what may be termed the functional or metabolic differences between these renal tumors and the adenomata of the adrenals, and lastly upon studies of the embryological development of the adrenals and kidneys.

From a histological study of the development of so-called hypemephromata of the kidney, Sudeck^^ as early as 1893 held that there was no connection between these tumors and adrenal rests, but that the former were derived from the tubular epithe-Hum itself. Stoerk^^ after the study of a large number of tumors of this type reached a similar conclusion, namely, that they are of

10 Drs. W. T. Councilman and F. B. Mallory, of Boston; Drs. T. M. Prudden, James Ewing and J. H. Larkin, of New York; Drs. Ludwig Hektoen and E. R. Le Count, of Chicago; Drs. W. M. L. Coplin and Joseph McFarland, of Philadelphia, and Dr. Horst Oertel of Montreal.

" Sudeck. Virchow's Archiv, 1893, 133, p. 407.

1* Stoerk. Beit. z. path. Anat. u. z. allg. Pathologie, 1908, 42, 393.


renal origin. This was based in part upon the location of these tumors, aways in the kidney, never in the adrenal itself nor in the liver, where so-called adrenal rests are so common; in part upon the histological study of the tumors; and, in part, upon the age at which they most conmionly appear, chiefly after middle life, imUke tumors from cell rests which have a tendency to appear in early life.

A further careful study of the subject was made by Wilson and Willis^' based not only upon the histological examination of 48 tumors of this type but also upon the embryological development of the suprarenal and kidney as shown in serial sections from 26 swine embryos and from 43 human embryos. Their conclusions from this study, namely, that the so-called hypernephromata are not of adrenal origin, are evidently based quite largely upon the complete separation at all periods of development between the adrenal and kidney. They say

The kidney and the adrenal come into final apposition only by the gradual atrophy of the Wolffian body, and between the two organs there is always interposed the mass of fibrous tissue which represents the stroma of the atrophic Wolffian body. Long before the two organs come into any close anatomic relationship with each other, each has formed a distinct and well marked capsule, which is greatly augmented between the surfaces of the two organs which are directed towards each other, by this dense mass of Wolffian-derived fibrous tissue Indeed it is difficult to conceive how any portion of the adrenal cortex can, during the process of embryological development, become imbedded within the kindey parenchyma without showing between its structure and that of the renal cortex, three distinct laminae of fibrous tissue, — the first derived from its own cortex, the second from the remains of the Wolffian body, and the third from the kidney capsule.

And further.

From what has been stated above, suprarenal inclusions within the kidney parenchyma must be exceedingly rare, if ever present. The only instances of conjugation of suprarenal and kidney tissue which I have ever seen were masses of suprarenal tissue which were attached to the kidney, projecting above its surface, and invariably separated from the kidney parenchyma by a thick capsule, which not only stripped readily from the kidney, but was also separable into a number of laminae.

" Wilson and Willis. Journal of Medical Research, 1911, 24, p. 73.


This necessary separation of the suprarenal gland from the kidney by a thick capsule of connective tissue appears to be one of the main reasons derived from their embryological study for concluding that the islands from which these tumors appear to arise may not be adrenal in origin. It is only upon this one phase of the subject, namely, the possible intermingling of suprarenal and kidney tissue without any separation by a definite capsule, that this abnormal union between suprarenal body and kidney, the subcapsular location of the adrenals, has a bearing.

In this condition the adrenal lies in part or entirely in direct apposition, on its under surface, with the kidney tissue, with in part at least, no more intervening connective tissue than there is between the kidney tubules of the normal kidney cortex.

The case of this kind which I had an opportunity to study was one in which each adrenal was directly in apposition with kidney parenchyma, extending dowil for a short distance into this. This direct apposition of adrenal and kidney did not involve the whole but only a part of each adrenal on its under surface. This condition as here described was found at autopsy in a coroner's case. Nothing was known of the previous history except that the person has been an alcoholic. The general autopsy findings were unimportant. The body was that of a small woman, 5 feet tall. She was about 30 years old. Aside from certain superficial bruises, the gross pathological findings were bronchopneumonia, obliteration of the pleural cavities by old adhesions, chronic gastritis, suprarenals on each side adherent to the kidney, and a slight degree of chronic interstitial nephritis. No other developmental errors besides those of the adrenals and kidneys were found. No careful search was made for adrenal rests in other structures than the kidneys but a superficial examination did not show any. The microscopic examination of the various tissues, aside from the suprarenal bodies and kidneys, requires no discussion as it merely confirmed the gross appearances.

On each side, the adrenal was found unusually adherent to the kidney. This was more marked on the right side than on the left. The right adrenal extended from the upper end of the kidney down over its anterior surface for about 4 cm. This suprarenal was 4^ cm. vertically by 5 cm. wide at its widest part. The upper third of the adrenal was separated from the kidney by areolar tissue. Below this it was firmly adherent to the kidney except for a few millimeters along its inner edge. A vertical section through the adrenal and kidney in this area not only showed in gross no connective tissue capsule separating the two, but in places the adrenal tissue extended down into the cortex of the kidney for a greater or less distance, the deepest extension being about 1 cm. (fig. 1). This adrenal at its upper part, where free from the kidney, was of the usual thickness and appearance. Below this it extended as a thin plate over the upper front part of the kidney. Its thickness here in general was only 1 to 1| mm. The darker color of the medullary portion in contrast to the cortex could be readily made out, though less marked than at the thicker portion at the upper part of the gland.


Fig. 1 Right kidney and adrenal, showing direct union between the two with extension of the adrenal down into kidney parenchyma.

Fig. 2 Shows lack of capsule between adrenal and kidney, with, in places, upward extension of small portions of kidney tissue into adrenal.


The left adrenal was also adherent to the kidney though to a much less extent than was the right one. It was not much larger than usual and was free from the kidney except for an area about 2| cm. by 1^ cm. at its widest part, where it appeared as a thin plate, adherent to the front part of the upper pole of the kidney, and extending a very short distance into the kidney substance in places. No capsule separating adrenal and kidney could be made out here.

Microscopic examination of sections of the adrenals where they were free from the kidney show nothing unusual. But where they lay as a thin plate attached to the kidney several departures from normal are seen (figs. 2 to 5). Both cortical and medullary portions are present. The part of the adrenals which in gross showed no evidence of a connective tissue capsule between it and the kidney are seen in general to lie directly against the kidney cortex with no connective tissue intervening. This is not so in all places, as here and there a distinct band of connective tissue can be made out separating the two. This is also true of the extensions of the adrenal down into the cortex of the kidney. The adrenal and kidney parenchyma are, as a rule, in direct apposition but in places there is a distinct microscopic band of connective tissue between them. It is noticeable that

74 C. J. BARTLETT

in those portions where adrenal and kidney are in direct apposition with no connective tissue separating them, the glomerular layer of the adrenal is lacking. On the other hand, where there is a distinct layer of connective tissue separating them, this glomerular layer is generally well made out. The medullary portion is also present in the epinephritic adrenal plates, and in


Fig. 3 Adrenal (above) and kidney (below) in direct apposition.

the largest downward extension of the right adrenal into the kidney cortex the medullary portion also extends down a short distance. In general, the line of union of the adrenal plate and the underlying kidney is readily made out and is fairly smooth. In places there are extensions of kidney tubules up into the adrenal. That these are kidney tubules can be made out here and there by

UNION BETWEEN ADRENALS AND KIDNEYS 75

finding direct extension of these from the kidney cortex into the adrenal. Similar structures found in the adrenal as far up as the medullary portion are probably from the same source. They consist of somewhat dilated glandular structures lined with a layer of cuboidal epitheUum and often containing a hyaline sub


Fig. 4 Area showing thin layer of connective tissue between kidney and adrenal . The glomerular layer of the adrenal is here seen, but is lacking in figure 3.

stance, staining red by the hematoxylin-eosin method. Where portions of the adrenal extend downward into the kidney, the line of separation is in places very irregular with intermingling of kidney structures and adrenal tissue (fig. 5).

In studying the effect of the direct apposition of suprarenal cells upon the kidney cells, none could be made out. The cells

76 C. J. BARTLETT

of kidney tubules lying directly against adrenal cells, with no more intervening connective tissue than is found between the tubules of the kidney cortex, appear as normal as elsewhere. They are not compressed by the adrenal tissue. Occasionally at the line of union a tubule may be filled with pale staining cells and


Fig. 5 Intermingling of adrenal and kidney parenchyma.

some doubt is felt as to whether one is here dealing with adrenal or kidney cells. This is, however, the exception.

That this unusual union of kidneys and adrenals is to be explained as a developmental error, and not in any sense as a beginning tumor growth or the result of an inflammatory process, appears to be self-evident. Where the adrenal Ues as a thin plate upon the kidney, the thickness from medullary portion to

UNION BETWEEN ADRENALS AND KIDNEYS 77

kidney is no greater than from the medullary portion to the upper border of cortex. And where there is a downward extension of adrenal into the kidney cortex there is no compression capsule of connective tissue to indicate a disappearance of kidney cells. This subcapsular location of the adrenal has in most of the reported cases, as in this one, been bilateral.

Although the condition is very rare, its occurrence at times is not in keeping with the conclusions of Wilson and Willis that the adrenal and kidney are necessarily at all times separated by distinct layers of connective tissue. It gives definite evidence of the possibiUty of portions of the adrenals becoming imbedded in the kidney parenchyma as claimed by Grawitz as the basis of his theory of the origin of the hypemephromata. THE VASCULARIZATION OF THE EMBRYONIC BODY

OF HYBRID TELEOSTS WITHOUT

CIRCULATION

FRANKLIN P. REAGAN and J. MONROE THORINGTON

Department of Comparative Anatomy , Princeton University and U. S. Bureau of Fisheries, Woods Hole, Mass.

PIPTEEN FIGURES

Within recent times it has been shown that certain unusual conditions may be imposed upon the developing vertebrate embryo which give a clue to the process by which such an embryo would normally come into possession of its blood vascular system. These unusual conditions have fallen, so far, into two categories: unusual mechanical relations with reference to the extra embryonic blastoderm, and chemically altered environment at an early stage of ontogeny.

Accounts of these unusual mechanical relations, consisting of partial separation of the embryonic body from the yolk-sac blastoderm, may be found in the writings of Hahn,^ Miller and McWhorter,^ who obtained blood-vessels on the operated side of partially excised embryos. In view of the fact that the results of these observers were not universally regarded as adequate proof of the local origin of the endothelium which developed under these experimental conditions, such experiments were extended further to a complete separation of the intraembryonic tissue from the yolk-sac blastoderm in the experiments of Reagan, who succeeded in obtaining blood-vessels in completely isolated portions of embryonic bodies. In addition to the advantage of excluding all possibility of an invasion of the intraembryonic tissue by yolk-sac 'Angioblast,' this latter work was based on mate 1 Hahn, H. Archiv ftir Entw., Bd. 24, 1907.

Miller, A. M. and McWhorter, J. E. Anat. Rec, vol. 9, no. 1, 1915. 79

80 FRANKLIN P. REAGAN AND J. M. THORINGTON

rial, the actual condition of the extraembryonic portion of which could be definitely determined; the extraembryonic blastoderm was removed at the time of operation so that the exact status of vascular development could be ascertained.

In his excellent monograph on the early development of the vascular tissue in the cat, Schulte^ not only furnishes conclusive morphological evidence for the local origin of vascular endothelium, but gives a most comprehensive r6sum6 of the two opposing theories of vascular development and the facts upon which each finds its support. On page 22 he reviews the results of Loeb^ on the inhibited circulation in chemically treated and hybrid teleost embryos, pointing to the probability that embryos possessed of beating hearts but devoid of circulation, might have non-continuous vascular anlagen. Since the publication of Schulte's work, it has been shown by Stockard*^ and by Werber* that in chemically treated embryos these anlagen arise locally and non-con tinuously in and from the mesenchyme, the failure of the separate anlagen to coalesce being caused by, or at least correlated with, the general arrest of development which the embryo suffers.

The existence of beating hearts in hybrid teleost embryos lacking circulation, as observed by Loeb, has recently been verified by Newman.' No author has yet examined such material for the histogenesis of vascular tissue. Since the external conditions of such embryos resemble those conditions in the chemically treated embryos described by Loeb, Stockard and Werber, it seems reasonable to suppose that a similarity of internal cond'tions may prevail. If this be true, it is evident that a study of hybrid material affords promise of another link in the chain of evidence, the significance of which points in no uncertain manner toward the local origin theory of endothelial development.

^ Schulte, H. von W. Memoirs of The Wistar Institute of Anatomy and Biology, no. 3.

Loeb, J. Pop. Sci. Mo., vol. 80, p. 5. Stockard, C. R. Proc. Am. Assn. Anatomists, Anat. Rec., vol. 9, no. 1. Werber, E. I. Anat. Rec., vol. 9, no. 7. ^ Newman, H. H. Journ. Exp. Zool., vol. 18, no. 4.

VASCULARIZATION OF BODY OF TELEOSTS 81

Of the several crosses tried, the most favorable seems to be that of Scomber scombrus X cf Fundulus heteroclitus 9 . About 30 per cent of the individuals resulting from this cross developed oedematous pericardia and string-like hearts. At the age of five days, such hearts showed little pulsation and gave no evidence of a blood content. In the course of an additional four or five days, more or less marked cardiac contractions could be observed. During this time there occasionally developed in the immediate region of, or as a part of, the distal (venous) end of the heart a large blood-island (^blood-island' in the sense in which Stockard uses the term), the blood contents of which attained a bright color. Such a blood-island always resembled closely those found on the yolk ventral to the tail in many other embryos. These blood-cells were evidently of local origin since they often arose before true blood cells could, by most careful observation, be found in the embryonic axis or on any other part of the yolksac; certain it seems from the observation of living material, that they were not passively washed in by the movement of bodyfluids, either as colorless erythroblasts or as differentiated blood cells. Neither does it seem probable that they could have originated from cells resembling the erythrocyte anlagen to be found in the 'Gefassstrang' of the embryonic axis nor from the erythroblasts on the yolk-sac ventral to the tail, although inference to the contrary would be possible. Morphologically, the immediate anlagen of such blood cells display no signs of capability of active amoeboid movement. If they possess at all the power of movement, it is very slight. As a matter of course, it is certain that the ultimate anlagen of such a blood-island have not always occupied their definite positions as observed after true blood-cells have differentiated. The anlagen of blood-cells do seem, however, to have occupied such a position before they could truly be designated as blood-cells.

The term 'blood-island,' as applied to such aggregates is to be accepted with some caution. In some cases they are not bloodislands at all. In normal development, mesodermal cells wander out or are proliferated out from the body of the embryo, some (destined, if you wish) to form endothelium, some to form pig 82 FRANKLIN P. REAGAN AND J. M. THORINGTON

ment cells, and some to form blood-cells In normal development, endothelium is formed rapidly enough and heart pulsations are sufficiently strong that blood-cells are generally though perhaps not always disposed of as rapidly as their differentiation proceeds. In chemically treated embryos, heart pulsations are often reduced to a minimum; endothelial cavities fail to unite and the anlagen of blood and endothelial cells congregate on the yolk, generally ventral to the tail. In such cases blood-islands might well be called vascular stagnations, or better still, *blood lacunae' of Werber. Embryos with complete circulation, but reared in more or less stagnant water show yolk-areas (usually accompanied by pigment cells) where blood-cells are certainly of local origin. In some embryos reared in running sea-water no such islands could be found.

When, however, one observes the actual passage of an erythroblast from the intermediate cell-mass to a region on the yolk-sac ventral to the tail, either by the motion of body-fluids or by independent amoeboid movement — if such a power be possessed by an erythroblast — he need not conclude that all the erythroblasts of that region reached there secondarily from that source by this means. It is conceivable that adjacent morphologically nonspecific intraembryonic mesenchyme not contained in or related to the intermediate cell mass had proliferated into the yolk-sac region and had there given rise to erythroblasts. If there is no such possibility, then so far as the yolk-sac is concerned there is but one blood-island and this is intraembryonic, while the 'yolk islands' are mere accumulated fragmentations or accumulated descendants of the one and only true blood-island — namely, the intermediate cell-mass.

The point which we wish to establish in this connection is the fact that islands of blood on the yolk-sac are not restricted, as Stockard maintained, to the region ventral to the tail; this point may or may not be of great significance with reference to the interpretation of normal ontogeny.

On page 216, Stockard states: **The vascular endothelium never gives rise to blood-cells. So that heart, aorta and vessels of the anterior end of the body, although invariably lined with

VASCULARIZATION OF BODY OF TELEOSTS 83

endothelium, do not contain a single corpuscle in embryos of any age/'

As we shall later demonstrate, embryos which have always been devoid of complete circulation may have red corpuscles in isolated and blindly-ending venous anlagen in the anterior end of the embryo.

If it be admitted that heart pulsations in the absence of complete circulation are sufficient to displace blood corpuscles from their locus of origin, or if we admit that erythroblasts possess properties of active migration — as one might be compelled to assume under the embarrassment of certain difficulties, or observe in the living condition — then we must admit that the emptiness of cardiac and aortic vessels has little significance so far as the potentiality of their endothelium is concerned, and we are again left without positive clue to the possible origin of blood-cells. Since an open endocardium may merge posteriorly into a loose and vacuolated mesenchyme, into which the aortic lumen may also open anteriorly, it is not at all improbable that the hydrostatic pressure of the beating heart through the body-fluid may be sufficient to remove blood-cells from their locus of origin to another region — an occurrence which may occasion confused interpretations somewhat analogous to the ancient conception that the arteries were air tubes. Even body movements and gravity are factors which may, and probably do lead to cell displacement. Such possibilities are at least worthy of consi:!eration.

If to the conditions to be found in hybrid embryos there may be attached any great significance, Stockard's generalizations will not universally hold good for vertebrate ontogeny, or at least for teleost development. There is good reason to believe that they will not even hold good for chemically treated homozygous embryos. In Fimdulus embryos obtained from cyanide and butyric acid treatment, the hearts may (rarely it is true) be so crowded with erythrocytes that they can scarcely pulsate even though there has been no completely established circulation. Occasionally, a large red blood-island may be observed in the head region, the erythrocytes of which seem certainly to have had a local origin. At least such blood-islands develop in some

84 FRANKLIN P. REAGAN AND J. M. THORINGTON

cases in the complete absence of a circulation. In amorphous masses of tissue which sometimes result from chemical treatment or from hybridization, blood-islands develop promiscuously and apparently without regard to organization, if such there be.

Less than 10 per cent of the hybrids resulting from this cross, (Scomber scombrus cf X Fundulus heterocHtus 9), developed complete circulation. All imaginable gradations from short lived masses of embryonic tissue to hatching embryos were obtained. Embryos which hatched did so at the age of twentyfive days.

A review of certain observations on normal teleost development may serv^e as a point of departure for the interpretation of certain conditions observed in hybrid development. Neglecting certain differences of opinion among these writers, it may be said that according to Oellacher,^ Swaen,^ Brachet,^® Mollier,^^ and others, the vessels of the greater axial portion of non-pelagic teleosts have their inception in or in close relation to a longitudinal column of mesodermal cells situated between chorda and gut. This vascular anlage has a double origin through the coalescence of two more or less massive colunmar cell aggregates derived from the median somitic surfaces. In the region of the first few (first five, perhaps) somites, the entire* Strang, 'smaller in diameter than in the posterior region, becomes transformed into dorsal aorta. In the remaining region posterior, characterized by the presence of the pronephric system, the prevascular column has a much greater diameter. Differentiated from its dorsal surface (Aortenstrang), or at least in close relation to that surface, there arises the posterior continuation of the dorsal aorta. The greater ventral portion of the column in this posterior region (Venenstrang) develops into erythrocytes. On the periphery of the Venenstrang, the endothelium of the postcard inal veins becomes differentiated. This peripheral endothelium may form a ramifying plexus which imbibes the erythrocytes (MoUier), or the postcardinal endotheUum may arise simply by the flattening of

» 9 10 11 References to the works of these observers will be found in Hertwig*8 Handbuch, erster Band, erster Teil, zweite Halfte, p. 1125 and following.

VASCULARIZATION OF BODY OF TELEOSTS 85

the peripheral cells of the column while the entu-e central mass differentiates into erythrocytes.

Felix'2 (p. 353) describes at length the schicksale des Venenstrang .... die Umwandlung seiner central gelegenen Zellen zu Blutkorperchen, seiner peripher gelegenen Zellen zu Gefassepithelzellen

That there is little fundamental difference between the endotheUum of the arterial and venous elements is indicated by the fact that they arise in connection with a common cell-strand and possess a common lumen at the anterior terminus of the cardinals, the latter vessels emptying into the aorta (MolUer). In hybrid embryos, furthermore, the entire 'Strang' may, and generally does, become surrounded by one endothelial tube, which sometimes by reason of its position and anterior connections appears to be the true dorsal aorta. In such cases, the postcardinals may fail to appear, or as such be represented by small, weakly developed independent vessels situated ventro-laterally which would seem to have arisen out of relation to the central'Strang.' The proof of this assumption is difficult at present but it seems to be the most logical explanation of the conditions observed (fig. 15). In cases in which the 'Strang' becomes surrounded by one tubular endotheUum besides which no separate aortic endothelium can be demonstrated, it might be possible to assume that the entire endothelium is venous and that the aorta had failed to form. Such a circumstance seems improbable from what we know of the normal differentiation of aorta and cardinals. A third possibiUty would be that the endothelium is mixed venous and arterial, whether it be regarded as differentiated from the 'Strang' itself or from the adjacent mesenchyme. A fourth and most remote possibiUty might be that the endotheUum figured by us is not real endotheUum; there is nothing to favor such a supposition.

The vessel which we have designated as aorta may in some cases be that which Stockard regarded as stem-vein of the conjoined postcardinals. In no case have we found an extensive 'stem " Felix, H. von W. Anat. Hefte, vol. 8, Heft 25, p. 346.

THE ANATOMICAL RECORD, VOL. 10, NO- 2

86 FRANKLIN P. REAGAN AND J. M. THORINGTON

vein' possessed of haemophoric properties coexisting with an aortic endothelium. On page 126, Stockard states that the blood forming portion of the 'stem-vein' is *' behind the anterior portion of the kidney, extending into the t il/' The derivative of the Gefassstrang anterior to the kidney would, according to Stockard, probably be interpreted as venous, or at least partly as venous. According to Swaen and Erachet, the most anterior portion of the vascular anlage is exclusively aortic; it is for this reason that we have designated the vascular cavity in question as aortic, reserving the right to suggest to the reader that he substitute the words 'stem- vein' for the word aorta, wherever we have made use of the latter term in descriptions of figures 2 to 13, providing he should feel more strongly inclined toward that interpretation. The point is, we have here an independently differentiated endothelial-lined vessel. We have not made a statement that the dorsal aorta is haemophoric. Certain it is, however, that in the normal ontogeny the venous derivative is predominantly haemophoric.

That we are here dealing with a true aorta is indicated by the fact that the endothelial surrounding the posterior portion of the 'Strang' may be a mere continuation of that surrounding the most anterior portion; the latter portion, in the light of normal development, may well be interpreted as aortic, even though it may in some cases contain erythrocytes. In addition to this great similarity of the 'aorta' of such hybrids to that of others more normally developed in which empty cardinal veins have also developed, apparently at a later time, renders a priori reasonable the assumption that we are here dealing with a true aorta. Be this as it may, we have taken liberty to designate this unusual vessel as aorta. Whatever may be its real nature, it has a local origin independent of all other vascular endothelium. This we regard as the central fr ct of the present work.

It now remains to consider the actual conditions observed in living embryos, whole-amounts and sections.

Figure 1 represents conditions typical for ten-day hybrids which have never had a circulation. The abnormal distention of the plasma-filled pericardium has stretched the heart out into

VASCULARIZATION OF BODY OF TELEOSTS 87

a slender tube, closed at either end. Numerous embryos of this sort were obtained. 1 he heart would pulsate and churn its fluid contents up and down. Contractions would sometimes pull the head downward with each pulsation. The reader is referred to Newman's recent work for adequate descriptions of living conditions in such embryos.

Figures 2, 3, and 4 are from cross-sections of a twelve-day hybrid which developed without circulation. The pericardium was distended with a plasma-like fluid so that the heart was stretched out into a long narrow tube between its 'point of origin' on the yolk and its 'point of insertion' on the ventral surface of the embryonic body. The endocardium is seen to be a solid cord of cells. In this section (fig. 2), there is a crescentshaped lighter area which, from its frequency in sections of such embryos, would seem to be a thinning or vacuolation of the mesenchyme preparatory to a linking of the endocardial tube with the dorsal aortic anlage present considerably farther back in the embryonic body (fig. 4, Endth). Eeading back in the series (fig. 3), it will be found that all signs of vascular tissue are absent. The notochord is in close proximity to the gut, so that very little mesenchyme can be found between them. Tracing still further (fig. 4), one encounters an independent and completely isolated vascular anlage in the periphery of which the cells have begun to flatten out to form endothelium. The central cells have acquired an oval or rounded contour; these cells still remain in a more or less syncytial relationship, or at least connected by protoplasmic bridges. They are to be regarded as intermediate in form between indifi'erent mesoderm and diff"erentiated erythroblasts.

Figures 5, 6, and 7 are cross-sections through the body of a ten-day hybrid. Although this embryo is slightly younger than the one just described, its difi*erentiations have proceeded mdre rapidly. This is not at all surprising when one considers the fact that the rate of differentiation varies with the degree of normality, which in turn, according to Newman, varies with the extent to which the hybrid displays maternal or paternal characteristics. The heart of this embryo was observed in the liv 88 FRANKLIN P. REAGAN AND J. M. THORINGTON


VASCULARIZATION OF BODY OF TELEOSTS 89

ing condition to be greatly stretched, solid in its distal extremity but hollow near its attachment to the embryonic body. No circulation was ever established. Figure 5 shows a section through the anterior end of this embryo. The heart (Ht.) lies ventral to the head tissue. As the section passes through the more distal portion of the heart, the latter appears as a solid cord of cells. Figure 6 is taken more posteriorly, the plane of section passing through the region where the heart joins the body — that is, through the aortic end. There is a rather indistinct endothelial lining in which may be seen rounded cells which may be transforming into erythroblasts. Tracing posteriorly a few sections as in the preceding figure, one finds (fig. 7) that the endocardium of the heart has ended and no vascular endothelium can be found in the section. The anterior end of the aortic anlage is first encountered about 275m back of the posterior limit of cardiac endothelium. Figure 8 is a section through the anterior end of the aortic endothelium which is quite distinct, bounding a dorso-ventrally compressed slit-like cavity. Traced anteriorly, this endothelium loses itself in the indifferent mesenchyme. Somites are not differentiated in this embryo, so that besides the somewhat abnormal Wolffian ducts there are no landmarks by which the boundary between piu*e arterial and mixed arterial

EXPLANATION OF REFERENCE LETTERS

Ht., Heart Endth., Endothelium

Y.S., Yolk-sac In.C.M., Intermediate cell-mass

Nek., Notochord Pr,C., Pre-cardinal vein

My.C, Myocardium. Erth., Erythrocytes

End'.C.f Endocardium. AorL, Aorta

N.T.f Neural tube Pec, Pericardium

W.D., Wolffian duct D.Cv., Duct of Cuvier

F.G., Fore-gut Mch.S., Mesenchymatous space

G.y Gut V.Aort., Ventral aorta

Fig. 1 Twelve day hybrid. Scomber scombrus cf X Fundulus heteroclitus 9 (cleared in xylol).

Figs. 2, 3 and 4 Sections through the body axis of a twelve day hybrid (same cross); 2, through the heart (X 180); 3, through the anterior end of the fore-gut (X 180); 4, through the trunk region (X 350).

90 FRANKLIN P. REAGAN AND J. M. THORINGTON ^^•^^-*--Ht.


7

Figs. 5 to 12 Sections through the body axis of a ten day hybrid (same cross) ; 5, through the distal portion of the heart (X 350); 6, through the proximal portion of the heart (X 350) ; 7, through the hind brain (X 350) ; 8, 9, 10, through the body axis anterior to the pronephros (X 350) ; 11, detail of ventral portion of figure 10 (X 700); 12, through the body axis in the region of the pronephros (X 400).

VASCULARIZATION OF BODY OF TBLEOSTS 91


8


9


10

92 FRANKLIN P. REAGAN AND J. M. THORINGTON


11


12


and venous portions of the intermediate cell-mass can be definitely located. From the fact that the pronephric tissues is situated a great distance posterior to the plane of this section, we may feel assured that we are here dealing with 'Aortenstrang' somewhere in the region of the most anterior potential somites.^' Tracing more posteriorly but still anterior to the pronephrogenous region, one finds the same slit-like cavity (fig. 9) situated

"The reader is requested to compare our figures 8 and 9 with figures 809 and 810 in Hertwig's Handbuch. Erster Band, erster Teil, zweite Halfte.

VASCULARIZATION OF BODY OF TELEOSTS 93

dorsal to a rather darkly staining mass of cells, bounded ventrally by a deeply staining membrane which is continuous with the lateral extremities of the aortic endothelium. This mass of cells might be interpreted as cells of the Venenstrang if the section were not so far anterior to the pronephric region. Tracing still more posteriorly (figs. 10 and 11), one finds continuous with the slit-like aorta, a large vascular lumen containing a few erythrocytes and traces of Strang-cells evidently being separated from the mesodermal column. Their nuclei are large and rounded; their cytoplasm is Ughtly staining. The nuclei are in a syncytial relationship. The cells are separated from the extra-vascular mesenchyme by a distinct endothelium which is less perfect adjacent to these cells than elsewhere. As before stated, this region is anterior to the pronephric anlage whether the section is through the 'Aortenstrang' alone, or through the anterio-venous portion of the intermediate cell-mass. Figure 12 is a section through the region of the pronephric ducts, a region which should normally contain both arterial and venous endothelium. There is, however, but one vascular cavity (which we designate arbitrarily as aorta) containing free rounded blood cells. Posteriorly, the erythrocytes and the endothelium merge gradually into indifferent mesenchyme where they lose their identities.

Unusually interesting conditions were exhibited by another ten-day hybrid from which figures 13, 14, and 15 are taken. The known history of this embryo lends interest to the conditions which its sections disclose. The embryo never developed a complete circulation, although the heart was almost normal in shape and the pericardium appeared but slightly distended. On the fifth day, the heart was pulsating slowly but rhythmically; its proximal (arterial) portion appeared, so far as could be observed in the living condition, to be an open tube possessed of a continuous lumen. The distal end of the heart appeared to end blindly. At the region of its 'origin' in the yolk-sac, just as the limit of the pericardial cavity, the cardiac anlage was perfectly solid. There could be detected no entrance of cellular elements into the heart, either by active or by passive means. Weak

End.O My.C


13


14

Fig. 13 Cross section through the body axis of a ten day hybrid (same cross), the plane of section passing through the distal extremity of the heart, showing vacant aorta but blood-containing heart and pre-cardinal (X 200).

Fig. 14 Detail of region of containing pre-cardinal and aorta from the right side of figure 13. Note th' emptiness of the aorta and the presence of erythrocytes in the precardinal (X S.50).

04

/ASCULARIZATION OF BODY OF TELEOSTS 95

pulsations continued in this manner, gradually becoming weaker perhaps, until the embryo was killed. The heart itself gradually became more distended and a well defined left-lateral flexure developed. Late on the eighth day, its contents began to display an orange hue. When the embryo was killed and sectioned on the tenth day, it was found that the heart contained numerous erythrocytes. The ventral (venous) end of the heart ended blindly at the pericardial limit, seeming never to have had ventral connection with a vascular cavity or continuous vessel. The endothelium of the heart faded gradually into a loose and vacuolated mesenchyme. The cardiac endothelium did not communicate with aortic endothelium, but the latter merged into the same vacuolated mesenchyme in which the posterior portion of the cardiac endothelium also lost itself. The two defined vascular cavities were well separated. The dorsal aorta was found to be completely devoid of blood cells. Posteriorly the aorta (fig. 15) communicates directly with the postcardinal vein which contains blood cells. Figure 13 shows the precardinals to be haemophoric, as had been previously inferred from observations on the living embryo. The ducts of Cuvier are likewise haemophoric. A reconstruction of this embryo (fig. 15) shows the precardinals terminating blindly in the anterior region. Traced back, they are found to course laterally and discontinuously as the ducts of Cuvier (D. Cv.) and to end blindly on the yolk-sac. In this embryo it is quite conceivable that the action of heart pulsation on the body fluids may have been communicated through the loose mesenchyme to the aortic cavity. That there was no complete circulation of cellular elements is indicated by the fact that the blood cells never left the cardiac tube to enter the aorta. Cardiac and aortic cavities never communicated but were separated by a loose mesenchyme. Also the heart had a blind ventral (venous) ending.

This embryo illustrates very well the fact that the aorta may be very well developed when postcardinals are practically absent. Thus it is seen that the interpretation of endothelial cavities in these embryos is often exceedingly difficult. For this reason we

96


FRANKLIN P. REAGAN AND J. M. THORINGTON 0) o

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> '^ eD

VASCULARIZATION OF BODY OF TELEOSTS 97

do not insist upon our interpretation of the ^ aorta' in figures 2 to 13.

It is difficult to formulate a possibility by which the erythrocytes contained in the precardinals of this embryo could have reached their present status unless these vessels are haemophoric. The action of the body fluids seems entirely inadequate to account for this phenomenon. In this and in many other instances, the ducts of Cuvier seem surely to be haemophoric channels; sometimes they are preceded by a densely staining 'Strang' of cells resembling the postcardinal 'Venenstrang.'^*

It might be added that the sub-intestinal veins may in some cases contain erythrocytes, which means either that these vessels are haemophoric^^ or that their contained cells have been washed in from the dorsal aorta. Lacunae of erythrocytes develop in various locations in the head region. Since erythrocytes may develop in practically any region of the body, it is not improbable that continued search may disclose endothelial cavities containing both leucocytes and erythrocytes, provided that true leucocytes are ever developed in embryos which have never possessed a circulation.

As before stated, the primary purpose of the present work was to determine whether hybrid material would furnish another basis of proof of the local origin theory of endotheUum. When we began the work, nothing was farther from our purpose than to trespass in the field of haematology. If our results do nothing more than stimulate a more minute and exacting study of the possibilities of error in the interpretation of conditions in embryos devoid of complete circulation, they would seem to be worth the time which has been expended on them.

A study of the 'Gefassstrang'of the hybrid teleost convinces us that aortic and postcardinal endothelium stands in no direct cell-lineage with that of the yolk-sac.

" This condition is quite striking in crosses between F. heteroclitus cf and F. majalis 9.

" The term haemophoric as employed by us denotes the condition of a vessel when it contains red blood cells which were not secondarily and passively carried there by body-fluid in motion.

98 FRANKLIN P. REAGAN AND J. M. THORINGTON

In conclusion, we wish to thank Professor C. F. W. McClure for his kind interest in our work and for many valuable suggestions. Acknowledgment of indebtedness is also due Professor P. H. Mitchell, Director of the U. S. Bureau of Fisheries, Woods Hole, Massachusetts, for numerous courtesies extended to us in his laboratories.

A FURTHER STUDY OF THE ORIGIN OF BLOOD VASCULAR TISSUES IN CHEMICALLY TREATED TELEOST EMBRYOS, WITH ESPECIAL REFERENCE TO HAEMATOPOESIS IN THE ANTERIOR MESENCHYME AND IN THE HEART

FRANKLIN P. REAGAN Department of Comparative Anatomy Princeton University

TWENTY-SIX FIGURES

As first shown by Loeb,* certain chemicals are capable of inhibiting, arresting, or preventing complete circulation in embryos of Fundulus heteroclitus. The same phenomena have since been recorded by Stockard^ and Werber.^ Stockard's highly interesting study is apparently based on the following postulate: In embryos '4n which the blood fails to circulate on account of the fact that the heart is either blind at one end or both, or fails to connect with the veins (p. 124) it is safe to conclude that any place in which blood-cells are found may be considered their locus of origin, even though in some cases the '* embryo develops in a fashion closely approximating the normal when the heart beat is fairly strong. Incidental to this main postulate his work relies on the proposition that tissues devoid of blood-cells under such conditions are proved to be, and always to have been, devoid of haematopoetic potentiality. Thus the demonstration of endothelial-lined cavities which contain no blood-cells demonstrates likewise the inability of all endothelium to produce blood-cells. The absence of blood-cells in the liver disposes of that organ as a blood producer in the bony fishes (p. 125). Another highly interesting observ^ation of Stockard's

' Loeb, J. Pop. Sci. Mo., vol. 80, p. 5.

- Stockard, C. R. Proc. Am. Assn. Anatomists, Anat. Rec, vol. 9, no. 1.

MVerber, E. I. Anat. Rcc, vol. 9, no. 7.

09

100 FRANKLIN P. REAGAN

(and very important if it is correct) is that the erythrocytes in Fundulus embryos have .... two distinct and limited places of origin, first in the stem vein or conjoined cardinal veins, and second from the blood islands of the yolk sac; the latter, according to Stockard, are '* always on the posterior and ventral yolk surface and do not extend over the anterior surface. Also in these embryos lacking circulation owing to the circumstances above outlined by Stockard the heart, aorta, and vessels of the anterior end of the body, although invariably lined with endothelium, do not contain a single corpuscle in embryos of any age."

There are, however, possibilities which should be thoroughly considered. Certain it is that heart pulsation in the absence of complete circulation may profoundly change the original and true picture of the actual genetic processes. At present I shall enter into this question no further than to state that I have observed in the living chemically treated embryo the actual passage of a fully differentiated erythrocyte from the intermediate cellmass to a large and bright red blood-lacuna on the yolk-sac ventral to the tail. I could see no evidence of active amoeboid movement but could observe the oscillation of such cells in the plasma-like fluid which contained them. Such oscillations followed the rhythm of heart pulsation. The heart was open at its arterial end, closed at its venous end so that circulation was incomplete. Concerning the possibility of the active migration of late erythroblasts I can say little from direct observation except that I have never yet been able to detect any such active movement.

I wish at present to consider certain groups of erythrocytes to be observed in a number of cases in the anterior end of living, chemically treated embryos which have always been devoid of circulation. These groups of blood cells are not very large or conspicuously red except in rare cases. They are, I believe, of sufficiently constant occurrence to be of significance for the normal development. Embryos which develop relatively normal eyes and fore-brains sometimes have a tendency to exhibit local and often bi-laterally symmetrical patches or erythrocytes at

BLOOD VASCULAR TISSUES — TELEOST EMBRYOS 101

an early state of their ontogeny, in the region of the eyes. The greater number of such cell groups which I have observed are located dorsal to and just median to the eyes. Often they may be anterior, posterior, ventral to, or even completely circumscribing the eye, or occupying a space in the optic cup. The time during which they display a content of haemoglobin is usually very short under the most favorable conditions of oxidation. I have quite often had the experience of studying such a cell group, setting the embryo aside for a few hours and reexamining it again only to find that the red color had entirely disappeared. Occasionally I have observed such a patch of cells on one side of the head dorsal and medial to the eye on that side. After sketching the position of this haematopoetic region I have re-examined such embryos a few hours later, sometimes to find that a similar group of cells had made its appearance on the opposite side and synmietrical with that previously studied and sketched, the group first studied having lost its red color. During such a study all possible efforts were made to detect the active or passive movements of such red colored cells, but careful examination gave no evidence of any sort of movement.

The region between the otic vesicles and also the middle anterior portion of the snout displayed such temporary patches. In still rarer cases groups of blood-cells could, at a later stage of ontogeny, be observed in the mandible and occasionally in practically any position in the mesenchyme of the anterior end of the embryo. Blood was sometimes, though rarely, observed in hearts having solid cord-like venous and arterial extremities, and dilations in their middle portions containing bright red erythrocytes. In some, instances there was a series of such dilations (fig. 15).

It is interesting to note that in certain dishes of experimental embryos and at a very definite age, with conditions under which I made my study, many embryos which had been subjected to the same sort of treatment would exhibit a large number of peculiarities in conmion. It is not yet safe to say that definite substances produce specific results. The tendency is, I believe, for writers on the subject to lump all peculiarities and abnor THE ANATOMICAL RECORD, VOL. 10. NO. 2

102 FRANKLIN P. REAGAN

malities into the category of developmental arrest. It is difficult for me to believe, when a given egg is subjected to the action of a protein-precipitant (such as alcohol), or to the action of a fatsolvent (such as butyric acid) that the resulting embryo would be afflicted in precisely the same manner as if, instead, it had at the same time been subjected to the lecithin-precipitant acetone. The relatively great variability of different eggs and the relatively great difference in the effects which a given substance has at different stages of ontogeny are generally sufficient to overshadow whatever specific action a chemical may produce.

It will be sufficient for the present work to figure certain of the conditions found in chemically treated embryos. Let us first consider some of the cases of haematopoesis in the mesenchyme of the anterior end of the embryo. Figures 1 to 10 show sketches of the heads of chemically treated living embryos in which erythrocytes have differentiated locally. In none of these embryos was there a blood circulation. In most cases the hearts were solid, both at their venous and arterial ends, and in some cases solid throughout. The stippled areas represent aggregates of erythrocytes. In all cases in which the embryo was killed at the time when these haematopoetic areas displayed a red color, the blood-cells would stain brightly red with eosin in my modification of Mann's methyl blue-eosin stain.* In many cases the area thus observed was not quite coextensive with the strongly eosinophilous and haematopoetic region revealed by the stained sections. I interpret this to mean that the developing bloodcells may contain sufficient haemoglobin to stain differentially without having a sufficient amount of that pigment to be discernible in the living embryo. I have never yet found the eosinophilous area to be smaller than the visible red area of the living tissue.

The method of chemical treatment of each embryo here described is given in connection with the explanation of figures. The first eight figures represent dorsal views. Figures 9 and 10 are lateral views showing especially well the distended peri

Reagan, F. P., Anat. Rec, vol. 8, no. 7. BLOOD VASCULAR TISSUES — TELEOST EMBRYOS 103

cardiiun and string-line hearts. In the embryo represented in figure 1, blood lacunae were observed in the living conditions dorso-medial to the eyes, dorso-posterior to the eye, and a single unpaired median dorsal lacuna was found just beneath the ectoderm in the repon sUghtly anterior to the otic vesicles. The position and nature of these lacunae is shown in section in figlu^es 19, 20, 21, and 22. All these lacunae displaying a bright red color in the living condition were found correspondingly located in the embryo when it was subsequently sectioned and stained. Certain haematopoetic areas could also be found in section which were not visible in the Uving tissue. The heart of this embryo was, from the beginning, solid at both extremities.

In figure 2 there are seen to be two lacunae, medial and dorsal to the optic cups. The red color disappeared from these regions within four hours after they were discovered. Figures 3 and 4 are sketches of the dorsal cephaUc surface of another embryo, the sketch for figure 4 having been made at a stage 14 hours older than that of figure 3. The embryo from which these sketches were made has the following history. At its four-cell stage on July 18 it was placed in a solution of 50 cc. sea-water containing 10 cc. of f2 molecular butyric acid, where it remained for 24 hours. At 10 a.m. July 27, the red lacuna over the left eye was observed and sketched. At 2 a.m. the following morning a red lacuna was observed over the right eye while that over the left eye had lost its color. The embryo was subsequently sectioned. Eosinophilous areas surrounded by endotheUiun and corresponding respectively to these lacunae were observed in section, their staining capacities being about equal. Figures 5 to 9 show a variety of positions in which similar lacunae were found in various embryos and later verified by sections. In some sets of experiments over 10 per cent of the embryos in a given dish might at a given time display lacimae comparable to these. Unfortunately my attention became attracted to these particular phenomena rather late in the spawning season of Fundulus, so that I am at present imable to offer statistics on their constancy. I am led to believe that if one could observe such experiments in 104 FRANKLIN P. REAGAN

cessantly, the embryos found to exhibit these conditions would not compose a small minority.

Figure 10 shows a side view of a rather remarkable embryo. At its four-cell stage it was treated for 24 hours with a mixture of equal parts of 4 per cent alcohol (sea-water solution) and a solution of potassium cyanide made from 2 cc. of i J o per cent KCN in 50 cc. sea-water. The embryo developed without blood circulation. On the twelfth day it exhibited haematopoetic areas conveniently viewed through the translucent otocysts. Within the next three days a single median haematopoetic area was observed in the mandible. These conditions continued for some time; on the twenty-fifth day the sketch for figure 10 was made. Blood cells were then observed scattered over the floor of the pericardimn. Within the next five days the red color had practically disappeared from the anterior haematopoetic areas. The embryo was allowed to Uve in running sea-water until it was forty-three days old; when it was sectioned there were found columnar longitudinal cell-aggregates medial and ventral to each otic vesicle undoubtedly representing precardinal lines. They could be traced discontinuously back to their junction with the postcardinals. The central cells of these columns were very weakly eosinophilous, staining reddish purple rather than bright red as do normal erythrocytes. The surrounding mesenchyme took the usual blue color. The cell coliunn in the mandible, the scattered blood-cells in the floor of the pericardium and those of the posterior axial region stained similarly to those of the precardinal line. Descriptions of sections of this embryo are reserved for a later work.

The conditions in figures 11 and 12 are of interest in that they demonstrate the fact that blood cells may develop on any part of the yolk-surface under conditions in which heart pulsation could not have been a factor in determining their location. In both these embryos the heart has failed to develop. Figure 11 shows an embryo where a small portion of the body axis abuts upon a much dilated pericardium in which no heart is to be found. On the floor of the pericardium near the junction of pericardium and yolk-sac will be seen numerous scattered erythrocytes.

BLOOD VASCULAR TISSUES — TELEOST EMBRYOS 105

Some have even developed on the inner surface of the pericardium. It is preeminently (though I should not say universally) characteristic of such embryos that the blood cells of the yolksac develop in this scattered manner when the heart fails to develop.

In figure 12 we have a condition in which the embryonic body is entirely lacking. The entire yolk-siuface is covered with blood cells more or less scattered, but in some regions rather densely aggregated. In the center of the region towards the observer, the blood cell mass is enclosed by endothelimn. In some instances in which the embryonic body fails to differentiate, these endothelial haemophoric cavities are relatively numerous and may even join to form intricate plexuses.

The embryo upon which figures 13 and 14 are based is of extraordinary interest. At the four-cell stage this embryo was treated for 20 hours with 3.5 per cent alcohol. On the sixth day there was noticed an elongated blood lacuna on the antero-ventral yolk-surface, very conspicuous for its fiery red color. No other erythrocytes could be found in the yolk-sac or body axis. The heart seemed to be closed or obstructed at the venous end but was otherwise apparently normal. There was no circulation though the heart was pulsating. On the tenth day a decided tremor could be observed in this 'lacuna' as a wave of contraction starting from the end nearer the venous end of the heart and passing to the opposite end. There had also developed a slight flexure, the convexity of which was dorsally directed (fig. 13). At this time the churning movement of the heart could be seen to loosen the closely packed cells in the intermediate cell mass which had begun to acquire a faint orange color. On the fifteenth day I was able to follow certain of these dislocated and roimded erythroblasts as they were dispersed to their later position on the yolk-surface, where they were accumulating in great numbers and apparently multiplying rapidly. I am convinced that those 'blood-islands' so often observed on the yolk-sac ventral to the tail are occasionally of this sort of formation. At a stage earlier than either here described certain observations (which I shall consider at another time in greater detail) were

106 FRANKLIN P. REAGAN

made on the formation of the endotheliimi on the yolk-sac. Single mesenchymal cells were observed in active migration. They would aUgn themselves into short solid or hollow cylindrical aggregates of isolated endothelimn, many of which joined each other. At the age of ten days, this activated 'lacuna/ accessory heart, or whatever it may have been, was pulsating feebly. On the nineteenth day (fig. 14) it was pulsating seventy times per minute, while the real heart, seemingly normal in shape was pulsating one hundred times per minute. The yolk-sac vessels at this stage were still discontinuous. So long as this embryo was observed, no vessels connected with, or developed immediately adjacent to the accessory blood-filled heart. In later stages the accessory heart gradually increased its pulsation rate when on the twenty-fifth day its rate was 136 pulsations per minute, whereas the actual heart-pulsation went at a rate of 126 times per minute. At this time the vitelline circulation was established. The embryo was killed on the twenty-sixth day when the accessory heart had begun to slow down considerably. I have no explanation to offer for this extraordinary state of affairs. Especially puzzling is the direction of pulsation in the 'accessory heart.' I was unusually fortunate in being able to do my work in a laboratory where I could avail myself of the counsel of many observers — the Marine Biological Laboratory, Woods Hole, Massachusetts. It was with reference to this embryo that I consulted many biologists. At the time when the sketch for figure 14 was made, the embryo was esxamined by Profs. E. G. Conklin, H. McE. Knower, E. I. Werber, and E. V. Cowdry. At a somewhat later time, when the vitelline circulation had been established, the embryo was examined by Prof. C. R. Stockard and others. I regret very much to be unable to figure sections of this interesting embryo owing to the fact that it became in some way mislaid.

Figures 16, 17 and 18 represent cross sections of a nine-day embryo, which, at the four-cell stage, had been treated with butyric acid. It will be seen that certain mesenchyme cells have been transformed into erythrocytes. This is especially striking in the optic cup (at least the invaginated portion of the brain

BLOOD VASCULAR TISSUES — TELEOST EMBRYOS 107

tissue on the reader's left) in figures 16 and 17. Likewise there is a region of haematopoesis dorsal to the optic tissue as shown on the right side of figure 17. These erythrocyte lacunae are completely isolated, having absolutely no connection with the conjoined cardinals. They were observed in the Uving embryo as red patches of blood tissue. Anteriorly the heart is solid (fig. 17); more posteriorly it possesses a small lumen (fig. 16). The heart again becomes solid at its arterial 'insertion' in the embryonic body.

In figure 18 the synmietrically situated precardinals are conspicuously haemophoric. The cells in these vessels were observed in the living condition to possess a content of haemoglobin; nevertheless the cells are so closely packed that their contours are not rounded but present plane surfaces. I have often observed conditions similar to this — that is, a haemoglobin content in cells not yet typically erythrocyte in shape, owing to their closely crowded condition. These erythroblasts are surrounded by endothelimn.

This embryo is, I think, of very great importance. Owing to the curious eye-formation it attracted my attention more than most other embryos which I studied. I feel quite certain that the erythrocytes in the anterior end of this embryo were not carried there by any sort of circulation or movement of body-fluids. I kept close observation on the yolk-sac and in' observing the living condition never found a single moving or stationary erythrocyte or any formation of endothelimn in the anterior hemisphere of the yolk-sac. Figure 16 shows a section in which the yolk-sac is included. In this section there is not a sign or trace of vascular tissue. The same is true of all sections of the yolk-sac anterior to this, and of many which are posterior. In this embryo the solid venous end of the heart ends blindly on the yolk-sac, the anterior portion of which is, and always has been, devoid of any sort of vascular tissue, endothelial or corpuscular, yet the anterior vessels do contain erythrocytes and the anterior rmsenchyme is haematopoetic.

It may be profitable to divert our attention long enough to consider the eye abnormalities in this embryo. On the left side

108 FRANKLIN P. REAGAN

of figure 16 there is what I would interpret as an optic cup with more or less distinct choroid and retina. The space normally occupied by a lens seems to have been filled with haematopoetic mesenchyme which gave rise to a blood lacuna that is more (fig. 16) or less (fig. 17) completely surrounded by endothelimn. It was this lacuna which induced me to continue unusually careful study of the living embryo. The plane of section in figure 16 is almost tangential to the more dorsal lens of the opposite side. On the right side of figure 17 it will be seen that there are two optic cups and two lenses, each of the latter appearing to be normal in size. It is not my place to advance an explanation of this ophthalmic dilemma; I merely present the facts as of possible importance to those engaged in the highly interesting controversy over the explanation of cyclopia. If, however, both eyes originate and diverge from a median anlage, and if cyclopia is an expression of a weakening of this anlage by toxic substances, as Stockard maintains, the conditions here observed could, so far as I can see, be explained only by assuming that the anlage, instead of becoming stupefied or stunted, had been stimulated to an unusually great activity, rivaled only by the conditions in Werber's figure 8 (loc. cit).

In figures 19 and 20 we have sections of certain of the haemopoetic areas sketched grossly in figure 1. Figure 19 is taken from a section just posterior to the optic vesicles and shows the anterior limits of the two areas dorsal and posterior to these optic vesicles. The blood-producing regions are roughly indicated in this figure by the areas containing the large black dots; the two small bloodproducing areas on the right side of figure 19 are shown at a considerably higher magnification in figure 21. The erythrocytes, represented by the cells with un-stippled cytoplasm, are large and rounded. Their large nuclei are very strongly eosinophilous while their peripheral cytoplasm remains almost colorless. Judging from their morphological characterictics and microchemical reaction they are absolutely identical with the undoubted erythrocytes of the postcardinals, of the yolk-sac, and with those of local origin in figiu-e 24. The adjacent mesenchyme cells have very small nuclei and take a bluish purple stain. Certain of these

BLOOD VASCULAR TISSUES — TELEOST EMBRYOS 109

mesenchyme cells seem to be transforming into erythrocytes. Their nuclei have become larger, their staining reaction less basophilic, and their contour is becoming more rounded. Not all of the strongly eosinophilous cells have yet become rounded. Especially in the lower area has the cavity of a potential bloodvessel made its appearance; the mesenchyme cells around this cavity are relatively more crowded than those more distant from the vessel, and some of them have begun slightly to flatten out to form endothelium.

Figiu-e 20 represents roughly the position of the posterior dorsomedian blood-cell group of figure 1. This cell-group is shown in accurate detail in figure 22. The differentiating erythrocytes are quite strongly eosinophilous, but do not have roimded contours by reason of their closely crowded quarters. Their status is similar to that of the erythrocytes in the precardinals in figure 18, and of erythroblasts often seen in the intermediate cell mass.

Let us now consider the possibility of haematopoesis in the heart. As has already been noted, blood-cells may appear in the dilated portion of a spindle-shaped heart which has always been solid at both ends, and erythrocytes were observed to develop locally in the accessory heart of figures 13 and 14. The possible anlagen of such cells might be endotheUal, myocardial, or mesothelial. It is not highly probable that mesenchymal cells have wandered there from the embryonic body (after the heart had formed), much less likely from the distantly located intermediate cell-mass. Whatever may have been their ultimate source, certain it is that blood-cells may be foimd in the hearts of embryos which have never had a blood-circulation, although Stockard has made statements to the contrary. An unusually interesting case is that of an embryo kindly placed at my disposal by Dr. E. I. Werber. Here, instead of one haemophoric dilation or even two as I had sometimes found, there are several, all separated by constricted solid regions. This embryo had never developed a circulation.

I have postponed so far an account of the rather interesting history of the heart of the embryo from which figure 1 is taken. On the fifth day it was observed that the heart of this embryo

110 FRANKLIN P. REAGAN

possessed solid extremities. Prior to that time it was definitely known that there had been no blood-circulation. At this time it was decided that a procedure might be resorted to by means of which one might be assured that no circulation would take place. The very feebly pulsating heart possessed a flexure and was not at all stretched, yet the solidity of its venous end could not be doubted. A very fiine needle was introduced into the pericardium and the mid-portion of the heart was carefully severed. The cut ends then lashed aimlessly about. At the next observation it was foimd that by some happy chance the free ends had overlapped and fused laterally and heart-pulsation had ceased. When the embryo was sectioned the fusion was foimd to be quite intimate in places, in others rather loose, as in figure 23. The rounded portion in the left of figure 23, if traced anteriorly, is found to be the venous portion of the heart. It becomes solid, and contains here and there a few erythrocytes, singly or in small groups. The small rounded portion of the heart on the right side of figure 23, if traced anteriorly for about thirty micra, disappears. If it be traced posteriorly it becomes larger and soUd with intermittent groups of erythrocytes. In the 'venous portion' (left side of figure 23), the outline of the endocardium is evident. Its cells have, however, become cuboidal, and even colunmar. The cavity on the right side of the figure contains, as in figure 19, well differentiated erythrocytes; some of them are still in a process of differentiation and somewhat basophiUc.

Encouraged by the success of this experiment, I performed about fifteen hundred cardiectomies on normal Fundulus embryos, in all cases removing the heart anlage previous to its pulsation or even before a well defined heart could be made out. The percentage of fataUty was high indeed, but a few embryos survived to the age of nine to twelve days. The exceedingly gratifying results of these experiments are reserved for a later publication.

So far I have offered no suggestion as to the source of erythrocytes in hearts containing erythrocytes which were not carried there passively by body-fluids in motion. The conditions to be observed in figures 24, 25, and 26 give a clue as to the possible

BLOOD VASCULAR TISSUES — TELEOST EMBRYOS 111

source of the erythrocytes which originate locally in the heart. The sections are from the embryo of which figure 7 represents the dorsal cephalic surface on the eleventh day of the embryo's history. Not realizing the extraordinary future of this embryo, I did nothing further than to keep it segregated with certain other embryos which had received like chemical treatment and from which all embryos acquiring circulation were removed. I feel sure that no circulation was ever developed in this embryo. As soon as distinct endocardium could be made out, this embryo with several others was further segregated to a dish into which only those embryos were placed whose otherwise normal endocardia were solid at both extremities. On the eleventh day the upper portion of the heart had acquired a faint orange color which gradually deepened until the embryo was killed on the fourteenth day. Not only was the existence of blood-producing areas as seen in figure 7 satisfactorily evinced, but the conditions in the heart were of unusual interest.

Figure 24 represents a section through the heart of this embryo in which a distinct and complete endocardium can still be made out. The endocardial cells have become decidedly cuboidal in outline, as in figure 23; the contained erythrocytes are in an eosinophilous colunm continuous with those eosinophilous cells of figures 25 and 26.

In the right of figure 25 it will be noted that an active haematopoesis has taken place, and that both endocardium and myocardiiun have been involved, whereas the mesothelium surroimding the myocardium has remained unaltered. In figure 26 it will be seen that both the endocardium and myocardimn have in this region become completely transformed into strongly eosinophilous erythroblasts, the crowding of which has prevented the rounding of their contours.

From the foregoing considerations it is evident that red bloodcells can develop in the anterior mesenchyme or in the anterior vessels under conditions in which heart pulsations could not have accounted for their observed position, whether the heart had acted as a force-piunp or as a vacuum-pump. Especially is this

112 FRANKLIN P. REAGAN

true of the embryo from which figures 16, 17 and 18 were made. It is evident also that cardiac tissue can produce blood-cells.

If these foregoing observations are correct, their significance is so apparent as to call for little discussion; with the premises upon which Stockard bases his support of the polyphyletic theory these observations seem incompatible.

I am willing to admit that a very large percentage of the embryos, under certain sorts of chemical treatment,^ may yield conditions which would conform to Stockard's conclusions. But I find also that in order to suppress conclusions diametrically opposed to those of Stockard, I must fail to take account of the exceptions to his sweeping generalizations. The situation is quite simple; exceptions can undoubtedly be foimd. I cannot regard such exceptions as mistakes of nature* and devoid of significance.

I realize, as I did in my previous experimental work on chick blastoderms, that '*it is not always possible to determine the extent to which experimental conditions portray a truly normal process," but I do claim for the experimental method that it possesses true value in breaking down certain dogmatic and preconceived negations which we have been prone to attach to the potentialities of the mesodermal derivatives.

Once we have seriously canvassed the potentialities of a tissue we are then in position to determine, in an unbiased manner, the extent to which the prospective significance of that tissue normally coincides with its potentiality.

ADDENDUM

Since the preparation of the foregoing account, I have been so fortunate as to receive Dr. Stockard's latest publication in the American Journal of Anatomy, vol. 18, no. 2. So elaborate in

In running a series of many thousand embryos for Professor McClure I have never found a case in which an alcoholic embryo displayed this anterior haematopoesis in the living condition. It will be noted that practically all my embryos were treated with butyric acid or acetone; they were reared in runniog sea-water. Stockard has told me that his embryos were reared in a very small amount of sea-water in an environment much warmer than that of my experiments. The hatching-period of my embryos was relatively about two weeks later than those of Stockard's experiments, so that our time-relations, at least, are not comparable.

BLOOD VASCULAR TISSUES — TELEOST EMBRYOS 113

its natiu^e and so minute in its detail, this article seems at first to render rather futile my own work.

"In reply to the extreme monophyletic position/' Stockard (p. 315) asks the following questions: 1) Why are only erythrocytes present in old blood-islands on the yolk of non-circulating specimens? 2) Why is no cellular blood element present in the aorta and other endotheUal lined vessels in the anterior region of similar embryos? 3) Why are the wandering primitive blood-cells unable to form blood in the liver and other positions, while bloodforming power is present to a certain extent in certain regions of the same embryo but from a different anlage?

In reply to this equally extreme polyphyletic position I give the following answers: 1) It is possible to find in the yolk of certain chemically treated embryos disintegrating or at least abnormal cells which bear a striking resemblance to certain cells in the anterior end of the embryonic body which Stockard would probably call white blood cells, but which I consider as abortive attempts at erythrocyte formation and which have, or have had the potentiality of taking on a haemoglobin content under favorable conditions. 2) Embryos entirely devoid of complete circulation may have erythrocytes within or outside their endotheUum in the anterior end of the body. Why such blood cells often fail to enter or become surrounded by endothelium or are generally much less typical, and display such a very transient period of redness as compared with the erythrocytes of the intermediate cell-mass, is difficult to explain. It may be merely an expression of the extreme general susceptibility of the anterior end of the embryo to the action of toxic substances — a susceptibility which Child has ably considered under the term 'axial gradient.'^ Werber saw the application of Child's useful work to the explanation of cyclopia. It is evident that the erythrocyte anlagen share in this sensitivity. From this consideration, the failure of the anterior blood-cells to come to full realization of their potentialities should be an occasion for no greater surprise than the circumstance that the anterior nervous, alimentary, endothe • Child, C. M., Jour. Exp. Zool., vol. 13.

114 FRANKLIN P. REAGAN

lial, or skeletal tissue may be very completely upset while in the posterior region it is perfectly normal (see many of Stockard's figures). In about 100 per cent of the survivors of cardiectomies which I have performed, on normal embryos where the anterior region suffered no specific injury the erythrocytes in this region, so far as I can detect, are in no way morphologically different from those of the posterior region of the embryo, or those of the yolk-sac including those on the anterior portion of the yolk-sac. 3) In reply to this question I wish to place the following counterinterrogation: Why does Stockard admit that '*the only cells within the embryo which resemble lymphocytes and leucocytes in their general structure and staining capacities have been found in the anterior portions of the body and in the head region of young embryos" (p. 280)? What assurance beyond mere inference does Stockard give that these cells (of his figures 45-48) 'resembling leucocytes' are of a roving disposition and finally (p. 284) become scattered throughout the embryo's body? One must admit that he has effected a skillful transition from his limited facts on this point to his general conclusions. Concerning the haematopoetic potentiality of the liver, I shall later have some facts of interest to communicate. I regard the term 'haematopoetic organ' a misnomer as applied to early embryonic development.

Stockard's difl5culty may have arisen from a misinterpretation of the potential, abortive, or abnormal erythroblasts in the anterior end of the embryo. The word erythrocyte means red cell, while the word leucocyte means white (or colorless) cell. When I have been able to locate these cells by their red color in the anterior end of the living embryo which has never had a circulation, and can predict their position in section (a feat to which I am unequal in case of leucocytes) I feel fairly sure of my ground. Such cells correspond exactly in position to Stockard's leucocytes, lymphocytes, and leucocyte-like cells, and may resemble them in abnormal cases in their staining reaction.

I am not sure that any of Stockard's white blood cells are really such. I have never found a cell in these abnormal embryos which I have felt sure could be considered to be a white blood cell. From

BLOOD VASCULAR TISSUES — TELEOST EMBRYOS 115

the point of view of the monophyleticist it would be very desirable for these erythroblast anlagen to transform into leucoblasts. . It is of interest to note Stockard's dual attitude towards the time of appearance of white blood cells. On page 265 their very early appearance fits in nicely with his theoretical considerations, while on page 309 their late appearance in ontogeny is fraught with significance.

I regard as a vulnerable point in Stockard's line of reasoning the fact that he in no way proves that the erythrocytes of the yolk-sac are always descendants of the material of the future intermediate cell-mass but in a vague manner derives them from mesoderm related to the intermediate cell-mass." What assurance does he give us of the specificity of this mesoderm?

When Stockard finds a blood-cell not comforming to his ideals (p. 395) he regards its position as 'exotic' Given such license one can prove anything. Such reasoning from authority could even be rendered disastrous to our experimental 'proof of the local origin of intraembryonic endothelimn.

Stockard argues with perfect justice that the demonstration of diverse types of differentiating blood cells in close physical proximity does not necessarily indicate that the cell which gave rise to one sort of blood-cell could have ever given rise to a different kind of cell. One must admit, however, that the benefit of the doubt here falls to the monophyletic view. It might be argued with equal fervor that the precocious formation of cells of a given type in a definite region does not necessarily prove that the fate of those cells had been sealed ab initio. One must admit, however, that the benefit of the doubt under these circmnstances falls to the polsT^hyletic view (neglecting for the moment the fact that the segregation is by no means so precocious as Stockard's account would indicate). Any such segregated cells might have given rise to a different sort of cell if placed in a different environment at a suflSciently early period of development.

I have previously plead that the development of the bloodvascular system "furnishes an unproductive field for the solution of the problems of preformation and epigenesis." The

116 FRANKLIN P. REAGAN

point which I hope to help prove is that practically all regions are capable of furnishing their own vascular tissues, endothelial and corpuscular. Those regions which do so to an extreme degree are all important but not all-important sources of the vascular tissue. The process is essentially one of transformation of morphologically indifferent mesenchyme cells in loco. That there is a specific cause underlying each transformation follows as a matter of course. I am not at present concerned with the possibility of those mesenchyme cells having migrated from some other position; the present situation is relieved of such perplexing considerations when Stockard goes so far as to say that the heart, anterior vessels, anterior yolk, and liver are regions in which wandering blood anlagen never make themselves manifest; he also speaks of erythrocytes 'originating' on the yolk when it is certain that their ultimate anlagen did not occupy that position. Neither would an authoritative extension of the intermediate cell-mass to include the region of the eyes and snout affect the main question at issue — the question whether the anterior region and certain other regions are necessarily devoid of blood-cells if the embryo has had no circulation.

It is not impossible that the embryo represented in Stockard's figure 9 possessed haemophoric precardinals. A further study of this embryo might point to the possibility that the haemophoric vessel which he has labeled as cardinal vein is really the duct of Cuvier with its anterior continuation, the precardinal. In his figure 13 the haemophoric 'cardinal vein' runs as far anteriorly as the otocyst. One should certainly not expect to find the cardino-Cuverian junction farther cephalad than the posterior limits of the otocysts. It is possible also that erythroblasts not possessed of sufficient haemoglobin to render them visible in the living embryo might be found in sections of these specimens still farther anteriorly. If these inferences be correct it is obvious that Stockard's own material does not bear out his conclusions. Stockard does not give in detail the individual treatment of each embryo but from his account of a typical experiment (p. 234) it would seem that the embryos were, in some cases, left in a fairly strong alcoholic solution until twenty-four hours prior

BLOOD VASCULAR TISSUES — TELEOST EMBRYOS 117

to the time at which they were preserved for study. It does not seem probable that such an environment would be favorable to the elaboration of the complex haemoglobin molecule, which, even when once formed, is very susceptible to the action of alcohol. It is possible that some regions might, under favorable conditions of oxidation, be able to produce erythrocytes even under these circmnstances while other regions might not.

It is interesting to note that in my figure 8 (a condition rarely found) the posterior region seems to have been the part affected by toxic action; this part of the body is diminutive in size whereas the anterior end is relatively normal. Correlated with these conditions we have a complete absence of erythrocytes in the tailregion of this embryo and an abundance of them in discontinuous lines into the ophthalmic region.

Once the main fact of the local origin of vascular tissues has been established, it is perfectly agreeable to me that we speak of 'diffuse anlagen' if the incontrovertible facts are more acceptable under that phraseology. It would be desirable indeed to permit of all conceivable grades and degrees of 'diffuseness' to suit all occasions.

Still more recently I have received Stockard's communication in the American Journal of Anatomyj vol. 18, no. 3. In the main I can confirm his observations on the living yolk-sac and have watched the same process in the tail of the living embryo. My metaphysical deductions differ to a certain extent from those of Stockard as may be seen by comparing his preformistic conclusions with a discussion which I have submitted for publication in the proceedings of the American Association of Anatomists for the current year.

I wish to call attention to the seemingly exotic vitelline vein of Stockard's figures 23 and 24 in his latest publication. Here is shown a vitelline vein undoubtedly comparable to that of his figure 26 which arises in the cephalic region. I strongly suspect that this vitelline vein is a duct of Cuvier. In this connection see p. 116 of my present publication, and Reagan and Thorington, figure 15, in the same number of the Anatomical Record. To anyone confused on this point I recommend a careful study

THE AKATOUICAL RECORD. VOL. 10, KO. 2

118 FRANKLIN P. REAGAN

of Wenckebach's excellent diagrams in Hertwig's Handbuch, Erster Band, Erster Teil, zweite Halfte, p. 1131.

I note with especial satisfaction the contents of Stockard's page 586, American Journal of Anatomy , vol. 18, no. 3, where he states that a loss of circulation has little or no tendency to cause a vessel to collapse or to become obliterated. I have removed the hearts of embryos which possessed a circulation; later I have been greatly surprised to find that the main endothelial tubes had persisted in a remarkable manner as long as the tissue of such embryos remained alive. It is not a rare thing to find embryos which, in addition to having blood in their anterior ends, display the following conditions : ventral and dorsal aorta and aortic arches absent; heart solid; anterior end of the body and anterior yolk devoid of endothelium. From these considerations it seems that Stockard's precautions on pp. 578-9 (ibid) are superfluous if the tissue be well preserved and sectioned.

A major claim of Stockard's latest work is that all the wandering cells on the yolk-sac have the same environment. I regard such a claim as thoroughly unscientific even when it is applied to any two cells in any stage of ontogeny.

PLATES

PLATE 1

EXPLANATION OF FIGURES

1-9 are dorsal views of the cephalic portions of chemically treated embryos. Figures 9 and 10 are lateral views. The stippled areas in these figures indicate the location of erythrocytes as observed in the living embryo.

1, 2, 5, and 7 represent the heads of fourteen-day embryos, which at their four-cell stage were treated for twenty-four hours with a solution of 50 cc. seawater to which had been added 16 cc. of m/12 butyric acid. They are all from the same experiment, one-fourth of the embryos in which yielded similar results. Owing to the evanescence of the red coloring, it is possible that many others may have passed through this critical stage without being observed.

3 and 4 are described with sufficient detail on page 103.

8 and 9 are from embryos obtained from the same experiment as that of figures 3 and 4.

10 is from an embryo, the treatment of which is described on page 104.

11 represents an embryo, only a small portion of the body axis of which developed. No heart formation has taken place. There is a large dilated pericardium in the region of which are many isolated erythrocytes. Embryo from same experiment as that from which the embryo of figures 1, 2, 5, and 7 were obtained. It seems improbable that the 'pericardium' of this embryo should be interpreted as Kupfifer's vesicle. Even so, there are blood cells of local origin on what would then be the anterior yolk, not shown in this figure.

12 represents a case in which the embryonic body has failed to develop, or as such has been transformed into mesodermal and vascular tissues which are scattered entirely over the yolk. The embryo at the four-cell stage was treated for ninety-six hours with a solution of 50 cc. of sea-water to which was added 15 cc. of a molecular solution of acetone.

ABBREVIATIONS

Erlh.f Erythrocytes Ht.j Heart


120

BLOOD VASCLLAR TISSUES-TELEOST EMBRYOS

FRANKLIN P. REAOAN


PLATE 1


6 ) (



8



121

PLATE 2

EXPLANATION OP FIGURES

13 and 14 are described in detail on page 106. The small arrows indicate the direction of pulsation in the 'accessory heart.' Note the apparently normal condition of the actual heart.

16 is a lateral view of a twelve-day embryo treated for twenty-four hours subsequent to the four-cell stage with a solution similar to that employed in case of the embryos represented by figures 1, 2, 5, 7 and 11 — kindness of Dr. E. I. Werber.

16 is a transverse section through the optic region of a nine-day embryo which at the four-cell stage was treated with a solution of 50 cc. sea-water, to which had been added 15 cc.m/12 butyric acid. The yolk-sac is devoid of vascular tissue. The space in the optic cup (on the left side of the figure) normally occupied by a lens, contains a blood lacuna. On the right side is seen the attachment of the upper optic stalk and the base of the lower.

ABBREVIATIONS

A.Ht.f * Accessory* heart L., Lens

Bl.l.f Blood lacuna My.C, Myocardium

End.C, Endocardium O.C.f Optic cup

Erth.f Erythroc3rtes O.st., Optic stalk Ht, Heart


122

BLOOD VASCULAR TISSUES-TELEOST EMBRYOS

FBANKLIN P. REAGAN


PLATE 2 A.Ht



123

PLATE 3

EXPLANATION OP FIGURES

17 is a transverse section of the same embryo from which figure 16 is taken; it is a section passing through the optic anlagen, showing isolated erytrocytes, solid heart, and unusual eye-formation.

18 is a section of the same embryo slightly posterior to the optic stalks, showing the pre-cardinals as haemophoric vessels. The erythrocytes are angular in contour (especially in the vessel on the right of this figure) due to their crowded condition A few of the erythrocytes in the less crowded vessel on the left have become rounded.

ABBREVIATIONS

Erth., Erythrocytes Ht,, Heart

L., Lens


124

BLOOD VASCULAR TISSUES— TELEOST EMBRYOS PLATE 3

ITRANKLIN P. REAGAN


Erth. -'


17 ^^Hi


Erth.

IS


125

PLATE 4

EXPLANATION OF FIGURES

19, 20, 21, 22 and 23 are transverse sections of the embryo from which figure I was drawn. Figure 19 is a section just posterior to the optic cups passing through the anterior ends of the haematopoietic areas of this region as shown in figure 1. In this figure, as in figure 20 the position of the blood-cells is roughly and arbitrarily indicated by heavy black dots.

20 is a section passing through the posterior portions of the optic cups, and shows the position of the median blood anlage of figure 1. The heart in this figure is a solid continuation of the left portion of the double body labeled heart in figure 19.

21 is an accurate detail of the blood-producing areas seen on the right side of the head in figure 19. Conditions in the lower lacuna seem to be farther advanced. Certain mesenchyme cells (lightly stippled) are in a stage of transition to erythrocytes (cytoplasm clear). A blood space is forming while the mesenchjrme cells bounding it seem to be receding to form endothelium. The free erythrocytes are rounded in contour,

22 is an accurate detail of the median dorsal blood anlage of figure 20. The blood-cells are very strongly eosinophilous but are crowded and possess angular contours.

ABBREVIATIONS

Erth., Erythrocytes //<... Heart


126

BIX)OD VASCULAR TISSUES— TELEOST EMBRYOS

rRANKUN P. BEAOAN


PLATE 4


Erth.


/ Erth.


19


Erth.


Ht.


20


127


Erth.


22

PLATE 5

EXPLANATION OP FIGURES

23 is a section through the heart of this same embryo near the plane of section of figxire 19. The connection of the fused portions as described in the text, is looser than in the plane of figure 19. The right 'arterial' side contains no endothelium in this section. Erythroytes in various phases of development are observed. Note the cuboidal nature of the endocardium in the left side of the figure.

24 is a section through the heart of the embryo from which figure 7 is made. In this figure the endocardial cells are cuboidal and rather deeply staining. The endocardial cavity contains erythrocytes which are in a column continuous with the erythrocytes of figures 25 and 26. There is a more or less distinct myocardium.

25 shows a section of the same heart in which a portion of the endocardium and myocardium are transforming into eosinophilous blood-cells.

26 is from the same heart The plane of section is near the upper end of the heart. The entire endocardium and myocardium of this region has transformed into eosinophilous blood-cells which, as in many instances already noted, are unable to round themselves out for lack of space.

ABBREVIATIONS

End.C, Endocardium Erih.y Erythrocytes

My.C, Myocardium


128

BLOOD VASCULAR TISSUES— TELEOST EMBRYOS PLATE H

FRANKLIN P. REAQAN


End.C.


25


Erth.


129


^O

BOOKS RECEIVED

The receipt of publications that may be sent to any of the five biological journals published by The VVistar Institute will be acknowledfced under this heading. Short reviews of books that are of special inter^t to a large number of biologists will be published in this Journal from time to time.

ANATOMY OF THE BRAIN AND SPINAL CORD WITH SPECIAL REFERENCE TO xMECHANISM AND FUNCTION. For students and practitioners Harris E. Santee, A.M., M.D., Ph.D., Professor of Nervous Anatomy in Chicago College of Medicine and Surgery.. Medical Department of Valparaiso University; Professor of Anatomy in Jenner Medical College, Chicago. Fifth edition, revised and enlarged with 158 illustrations, 46 of which are printed in colors. $4.00. Philadelphia: P. Blakiston's Son & Co., 1012 Walnut Street. '

GRUNDZUGE DER VERGLEICHENDEN GEWEBELEHRE, Dr. Med. Friedrich Maurer, O. Professor der Anatomic und Direktor der Anatomischen Anstalt in Jena. 486 pages including Index, 232 figures. Leipzig: Verlag von Emmanuel Reinicke. 1915.


Charles Sedgwick Minot

An address by Frederic T. Lewis, Vice-President of the American Association of

Anatomists, delivered at the New Haven meeting of the Association,

December 28, 1915.

The thirty-first session of the American Association of Anatomists, notwithstanding its large attendance, the excellence of the papers presented, and the notable hospitality of Washington University, was characterized by a pervasive sense of depression, due in part to the blighting effect of the European war and in part to the recent death of our leader — by common consent the foremost American anatomist. It is fittingly recorded in the minutes of that meeting, that as President and Member of the Executive Conamittee, his brilliant and constructive mind has guided the affairs of the Society with marked success, directing forward the long advance of national science. So broad were his biological interests that the physiologists also regarded him as of their nmnber, and at the opening meeting of the American Physiological Society in St. Louis, Prof. Frederic S. Lee delivered a memorial address. At the same time, in Philadelphia, the American Association for the Advancement of Science expressed its sense of irreparable loss. These resolutions, and the impressive record of Dr. Minot's achievements as presented by Professor Cattell, the sensitive personal tributes of Professor Donaldson, the keen analysis of his work by Professor Porter, and the exposition of the mental and moral qualities which made him what he was, by President Eliot, are all before us; and yet this Association gladly sets apart a time for grateful reminiscence and informal consideration of one who was peculiarly our own.

Among the books which Dr. Minot read throughout, marking many passages, was Galton's Hereditary Genius. As recently as 1909, he wrote what is essentially a review of it, for publication in the Youth^s Companion, 'ability of all orders tends to be inherited," he states at the outset, adding, We commit, perhaps, no injustice towards Mr. Galton if we surmise that the theory first arose in his mind on the contemplation of his own family, many members of which are distinguished/' Similar considerations may very reasonably have led to Dr. Minot's acquiescence, and doubtless he contemplated, with no little curiosity, .the references to his own family history in the current magazines and works on eugenics.

Professor Minot's most distinguished ancestor was Jonathan Edwards (1703-1758), a graduate of Yale in the class of 1720. The historian, Fiske, declares that the more one considers Edwards the more colossal and astonishing he seems. He regards him as one of the wonders of the world, probably the greatest intelligence that the Western Hemisphere has yet seen.

Although Professor Minot is in the fifth generation from Jonathan Edwards, their features, a^ seen in familiar portraits, have certain resemblances. Holmes describes Edwards as possessing "a high forehead, a calm steady eye, and a small rather prim mouth with something about it of the unmated and no longer youthful female." No reference is made to the rather long well-modelled nose, which is much like that of his descendant. Even though a beard conceals the mouth in Dr. Minot's portrait, there is altogether a comparable primness. But whether or not this facial resemblance is objective, these two relatives are alike in possessing the inquiring analytical mind of the naturalist. Professor Minot's early papers on insects may be compared with Edwards's astonishing paper on spiders, believed to have been written when he was not more than twelve years old.

On clear autumn days Edwards saw the air filled with shining webs and observed that — Very Often there appears at the end of these Webs a Spider floating and sailing with them." In order to explain this flight he secured spiders of various sizes and provoked them to let out their webs. In all Probability," he writes, "the web while it is in the Spider is a certain liquor with which that Great bottle tail of theirs is fiUd which immediately upon its being exposed to the air turns to a Dry Substance and very much rarifies." He saw the way in which the air currents caught the webs as they were let out and finally snapped them, so that from sticks held in his hand, the spiders "mounted away into the air with a Vast train of Glistening web before them:'

These beautifully accurate observations and experiments, recorded with sketches, amply justify Professor Packard's opinion that in another age and under other training Edwards might have been a naturalist of a high order.

Among all the available records left by Dr. Minot's ancestors, this manuscript of Edwards's alone shows the same type of mind, coupled with extraordinary ability and identity of interests. But is this more than a remarkable coincidence, of which the maze of genealogy presents so many examples? It must be admitted that Huxley's early dictmn that the production of men of genius becomes hereditary, not by physical propagation, but by the help of language, letters, and the printing press," pounds strangely antiquated. The descendants of Jonathan Edwards have been so conspicuously talented and intellectual that their family history has been carefully studied by genealogists. If their conclusions are to be accepted, the source of Professor Minot's ability should be sought not only in Jonathan Edwards but in Edwards's antecedents. Some assert that it came from Jonathan's mother Esther, but others revert to his grandparents — ^Richard Edwards, a merchant of Hartford, Conn., and Elizabeth Tuttle, his wife, who is credited with the nervous, sensitive and excitable temperament of genius." Thus Dr. Davenport declares — "Had Elizabeth Tuttle not been, this nation would not occupy the position in culture and learning that it now does." (Heredity in Relation to Eugenics, p. 228.)

It is surprising that in order to show that the production of gifted men depends upon careful marital selection, this union which no eugenist could recommend is the most frequently cited American example. Without considering further the story of Elizabeth Tuttle, we may venture an opinion that her importance has been overestimated, and in regard to Dr. Minot we may ascribe to her that proportion of his inherited qualities which, according to Galton's law, she was entitled to transmit.


In addition to the Edwards family, Dr. Minot is descended from many others which are old and highly honored in New England. From the time when Col. Stephen Minot was selectman of the town of Boston and member of a committee to draw up its charter of incorporation, the Minots have been prominent as merchants and lawyers of Boston, always active for civic betterment. Dr. Minot's grandmother Minot was the daughter of Daniel Davis, Solicitor-General of Massachusetts, and granddaughter of Judge Davis of Barnstable, member of the Provincial Congress. On his mother's side, his grandfather, Charles Sedgwick, was a lawyer of Lenox, Mass., the son of Theodore Sedgwick, a friend of Washington, Senator from Massachusetts, and Speaker of the national House of Representatives. Dr. Minot's grandmother Sedgwick was the daughter of Hon. Josiah Dwight of Northhampton, State Treasurer of Massachusetts.

It may be of interest to note that although there were no anatomists among Dr. Minot's antecedents, Prof. Thomas Dwight was his fourth cousin; Dr. Leonard Williams and Dr. Minot were both descendants of Timothy Edwards; and George Lewis was a common ancestor of Dr. Minot, Dr. Winslow Lewis (an early demonstrator of anatomy). Dr. Warren Lewis, and the writer. Professor Kingsbury also is remotely a kinsman of Dr. Minot. But among Dr. Minot's progenitors there were neither anatomists nor physicians. Their predominant legal training and legislative services are very striking. Dr. Minot's deviation from the traditional occupation may be partly explained by the fondness for nature manifested by both of his parents.

Professor Minot's father, William Minot, was born in Boston. The tide-waters of the Charles afforded him excellent fishing and the occasional excitement of seeing a seal, as he has carefully recorded. In his biographical notes he writes — I had a natural fondness for shooting, and as soon as I was old enough, I procured a small gun; adding — I am fortunate in that the taste for it has continued unabated in my old age. At Nantucket he once shot sixty-seven black-breasted plovers in a day, and at Swan Island, of ducks, one hundred and eight. Then in 1880, he published an admirable paper on game protection, advocating national as well as state legislation, which should be based on scientific knowledge and observation. He desired to see a gradual extinction of the instinctive habit of pursuit and destruction." With evident satisfaction, he states that all his children acquired an early fondness for nature and out-of-door life. '* My children's love of nature," Mr. Minot continues, was developed by their mother's tastes. No day passed without her getting interest and pleasure from its out-of-door changes of expression and character. In our holidays she was our constant companion."

So it happened that Mr. and Mrs. Minot selected for themselves and their children a beautiful and extensive estate in West Roxbury. The place, a high plateau covered with a pine forest, was very secluded and quite in the country. For four miles, toward Dedham, the woods were almost continuous, and hardly a house was to be seen; birds of all kinds were abundant and in some simmiers, as Mr. Minot has recorded, forty or fifty nests could be counted in our grounds." At Woodbourne, as this estate, was named, Dr. Minot was born on the twenty-third of December, 1852.

It is interesting to note the varying degrees in which Mr. Minot's sons responded to their opportunities for studying and enjoying nature. All of them liked the country and William, the eldest, was a keen sportsman and his father's companion on many expeditions. Henry, who was younger than Charles, did not care to shoot or collect birds, but he studied them with great ability, and at seventeen, had completed his well-known book entitled Land-Birds and Game-Birds of New England. He was an accurate observer, and gave promise of a notable career in science, had such been chosen. But he became interested in the construction of railroads and was soon the youngest railroad president in the United States. To Charles alone did the beauty and the problems of organic life appeal with irresistible compulsion — not as a mere source of recreation, nor as an occupation which brooked a rival, but as the one great theme worthy of life-long study and devotion. This was more than his father had anticipated, and apparently with some apprehension, the family obi^erved what IVIV-^- i \)in\- id-on describes as the sorious wa}'* in which ho tf>' ^ !u^ Snji'^uyj and his adoption of a precarious profe*^sioii.

It may be said that Dr. Minot began his scientific career in July, 1868, by joining the Boston Society of Natural History, then under the presidency of Jeffries Wyman, and including Scudder, Putnam, Hyatt, Packard, Verrill, Wilder and Morse among its officers. Although but fifteen years old, he became at once an active member, and the report of the September meeting of the Section on Entomology includes the following brief but correct communication :

Mr. C. S. Minot stated that there were three broods of Chrysophcmus americanusy one appearing early in May, the second in July and the third the last of August. The insects of the first brood cUffer from those of the other two in wanting the row of red spots on the imder side of the secondaries.

In the following year he described the previously unrecorded male of Hesperia metea Scudd., a small butterfly which he had collected in Dorchester; and in another brief paper he considered the great difference in the number of species in various genera of insects, regretting that there were so many which contained only one, two, or three species. At this time his father wrote:

I must add that Charles is thoughtful and industrious about the place and has worked some days like a beaver, doing a deal of sodding and re-graveling, and generally getting the place in good order. In fact he has had an unexpected eye to everything and is an argus after bugs, of course; but what did surprise me were two modest well-expressed and mature articles on scientific subjects published in one of the scientific journals. I saw them by accident; hedidnH tell of them. I could scarcely believe he wrote them, they were so good.

In 1868, the year in which Dr. Minot joined the Society of Natural History, he was admitted to the Massachusetts Institute of Technology. It was required for admission that candidates should be sixteen years of age and should pass satisfactorily in arithmetic, algebra, plane geometry, English grammar and geography. Dr. Minot was admitted before he was sixteen. He could not have entered the Scientific School of Harvard Uni CHARLES SEDGWICK MINOT 139

versity until he was eighteen; and although there was no age requirement for Harvard College, it is improbable that he could have passed the entrance examinations without longer preparation. These examinations included not only all the subjects required by the Institute (with the substitution of '^ reading English aloud" for English granamar), but also ancient history, both Latin and Greek granmiar and composition, parts of the Iliad and the Anabasis, Caesar's Conmientaries, selected orations of Cicero, and the whole of Virgil. Therefore, although natural history was such a flourishing department at Harvard, with Asa Gray and Louis Agassiz at its head, its approach was so guarded by requirements in classics, both before and after admission, that Dr. Minot chose the Institute; and since a natural history curriculum had not been established there, he selected chemistry as his major subject. Perhaps his most influential teacher at the Institute of Technology was Edward Pickering, the astronomer, who was then professor of physics. In his department Dr. Minot prepared a simple apparatus for micro-photography and made a number of pictures of the parts of insects. An account of this work is included in the Reports of the President and Departments of the Institute for 1871-72. Dr. Minot completed his course with a good record and received the degree of Bachelor of Science in 1872, being the youngest member of his class. He was always a loyal alumnus, and never approved of the Harvard A.B. as a preparation for scientific studies '* unless that degree represents adequate courses in chemistry, physics, biology, French and German. In fact these courses, without the A.B. degree, seemed to him sufficient.

While an undergraduate. Dr. Minot continued to publish notes on entomology, including the description of several new species of geometrid moths, which were, perhaps, his favorite group. At the same time Dr. Minot was reading very carefully the great works on evolution. Huxley's Man^s Place in Nature was given him by an aunt at Christmas, in 1869. On the blank pages at the end, he wrote a terse summary, beginning Herein is shown — " and concluding " In fine, man, tho' at the head of the family, is an ape;" but he adds — "The structural formation of the organs of speech is not herein treated of and exandned/^ and he notes that the human brain-size consistent with sanity is greater than that of the gorilla and asks how did man bridge this hiatus?" Doubtless Dr. Minot had read Darwin's Origin of Species and many other scientific works before this. Beginning in 1870 he kept a record, showing that with much of the best general literature, he read while at the Institute many numbers of Nature, Huxley's Lay Sermons and Elementary Physiology, Darwin's Descent of Man, Lubbock's Origin of Civilization, Tyson's Cell Doctrine and Alexander Agassiz's Marine Animals and the Embryology of the Star Fish. The effect of this early reading is evident throughout Dr. Minot's career. It were wiser," he said, "to take out the mainspring from a watch than to eliminate evolution from biology." Of Darwin, he writes (1885) "We are already able to appreciate the directness and force of his intellect, his noble candor, and above all, his insatiable love of knowledge and research;'*' and Huxley, he declares, "has carried scientific writing to unsurpassed excellence. His Lay Sermons are masterpieces.

In the fall of 1872, having obtained the degree of B.S., Dr. Minot could enter the graduate school of Harvard College. There he became one of three candidates for the degree of S.D. in natural history, the others being his friend Faxon, the zoologist, and Shaler, the geologist. Unfortunately the records do not show what studies were taken. There was some work with Louis Agassiz, who had just returned from the Hassler expedition with an abundance of material, and at that time Dr. Minot read his Essay on Classification. In the following summer both Faxon and Minot were with Agassiz at Penikese. Apparently some botany was studied, for Gray's Lessons were read in February, but descriptive phanerogamic botany did not meet with Dr. Minot's approval. It happened, however, that Prof. Henry P. Bowditch had returned from Europe in 1871 and had established his physiological laboratory at the Harvard Medical School. Although Dr. Bowditch was considerably older than Dr. Minot, the families had always known each other, and perhaps it was personal acquaintance which led Dr. Minot to become Bowditch's first research pupil. Admission to his laboratorywas a revelation, and teacher nnd pupil became the warmest friends. Ever afterwards, Dr. Minot enjoyed Bowditch's sympathy, interest, and appreciation, to which he responded with life-long respect and admiration. The experiments which they performed dealt with the influence of anaesthetics on the vasomotor centres," and the results were published in a joint paper in 1874. The experiments were probably largely by Minot, but as Dr. Porter states, the publication itself bears unmistakably the marks of Bowditch's style and hand.

Dr. Bowditch had recently returned from the laboratory of the renowned Carl Ludwig, and it can readily be inferred why Dr. Minot gave up, for the time being, his candidacy for the S.D. degree and set out for Leipzig in 1873. He had fulfilled one of the required three years of study.

At Leipzig in October, he began a long course of German reading, and entered at once upon his physiological studies under Ludwig. At Ludwig's suggestion, he investigated the formation of carbon dioxide in resting and active muscle, the results of which were published in 1876; and in later years he referred to Professor Ludwig as the greatest teacher of the art of scientific research whom he had ever known.- But he did not limit his studies to physiology. In December of 1873, he had apparently begun his investigations with the distinguished professor of zoology at Leipzig, Rudolf Leuckart. These studies were chieflj'^ on the structure and classification of the lower worms, and led to several publications. In December of this eventful year the death of Agassiz occurred in Cambridge, and Dr. Minot received a letter from his father containing the following interesting comments:

You were among his last pupils. It is too soon for any just estimate of Agassiz's life and service to science, but no doubt he did more than any one else in this centiuy to transplant to America enthusiasm* for scientific investigation. Your own choice of life is probably as much due to his indirect influence as to any other source, except your predisposing tastes.

Next Tuesday is your birthday — your majority. How thankful I am for your good character, high aims, and excellent promise, for your affectionate disposition, love of home, ajid generous ambition! You have a bright prospect before you. , Your labors will always have your heart in them, and your acquisitions will every day enlarge your horizon over the illimitable kingdom of Nature.

Throughout the spring of 1874 Dr. Minot remained at Leipzig, continuing his physiological and zoological studies, and reading von Baer's Entwickelungsgeschichte, which served as the foundation for his embryological work. Later, while in Germany, he read von Baer's autobiography and addresses, and regarded him henceforth as "the greatest embryologist." In the summer, however, work was laid aside for a pedestrian trip in Switzerland, with Mr. Faxon. Professor Mosso, a fellow student under Ludwig, was another of Dr. Minot's companions on such expeditions, and a close friend. This friendship doubtless led to the deep interest which Dr. Minot took in Italian literature, both general and scientific.

After another term at Leipzig, during which Dr. Minot read His's Unsere Korperform, he went to Paris in the spring of 1875, and studied some months with Ranvier. Here he learned important methods in histological technique which he applied to the study of the water-beetle Hydrophilus, and this work was published in the following year. He then returned to his headquarters at Leipzig, but spent the winter of 1875-76 at Wurzburg in Professor Semper's laboratory. Here he studied Leydig's ^'invaluable Lehrbuch der Histologic and made serial sections of worms, thus perfecting his training in histological technique. Finally he returned to Leipzig for the summer of 1876, and then, after three years of European study, back to Boston in the fall.

No time was to be lost. Dr. Minot proceeded to publish the results of his investigations in a variety of papers. An abstract of Dr. Lessor's lecture on auto-transfusion was sent to the Medical and Surgical Journal. The Society of Natural History listened to a paper on the classification of worms. In the American Naturalist, Leyser's primitive sliding microtome was described under the title. The sledge microtome; and two papers were devoted to an enthusiastic account of the study of zoology in Germany, with sweeping criticisms of American institutions. To obtain the degree of S.D., however, another year of resident study was required, and it was specified that Dr. Minot should take a course in anatomy at the medical college and do further work in Dr. Bowditch's laboratory. It is probable that he took the course in gross anatomy, with human dissection, under Dr. Oliver Wendell Holmes, but the record is not definite, and it is evident that Dr. Minot did very little work in gross himian anatomy. With Dr. Bowditch he performed experiments on tetanus, which were published in full. This research is characterized by Professor Porter as " ingenious, laborious, meticulous, a conscientious collection of crumbs left by those earlier at the feast. With this array of publications, in place of the usual thesis, Dr. Minot took his final examination in 1878, and received the degree of Doctor of Science from Harvard University. Unfortunately the record of that interesting occasion was never written out by the secretary.


Fig. 1 "Anatomy of the Gunner, male." Drawn by C. S. Minot for Packard's "Zoology." (Henry Holt and Company, 1879.)


For two years after receiving his degree, Dr. Minot remained without any position and apparently was still uncertain as to what he should do. This interval, however, was spent in active scientific work. Professor Packard in 1879 published his Zoology, and to this Dr. Minot contributed a series of engravings of the anatomy of vertebrates, one of which is here reproduced as an example of his early drawings. They were accompanied with short accounts of the viscera shown m the dissections. Dr. Minot likewise collaborated with Professor Packard in writing the reports on the Rocky Mountain Locust, issued by the Entomological Commission at Washington. Dr. Minot's part was to describe the histology of the locust and cricket, and this is said to be his most important entomological work, still being used as a laboratory guide and book of reference.

In these years Dr. Minot began to formulate the great problem which was at the center of all his later work — an insoluble problem, but one which, as he declared, should be regarded as the object of all botanical and zoological studies. This is no less than the ultimate and essential nature of life. It was approached, in 1879, by stating the conditions to be filled by a theory of life. Consciousness, growth, senescence and rejuvenation, and heredity must all be explained in conformity with the cellular structure. Since this involves physiological and psychological studies, as well as those purely morphological. Dr. Minot made frequent excursions into other fields. But morphology was now his chosen science. He read scientific memoirs incessantly and announced that he was preparing a large work on comparative histology.

Up to the time of Dr. Minot's appointment as a lecturer at the Harvard Medical School (in 1880) his microscopic studies had been almost entirely of invertebrates. But he had proposed several new terms and radical hypotheses of general application. He had rejected Haeckel's gastraea theory, and had named the two-layered stage of the embryo, the diaderm (1877). In 1879, he stated that the primitive cells of the mesoderm are amoeboid in character, and for them he proposed the name of mesamoeboids. The sexual cells he called genohlastSj and his original hypothesis concerning them was illustrated by a diagram here reproduced as figure 2. All cells of the body were regarded as "hermaphrodite or neuter — sexless," since they contain both male and female elements (fig. 2, A). In producing a female cell, or thelyblast, Dr. Minot believed that the male elements, or arsenoblasts, were removed in several parts, which formed the polar globules (fig. 2, B). Adopting Kolliker^s conclusion of 1847 that spermatozoa arose in vesicles or cells, he considered that the male portion of such a cell became separated as several arsenoblasts or spermatozoa, leaving behind the nucleated remainder of the cyst as a female cell or thelyblast (fig. 2, C). By the imion of an arsenoblast and thelyblast, a neuter cell, the fertilized ovum, would be produced. According to Wilson, this ingenious view was independently advocated by Van Beneden in 1883," but for reasons now obvious it has been abandoned.

The appointments at the Harvard Medical School which Dr. Minot received in 1880 were "procured for him with some difficulty." Because of his scientific attainments he had the support of President Eliot. Professor Bowditch was his friend; and an uncle, Dr. Francis Minot, was Hersey Professor of the Theory and Practice of Physic. But there was opposition to the appointment on the Medical Faculty of one who was not a physician and who had no intention of becoming one, but who had for years been fitting himself to become a professional zoologist. Semper had taught Dr. Minot that medical men are all spoilt zoologists, and Dr. Minot said significantly of Huxley — '^He might have been a successful physician, but in that case what a rich vein of mental treasure would have been buried beyond recovery." So it resulted that Dr. Minot was given the absurd position of Instructor in Oral Surgery and Pathology in the dental school. At the same time he was appointed Lecturer on Embryology in the medical school, but without being admitted to the faculty. From the catalogue of the dental school it appears that Dr. Minot's first class consisted of four students, who were taught the use of the microscope and the preparation of sections, in connection with lectures on the finer structure and development of the teeth. At the medical school Dr. Minot gave a few lectures, but there was no separate examination in embryology. Instead, the subject was covered by the twentieth question on Professor Bowditch's paper, which reads — How is the pleuroperitoneal cavity formed in the embryo?


Fig. 2 ^Diagrams to show the relation of the sexual products to cell?. A, an ordinary cell; B, egg with polar globules; C, spermatocyst with spermatozoa." (American Naturalist, 1880, vol. 13, p. 106.)


Year by year Dr. Minot increased the number of his lectures, and in 1883 he was appointed Instructor in Histology and Embryology. In the following year, with Dr. Quincy, he was in charge of laboratory exercises in histology twice a week, and his work at the dental school was then discontinued. In his first lecture after his appointment as instructor, he announced that embryology would enable the student who has been seeking his way through the mazes of adult anatomy by sheer force of memory to have a mental picture which is at once clear, interesting and correct. '^ In later addresses (1899) he expressed his conviction that *'far more time is usually devoted to anatomy than is advantageous to the student ;^^ and in 1890 he declared that unless the student betakes himself to embryology, his anatomy will be no better than a stupid system of mnemonics. Doubtless enough has been said to show Dr. Minot's point of view toward medicine, and to explain why certain of his colleagues felt it their duty to prevent his control of the department of anatomy. Dr. Minot's first publication from the department of histology and embryology of the Harvard Medical School was on the seminal vesicles of guinea-pigs (in 1885), and the second was on the skin of insects. So Dr. Minot was reminded that the first duty of the medical school is to train practitioners of medicine. *' A platitude, he replied, but in conversation he added — Professors are very difficult to get along with; they not only have opinions, but have reasons for their opinions."


Gradually Dr. Minot's work changed, and conformed to a greater extent with that of a medical school. His studies on the classification of worms ended in 1885 with an account of the Vermes in the Standard Natural History, edited by his friend Professor Kingsley. In this same year Dr. Minot likewise completed his studies of the structure of insects, by writing an account of the anatomy of the cotton worm. Meanwhile most, but not all, his new species had 'gone into the synonymy' where he had helped to place others. In 1884, at the meeting of the American Association for the Advancement of Science, he urged a return to the Linnaean system of nomenclature, of which he says, We have retained the form but rejected the principle. He believed that the present method of determining species was "thoroughly unscientific" and that "species will all have to be redetermined." His farewell to systematic zoology was expressed to Professor Kingsley in the following "original poem."

Classification is vexation.

Taxonomy is as bad; Priority doth puzzle me,

And trinomials drive me mad.

An unexpected departure from anatomical studies occurred at this time. It was foreshadowed in 1880 in his review of Mosso's investigations, by means of a plethysmograph, of the changes in the circulation during cerebral activity. In this review Dr. Minot writes:

Although psychology is usually regarded as a department of philosophy, it is certainly more completely a natural science, since it deals with natural events, which are learned by direct observation, and which we coordinate by our reason .... During the new phase, into which psychology has apparently entered, the principal problems will probably concern the relation of mind to the substratum of matter in which it displays itself.

In 1884, he declared that to study scientifically the obscure and abnormal so-called psychical phenomena was a moral duty for those gifted with a clearer intelligence and purer moral sense. So he became a member of the organizing conunittee of the American Society for Psychical Research, and for the following review of his work in this direction we are indebted to Professor Yerkes.

To the study of telepathy, muscle reading, the number habit, superstitions, and other phenomena, Dr. Minot gave close and critical attention, bringing to bear upon the problem? the thoroughness and impartiality of method which characterize all of his research.


During the ten years from 1884 to 1894, Professor Minot for the Society conducted a number of special investigations, the reports of which are of obvious scientific value. It is from them clear that he long remained open-minded and hopeful that important truth concerning fundamental action* might be revealed. Especially interesting are his reports on diagram tests and number habits. These show his admirable spirit of scientific research most effectively.

During the decade in question, his writings indicate that his attitude toward psychical research gradually changed, and in an article entitled The Psychical Comedy, published in 1895, in the North American Review, we find him reacting vigorously against the unscientific methods of psychical investigators. It is foolish to search for marvels. The wise search for truth. Yet there are many who have done and are stUl doing the former, and in so far as they are seeking to find marvelous faculties of the human mind they are performers in the psychical comedy and their acts and opinions form the basis of this article."

Having become convinced by his contact with the members of the Psychical Research Society that their interest was not centered in the discovery of truth. Dr. Minot withdrew from psychical research, and our only evidence of the continuation of his interest in the problems of mental life is his presidential address before the American Association for the Advancement of Science, in Pittsburgh, in 1902. At this time he spoke of the problem of consciousness in its biological aspects in a way which at once revealed his keen interest in everything mental, and his conviction that the study of consciousness is an important duty as well as opportunity of biologists. It matters not that few psychologists, and still fewer biologists, can agree with all of his statements, for they are the utterances of one who thought honestly and vigorously along other lines than those of his daily work.

By his contributions to psychical research and to the general Uterature on consciousness (his papers number about a dozen). Professor Minot deserves to be ranked as an important contributor to our knowledge of mental phenomena. To the psychologist, most impressive of all is his insistence upon accuracy and reUability in observation and statement, and the evidence of his single-minded devotion to the truth.

In taking leave of psychical research, Dr. Minot published a characteristic statement, which involved him in amusing consequences. He said:

The failure of psychical research should teach us a profound lesson — the lesson that literary training sets hmits to the faculties. The leaders of the Psychical Society are Uterary men. . . .

To which Mr. Andrew Lang spicily replied in an article in the London News (Mar. 30, 1895) entitled "On a certain condescension in scientific men/' showing that literary training is not alone in limiting the faculties.'*

On many occasions Dr. Minot severely criticized his colleagues. When in 1883 he described Hubrecht's hypothesis of primogeniture as "pin-e speculation of that reckless quality which of late years has crept into zoology/' and in 1886 referred to Flemming's new terms (including mitosis) as "new-fangled" and a burden to science, he was approaching the bad-mannered faultfinding" which he perhaps repentantly denoimced in an anonymous article — ^Youthfulness in Science (1889). His characteristic intensity of conViction was frequently vigorously expressed, and not always in such a way as to facilitate his progress.

In the year 1883, when Dr. Minot was appointed Instructor and took charge of the Department of Histology, the Harvard Medical School moved to its new building on Boylston Street — "a noble edifice," as Dr. Holmes declared, in which *'you will find apartments devoted to microscopic instruction and study." These apartments included a welMighted students' laboratory on the top floor which, according to President Eliot, was of Dr. Minot' s own planning. It was equipped with eighteen Hartnack microscopes, and the department, we are told, was supported by an annual appropriation of fifty dollars, supplemented by a gift of six hundred dollars made personally to Dr. Minot and increased by his own generosity. Additional microscopes were purchased with money borrowed from the University and in time repaid through rental fees. This was done at Dr. Minot's suggestion, and the University was impressed not only with his vigorous teaching and many publications, but with his business qualities, so that in 1887 it was possible to promote him to an Assistant Professorship in Histology and Embryology."

His inventive qualities were also now apparent, for in 1886, he had designed the rotary microtome, familiar tp all histologists. Baltzer, the instrument maker for Professor Ludwij^'s laboratory, made the first one, but in 1888 they were being manufactured in Boston. The original form of this very valuable device is shown in figure 3. Its toothed wheel was small and therefore sections could not be cut thinner than 30 microns. Although the 'precision microtome/ likewise designed by Dr. Minot (figured in Science, 1897, vol. v, p. 862), has largely replaced the rotary microtome in his own laboratory, especially for cutting serial sections of embryos, the rotary microtome is generally more widely used and is still of great service.

In the five years of his assistant professorship. Dr. Minot accomplished his most important scientific work, ending in 1892 with the publication of his remalrkable treatise on Human Embryology, and his promotion to a full professorship. The Human


Fig. 3 Minot' 8 Automatic Microtome," as figured in the American Naturalist, 1888, vol. 22, p. 945.

Embryology, a volume of 815 pages, is described in the preface as the result of ten years' labor. It was an attempt "to present a comprehensive summary of embryology, as it bears upon the problems of human development, and it embodied the material previously published by Dr. Minot in a large number of papers. Two characteristics are most conspicuous. First, it is a masterly smnmary of an unwieldy literature, of which its author was in full conmiand; and second, it is a presentation of a continuous succession of problems on which the author passes judgment in a manner compelling attention and wholly his own.


These features may be illustrated in the following paragraph which shows also the characteristic but distracting boldface references to the literature.

The ORIGIN of the decidual cells (of the uterus) was long uncertain. Three views contended for acceptance: 1st, th^y are modified leucocytes (Hennig, Langhans just cited above, Sinfety 76.1); 2d, they arise from the connective-tissue cells of the mucosa (Hegar and Maier, Leopold); 3d, they are produced by the epitheUum. In favor of the first view, there has never been, to my knowledge, any evidence of importance. The second view has been definitely established by Minot, 98, 429.

Dr. Minot's own paper, to which he here refers, was entitled Uterus and Embryo and was of great practical importance. It included a thorough consideration of the human placenta and membranes, based upon an extensive series of original preparations, and led the way toward utilizing sections of these structures in courses in histology for medical students.

In discussing the nature of sex, Dr. Minot still held to his theory of genoblasts already described. The chapter on blood embraces his work previously published in both the Anatommischer Anzeiger and the American Naturalist for 1895, which he summarized as follows:

In the development of red corpuscles, we can distinguish three principal stages: 1, young cells with very Uttle protoplasm; 2, old cells with much protoplasm and granular nucleus; 3, modified cells, with shrunken nucleus, which colors darkly and uniformly. I do not know whether the first form occurs in any Uving adult vertebrate, although the assumption seems justified that it is the primitive form. On the other hand, the second stage is obviously characteristic of the Ichthyopsida in general, while the third form is typical for the Sauropsida. Therefore the development of the blood-cells in amniota offers a new confirmation of Louis Agassiz' law (HaeckeFs biogenetisches Grundgesetz).

This fundamental interpretation of the blood corpuscles appears to be well established. But it was followed by the adoption of Schafer's opinion that the non-nucleated red corpuscles of mammals are intra-cellular protoplasmic products, which Dr. Minot names plastids, (Often we have seen Dr. Minot consult his Embryology to find, as he remarked, "opinions which I once held".) In describing the liver, the accompanying figure was used (fig. 4) showing clearly the relations of the tubules to the large blood-channels which were later described as sinusoids, constituting another of Dr. Minot's far-reaching generalizations. The figure is from a drawing by Dr. Minot, and shows the simple nature of the illustrations used throughout the book.

The Human Embryology was immediately recognized as the most important work in its field which America had produced and the most noteworthy work in English since the publication


Fig. 4 Portion of a ^section of the liver of an Acanthias embryo of 29 mm. hpf hepatic cylinders; 6i, blood-channels (later termed sinusoids). From Minot's Human Embryology, 1892, p. 761.

of Balfour's Comparative Embryology. In 1894, it was published in German translation, and Professor His, to whom Dr. Minot was indebted for generous permission to use his unique embryological collection in Leipzig, wrote the preface. In it he says that the book is substantial throughout, with the facts everywhere in the foreground. He adds, "Minot's work is at present the fullest human embryology which we possess . . . . and even after its contents in many parts shall ha\'e become superseded, it will retain its value as a bibliographical treasure-house. Dr. Minot's subsequent embryological publications are overshadowed by this masterpiece. They include many well known papers, together with the Laboratory Textbook of Embryology. In this text-book pig embryos were so described as to become the commonest objects of study in courses on mammahan embryology. This was of great service, as was also the establishment of his embryological collection. This collection of two thousand selected vertebrate embryos, cut in serial sections, has served for many investigations, and is a rich mine of opportunity for further work. The careful methods which were used in preparing and preserving these extensive series have served as a model in many laboratories.

The progress which Dr. Minot made toward a theory of life, which he had so early set forth as the ultimate goal of all biological work, is recorded in a series of papers of great interest. Influenced by Professor Bowditch's studies of the growth of children, Dr. Minot undertook a more comprehensive study of the growth of guinea-pigs. He was greatly impressed with the senescence, or loss of power to grow, manifested in infancy. Pushing his inquiries further, he concluded that the rate of growth was greatest in the segmentation of the ovum, and that it declined at such a rate that at birth 98 per cent of this power had been lost; and the remaining 2 per cent was largely exhausted in infancy. But before senescence conquers, the germ cells are set free, effecting rejuvenation. Having determined that there was a tremendous power of growth in the germ cells, which was lacking in those of the adult tissues, he sought for some corresponding difference in their morphological characteristics, and he thought that it was to be found in the proportionate bulk of nucleus and cytoplasm. In the young cells there is but little cytoplasm and correspondingly little functional differentiation. In the specialized cells of the adult, cytoplasm is abimdant and differentiated, and death is the inevitable price which the organism must pay for the cytological differentiation on which all higher life depends. Cytomorphosis was the term which Dr. Minot used to designate these changes, and toward the close of his life he had planned further studies in this direction. Those which he had completed were published in his book on Age, Growth, and Death, which has received wide-spread recognition, and of which a Japanese translation has recently been issued.

Such are the contributions to science which established Dr. Minot's position as the foremost American anatomist of his time. But as Galton has found, some eminently scientific men have shown their original powers by Uttle more than a continuous flow of helpful suggestions and criticisms, which were individually of too little importance to be remembered in the history of Science, but which in their aggregate formed a notable aid towards its progress." In this respect also, it is important to record the distinguished services of Dr. Minot, for not only in private, through extensive correspondence, but as president of societies, through stirring addresses, and as an organizer of scientific activity, his influence was powerfully felt throughout the country. He exalted the work of the scientist as few scientists would venture to do, but this he regarded as a duty. He declares that until it is clearly recognized that the greatest crime of the French revolution was not the execution of the king but the execution of Lavoisier, there is no right measure of values, for Lavoisier was one of the three or four greatest men France has produced." It has been suspected that "in his heart. Dr. Minot concealed a regret that he could not become a philosopher," but this is far from true. "Philosophy," he says, "is ever a laggard and a follower after her swifter sister. Science." "Let us part company from the horde of foolish thoughts which have too long masqueraded under the false garb of philosophy." "Observation," he says "is the foundation of knowledge and no human knowledge is built on any other foundation." So Dr. Minot declares that "the applications of the invention of placing pieces of glass of particular shapes in the two ends of a brass tube have more profoundly influenced human thoughts and beliefs than any other single invention, excepting only printing. The telescope has revolutionized our conception of the universe, the microscope our conception of hfe."

The significance and diflSculties of correct microscopic observation he beheved to be very generally underestimated. Professor His has said that a keen mind is a common possession in comparison with keen vision; but Dr. Minot goes further. It is conceit, he declares, which leads one to ascribe his failure to observe to poor vision. The retina is good — it is the brain which * fails the poor observer. Accurate observation," he repeats is by far the most diflScult art which mankind ever essayed. In this spirit he met his colleagues in annual convention, always genial and happy, persuading them that they were the salt of the earth. So also he met his classes of students, showing them how differently science may be regarded. Schiller says of Science:

To one she is divine — a heavenly goddess; to another A good cow, providing him with butter.

Dr. Minot's message was always to those who looked upon medicine as more than a means of livelihood. For himself, science was enthroned on high, and faithful scientific research was Christian service.

•It is very gratifying that academic distinctions were so liberally conferred upon Dr. Minot. He received the honorary degrees of Doctor of Laws from Yale in 1899; Doctor of Science from Oxford in 1902, Doctor of Laws from Toronto in 1904, and from St. Andrews in 1911. In his exchange professorship with Germany in 1912-13, which was likewise an honor conferred upon him, he represented not only Harvard University, but the anatomists of America, and he took no less pleasure in presenting the work of his colleagues than in describing his own researches.

His death occurred on the 19th of November, 1914, and by vote of the Association of Anatomists at the following meeting, the foregoing memorial of Doctor Minot's personal and academitJ life, with due consideration of his education and scientific achievements, has been prepared. Of necessity it is a very imperfect record. But it is hoped that it may show how a great anatomist arose among us, and how as President Eliot has said, "by clear merit he made his way." Every encouragement should be given to any youth of similar possibilities, and this Association exists primarily for that purpose, — but where can we find him?

A CHRONOLOGICAL LIST OF PROFESSOR MINOT'S PUBLICATIONS

Description of the male of Hesperia meieay Scudder. Proc. Boston Soc.

Nat. Hist., vol. 12, pp. 319-320. Upon the limits of genera. Proc. Boston Soc. Nat. Hist., vol. 12, p. 380. American Lepidoptera. I. Geometridae, Latr. Proc. Boston Soc. Nat. Hist., vol. 13, pp. 83-85. ' Brief notes on the transformations of several species of Lepidoptera. Canadian Entomologist, vol. 2, pp. 27-29. American Lepidoptera. II. Phalaenidae, Latr. Proc. Boston Soc. Nat.

Hist., vol. 13, pp. 169-171. Cabbage butterflies. American Entomologist, vol. 2, pp. 74-76. 1870 Notes on the flight of N. E. butterflies. Proc. Boston Soc. Nat. Hist.,

vol. 14, pp. 55-56. 1872 Notes on Limochores bimacula, Scudd. Canadian Entomologist, vol. 4,

p. 150. 1874 Henry P. Bowditch and Charles Sedgwick Minot. The influence of anaesthetics on the vaso-motor centres. Boston Med. and Surg. Journal, vol. 90, p. 493-498. 4 plates.

1876 Recherches histologiques sur les trach^es de THydrophilus piceus. Arch.

de Physiol, norm, et path., 2e S^rie, T. 3, pp. 1-10. PI. VI-VII. • Die Bildung der Kohlensaure innerhalb des ruhenden und des erregten

Muskels. Arbeiten der physiol. Anstalt zu Leipzig, Jahrg. XI, pp.

1-24. Transfusion and auto-transfusion. (Abstract of a lecture by Dr. Lesser.)

Boston Med. and Surg. Journal, vol. 94, pp. 741-743. On the classification of some of the lower worms. Proc. Boston Soc. Nat.

Hist., vol. 19, pp. 17-25.

1877 Studien an Turbellarien. Beitrage zur Kenntnis? der Plathelminthen.

Arbeiten a. d. zoolog. -zoo torn. Institut in Wlirzburg, Bd. 3, pp. 405471. PI. XVI-XX.

The sledge microtome. Amer. Naturalist, vol. 11, pp. 204-209.

The study of zoology in Germany. I. The laboratories. II. The methods used in histology and embryology. Amer. Naturalist, vol. 11, pp. 330-336; 392-406.

Recent investigations of embryologists. Proc. Boston Soc. Nat. Hist., vol. 19, pp. 165-171.

1878 Experiments on tetanus. Journ. Anat. and Phys., vol. 12, pp. 297-339.

PI. III-VI.

A lesson in comparative histology. Amer. Naturalist, vol. 12, pp. 339347. PI. II.

On Distomum crassicole; with brief notes on Huxley's proposed classification of worms. Mem. Boston Soc. Nat. Hist., vol. 3, pp. 1-12. PI. I.

Report on the fine anatomy of the locust. First Annual Report of the V . S. Entomological Commission, for the year 1877. Washington, 1878. Pp. 273-*277. PL V.

CHARLES SEDGWICK MINOT 157

1879 Growth as a function of cells. Proc. Boston Soc. Nat. Hist., vol. 20, pp.

190-201. Preliminary notice of certain laws of histological differentiation. Proc.

Boston Soc. Nat. Hist., vol. 20, pp. 202-209. On the conditions to be filled by a theory of life. (Abstract.) Proc.

Amer. Assoc. Adv. Sci., vol. 28 (1880), pp. 411-415.

1880 A sketch of comparative embryology. I. History of the genoblasts and

the theory of sex. II. The fertilization of the ovum. III. Segmentation and the formation of the gastrula. IV. The embryology of sponges. V. The general principles of development. Amer. Naturalist, vol. 13, pp. 96-108; 242-249; 479-485; 871-880.

The lowest animals. (Review of Leidy's Fresh-water Rhizopods.) Intemat. Review, vol. 8, pp. 646-651.

Changes of the circulation during cerebral activity. Pop. Sci. Monthly, vol. 17. pp. 303-311.

Human growth. Boston Med. and Surg. Journal, vol. 103, pp. 79-82.

Review of Balfour's Comparative Embryology. Vol. 1. New York Med. Journal, vol. 32, pp. 630-635.

Histology oJF the locust (Caloptenus) and the cricket (Anabrus) . Second Report of the U.S. Entomological Commission, for the years 1878 and 1879. Pp. 183-222. PI. II-VIII.

Studies on the tongue of reptiles and birds. Anniversary Mem. Boston Soc. Nat. Hist., 20 pp. PI. 1.

1881 Some recent investigations of the histology of the scala media cochleae.

Amer. Journal of Otology, vol. 3, pp. 89-95. PI. I. Comparative morphology of the ear. Part I. The Medusae. Amer.

Journal of Otology, vol. 3, pp. 177-186. Mounting chick embryos whole, Amer. Naturalist, vol. 15, pp. 841-842. Review of Balfour's Comparative Embryology. Vol. 2. Boston Xled.

and Surg. Journal, vol. 105, p. 450. Comparative morphology of the ear. Second article. Amer. Journal of

Otology, vol. 3, pp. 249-263. A grave defect in our medical education. Boston Med. and Surg. Journal,

vol. 105, pp. 565-567. Huxley's writings. Internat. Review, vol. 11, pp. 527-537. Editors' table. (A paragraph on inviting the* British Association to America.) Amer. Naturalist, vol. 15, pp. 37^380. Is man the highest animal? Proc. Amer. Assoc. Adv. Sci., vol. 30 (1882),

pp. 240-242.

1882 Review of Balfour's Comparative Embryology. Vol.2. New York Med.

Journal, vol. 35, pp. 152-156. Comparative morphology of the ear. Third article. Amer. Journal of

Otology, vol. 4, pp. 1-16. Comparative morphology of the ear. Fourth article. Amer. Journal of

Otology, vol. 4, pp. 89-101. Charles Robert Darwin. (Editorial.) Boston Med. and Surg. Journal, vol. 106, pp. 402-403.

Report on general physiology. Boston Med. and Surg. Journal, vol. 106, pp. 440-444. Theorie der Genoblasten. Biol. Centralbl., vol. 2, pp. 366-367.

1883 Anatomical technology as applied to the domestic cat. By Burt G. Wilder and Simon H. Gage. (Review.) The Nation, Jan. 25, p. 89.

Criticism of Professor Hubrecht's hypothesis of development by primogeniture. Science, vol. 1, pp. 165-166.

Life-history of the liver-fluke. (Abstract of an article by A. P. Thomas.) Science, vol. 1, pp. 330-331.

The foetal envelopes. (Opening lecture in the course on embryology at the Harvard Medical School in 1883.) Boston Med. and Surg. Journal, vol. 108, pp. 409-411.

Report on general physiology. Boston Med. and Surg. Journal, vol. 108, pp. 440-442.

Retrogressive history of the foetus. (Second lecture in the course on embryology at the Harvard Medical School.) Boston Med. and Surg. Journal, vol. 108, pp. 529-531.

Heitzmann's microscopical morphology. Science, vol. 1, pp. 603-605.

National traits of science. (Editorial.) Science, vol. 2, pp. 455-457.

1884 The laboratory in modern science. (Editorial.) Science, vol. 3, pp. 172 174. An international scientific association. Science, vol. 3, pp. 245-246. The organization of an international scientific association. Science, vol. 4, pp. 80-81. Proceedings of the section of histology and microscopy. (A. A. A. S.

Phila., 1884.) Science, vol. 4, pp. 342-343. "Comment" on microscopical technique. Science, vol. 4, pp. 350-351. Psychical research in America. Science, vol. 4, pp. 369-370. Death and individuality. Science, vol. 4, pp. 398-400. Comments*' on cooperation in science. Science, vol. 4, p. 411. Researches on growth and death. Proc. Soc. Arts, Mass. Institute of

Technology, 310th meeting, pp. 50-56. Researches on growth and death. (Abstract.) Biological Problems.

(Abstract.) Vesiculae seminales of the guinea-pig. (Abstract.) On the skin of insects. (Abstract.) Proc. Amer. Assoc. Adv. Sci., vol. 33 (1885), pp. 517-521.

1885 Report on the anatomy of Aletia xylina. By Charles Sedgwick Minot and Edward Burgess. Fourth Report of the U.S. Entomological Commission, pp. 45-58. PI. VI-XI.

Zur Kenntniss der Samenblasen beim Meerschweinchen . Arch. f. mikr. Anat., Bd. 24, pp. 211-215. Taf. 12.

American society for psychical research. The Evening Post, New York. Jan. 10.

Branch V. Vermes. "Standard Nat. History," edited by J. S. Kingsley, vol. 1, pp. 185-235.

The effects of cold on living organisms. (Review of Coleman and McKendrick.) Science, vol. 5, pp. 522-523.

The formative force of organisms. Science, vol. 6, pp. 4-6.

Report on histology and embryology. Boston Med. and Surg. Journal, vol. 113, pp. 30-34.

A new endowment for research. Nature, July 30, pp. 297-298. Also, Science, vol. 6, pp. 144-145.

Some histological methods. Amer. Naturalist, vol. 19, pp. 828-830 and 916-917.

Organization and death. (Abstract.) A new membrane of the human skin. (Abstract.) The structure of the human placenta. (Abstract.) Morphology of the supra-renal capsules. (Abstract.) Evolution of the lungs. (Abstract.) Proc. Amer. Assoc. Adv. Sci., vol. 34, 311313.

The early stages of human development. Part 1 . Ova of the second week of pregnancy. New York Med. Journal, vol. 42, pp. 197-2(X).

Review of Behren's "The microscope in botany," translated by A. B. Hervey. Boston Med. and Surg. Journal, vol. 113, p. 235.

Darwin's biography. (Review of Krause's Charles Darwin.) Science, vol. 6, pp. 276-277.

Reference Handbook of the Medical Sciences, edited by A. H. Buck. N. Y., Wood & Co., vol. 1: Articles on Age; Allantois; Ammion; Area embryonalis; Bioplasson; Blastoderm; Blastojiore.

The early stages of human development. Part II. Embryos of the third week. New York Med. Journal, vol. 42, pp. 396-401 ; 426-431.

1886 Reference Handbook of the Medical Sciences, vol. 2: Chorion; Coelom;

Decidua; Ear, Development of; Ectoderm; Embryology; Entoderm;

Evolution of man. The rotifera.

Structure of the human skin. Amer. Naturalist, vol. 20, pp. 675-678. Report on histology and embryology. Boston Med. and Surg. Journal,

vol. 114, pp. 460-463. The physical basis of heredity. Science, vol. 8, pp. 125-130. Notes on histological technique. Zeitschr. f. wiss. Mikroskopie u. f . mikr.

Technik, Bd. 3, pp. 173-178. The nimiber habit. Proc. Amer. Soc. Psych. Research, vol. 1, pp. 86-95. Reference Handbook of the Medical Sciences, vol. 3: Foetus, Development

of; Gastrula; Germ layers; Growth. Zur Kenntniss der Insektenhaut. Arch, f . mikr. Anatomie, Bd. 28, pp. 37 48. Taf. VII. W. A. Locy's Embryologie der Spinnen. Biol. Centralbl., Bd. 6, pp. 569 562. Muscle-reading by Mr. Bishop. Science, vol. 8, pp. 506-507. Researches on snake-poison. Boston Med. and Surg. Journal, vol. 115,

pp. 554-555. Whence come race characters? Science, vol. 8, pp. 623-624.

1887 Bemerkungen zu dem Schroder' schen Uteruswerke. Anat. Anzeiger, Bd.

2, pp. 19-22. American society for psychical research. Science, vol. 9, pp. 50-61. Youthfulness in science. Science, vol. 9, pp. 104-105.

Reference Handbook of the Medical Sciences, vol. 4: Impregnation; Longevity; Meconium; Mesoderm. Vol. 6: Notochord; Ovum; JJeurenteric canals; Placenta, Anatomy of.

Report on histology and embryology. Boston Med. and Surg. Journal, vol. 116, pp. 520-523.

American microscopes — a complaint. Science, vol. 10, pp. 275-276.

First report of the committee on experimental psychology. (Prevalence of superstitions.) Proc. Amer. Soc. Psych. Research, vol. 1, pp. 218223.

1888 Tricks in mind reading. Youth's Companion, vol. 61, p. 122.

The mounting of serial sections. The Microscope, vol. 8, pp. 133-138.

Growth and age. Annual of the Medical Sciences, edited by C. E. Sajous, vol. 5, pp. 359-366.

Reference Handbook of the Medical Sciences, vol. 6: Proamnion; Segmentation of the body; Segmentation of the ovum; Senility; Sex; Spermatozoa.

1889 Reference Handbook of the Medical Sciences, vol. 7: Umbilical cord.

Vol. 8: Yolk-sac. Growth and age. Annual of the Medical Sciences, vol. 2, Section L, pp. 1-2. Second report on experimental psychology: Upon the diagram tests.

Proc. Amer. Soc. Psych. Research, vol. 1, pp. 302-317. Open letter concerning telepathy. Proc. Amer. Soc. PsycA. Research, vol.

1, pp. 547-548. Uterus and embryo. I. Rabbit. II. Man. Joum. Morphol., vol. 2, pp.

341-462. PI. XXVI-XXIX. Segmentation of the ovum with especial reference to the mammalia. Amer.

Naturalist, vol. 23, pp. 463-481 ; 753-769. Evolution of the medullary canal. Amer. Naturalist, vol. 23, pp. 1019 1021.

1890 The use of the microscope and the value of embryology. Canadian Prac titioner, vol. 15, pp. 43-46. National medical dictionary by John S. Billings, assisted by Dr. C. S.

Minot and others. 2 vols. Philadelpnia, Lea. 1890. Die Placenta des Kaninchens. Biol. Centralbl., Bd. 10, pp. 114-122. Die Entstehung der Arten durch r&umliche Sonderung. Von Moritz Wagner. (Review.) Science, vol. 16, pp. 305-306. Growth and age. Annual of the Medical Sciences, vol. 2, Section N, pp.

1-4. The concrescence theory of the vertebrate embryo. Amer. Naturalist,

vol. 24, pp. 501-516; 617-629; 702-719. The mesoderm and the coelom of vertebrates. • Amer. Naturalist, vol. 24,

877-898. Zur Morphologie der Blutkorperchen. Anat. Anzeiger,Bd. 5, pp. 601-604.

Translation of the same, Amer. Naturalist, vol. 24, pp. 1020-1023. About worms. Youth^s Companion, vol. 63, p. 681. On the fate of the hu^ian decidua reflexa. Anat. Anzeiger, Bd. 5, pp.

639-643. On certain phenomena of growing old. Proc. Amer. Assoc. Adv. Sci., vol. 39, 21 pp.

1891 A theory of the structure of the placenta. Anat. Anzeiger, Bd. 6, pp.

125-131. Senescence and rejuvenation. First paper: On the weight of guinea pigs.

Joum. of Physiol., vol. 12, pp. 97-153. PI. II-IV. Growth and age. Annual of the Medical Sciences, vol. 2, Section N, pp. 1-7.

1892 Human embryology. New York. William Wood and Company. 8°.

XXVI + 815 pp., 463 figs. (Also the Macmillan Company, 1897).

1893 Structural plan of the human brain. Pop. Sci. Monthly, vol. 43, pp.

372-383. Bibliography of vertebrate embryology. Mem. Boston Soc. Nat. Hist., vol. 4, pp. 487-614.

1894 Gegen das Gonotom. Anat. Anzeiger, Bd. 9, pp. 210-213.

Lehrbuch der Entwickelungsgeschichte des Menschen. Deutsche Ausgabe mit Zusatzen des Verfassers von Dr. Sdndor Kaestner. Leipzig. Veit und Comp. XXXVI+844 pp., 463 figs.

1895 The psychical comedy. North Amer. Review, vol. 160, pp. 217-230.

If microscopes were more powerful. Youth's Companion, vol. 69, p. 78. The fundamental difference between plants and animals. Science, N. S.,

vol. 1, pp. 311-312. The work of the naturalist in the world. Pop. Sci. Monthly, vol. 47, pp.

60-72. Ueber die Vererbung und VerjUngung. Biol. Centralbl., Bd. 15, pp. 571 587.

1896 On heredity and rejuvenation. Amer. Naturalist, vol. 30, pp. 1-9 ; 89 101. The microscopical study of living matter. North Amer. Review, vol.

162, pp. 612-^620. Microtome automatique nouveau. Comptes rendus Soc. Biologie de Paris,

lOme S^r., vol. 3, pp. 611-612. The theory of panplasm. (Abstract.) Report of the Brit. Assoc. Adv.

Sci., vol. 66, pp. 832-833. The olfactory lobes. (Abstract.) Report of the Brit. Asso. Adv. Sci. vol. 66, p. 836.

On the principles of microtome construction. Report of the Brit. Assoc. Adv. Sci., vol. 66, pp. 979-980.

1897 Our unsymmetrical organization. The Harvard Graduates' Magazine, vol.

5, pp. 485-491. On two forms of automatic microtomes. Science, N. S., vol. 5, pp. 857 866. Bibliography — A study of resources. Biological lectures delivered at the

Marine Biological Laboratory of Wood's Hole in the summer session

of 1895. Boston. Pp. 149-168. Cephalic homologies. A contribution to the determination of the ancestry

of vertebrates. Amer. Naturalist, vol. 31, pp. 927-943. Die friihen Stadien und die Histogenese des Nervensy stems. Ergebnisso

der Anat. u. Entwickelungsgeschichte, Bd. 6, pp. 687-738.

18d8 Contribution k la determination des ancetres des vertebras. (Traduction de M. E. Brumpt, des Hautes £tduto.) Arch. Zool. exper., S^r. 3,

vol. 5, pp. 417-436. On the veins of the WolflSan body in the pig. Proc. Boston Soc. Nat. Hist.,

vol.28, pp. 265-274. PL I. A memento of Professor Edward D. Cope. Science, N. S. vol. 8, pp. 113 114.

1899 Knowledge and practice. Science, N. S., vol. 10, pp. 1-11.

1900 On a hitherto unrecognized form of blood circjulation without capillaries

in the organs of vertebrates. Proc. Boston Soc. Nat. Hist., vol. 29, pp. 185-215.

On the solid stage of the large intestine in the chick with a note on the ganglion coli. Journ. Boston Soc. Med. Sci., vol. 4, pp. 153-164.

Ueber mesotheliale Zotten der Allantois bei Schweinsembryonen. Anat. Anzeiger, Bd. 18, pp. 127-136.

The unit system of laboratory construction. Philadelphia Med. Journ., vol. 6, pp. 390-391.

The study of mammalian embryology. Amer. Naturalist, vol. 34, pp. 913941.

1901 Notes on Anopheles. Journ. Boston Soc. Med. Sci., vol. 5, pp. 325-329.

PI. XXXI. Further study of the unit system of laboratory construction. Science, N.

S., vol. 13, pp. 409-415. The embryological basis of pathology. The Middleton Goldsmith lecture

delivered before the New York Pathological Society, March 26, 1901.

Science, N. S., vol. 13, pp. 481-498. Also Boston Med. and Surg. Journal, vol. 144, pp. 295-305. SoUen die Bezeichnungen ** Somatopleural und " Splanchnopleura" in

ihrem ursprUnglichen richtigen oder in dem in Deutschland gebrauch lich gewordenen Sinne verwendet werden? Anat. Anzeiger, Bd. 29,

pp. 203-205. Improved automatic microtomes. Journal Appl. Microscopy, vol. 4, pp.

1317-1320. Remarks (made at the opening session of the A. A. A.S. at Denver, Aug.

26, 1901). Science, N. S., vol. 14, pp. 357-360. On the morphology of the pineal region, based on its development in Acan thias. Amer. Journ. Anat., vol. 1, pp. 91-98.

1902 The relation of the American Society of Naturalists to other scientific

societies. Science, N. S., vol. 15, pp. 241-244.

Convocation week. Harvard Graduates' Magazine, vol. 10, pp. 348-351.

The distribution of vacations at American universities. Science, N. S., vol. 15, pp. 441^444.

The problem of consciousness in its biological aspects. Science, N. S.. vol. 16, pp. 1-12. Also, Nature, vol. 66, pp. 300-304; Proc. Amer. Assoc. Adv. Sci., vol. 51, pp. 265-283. Translation, Revue Scientifique, S^r. 4, vol. 18, pp. 193-200.


1903 A laboratory text-book of embryology. Philadelphia. Blakiston. XVII +380 pp., 218 figs. The history of the microtome. Journ. of Applied Microscopy, vol. 6, pp. 2157-2160; 2226-2228.

1905 The implantation of the hmnan ovum in the uterus. Trans. Amer. G3me cological Soc, 1904, pp. 395-402. Genetic interpretations in the domain of anatomy. Presidential address

before the Association of American Anatomists. Amer. Journ. Anat.,

vol. 4, pp. 245-263. The Harvard embryological collection. Journ. Med. Research, vol. 8,

pp. 499-522. PI. XXXIX. Charles S. Minot and Ewing Taylor. Normal plates of the development

of the rabbit (Lepus cuniculus, L.). KeibeFs Normentafeln zur

Entwickelungsgeschichte der Wirbelthiere, Heft 5, 98 pp. PI. I-III. Jena. 4**.

1906 The relations of embryology to medical progress. Pop. Sci. Monthly, vol. 69, pp. 5-20.

1907 The segmental flejoires of the notochord. Anat. Record, vol. 3, pp. 42-50. The problem of age, growth and death. Pop. Sci. Monthly, vol. 70, pp.

481-496; vol. 71, pp. 97-120; 193-216; 359-377; 455-473; 509-523.

1908 Theproblemof age, growth, and death. A study of cytomorphosis. Based

on lectures at the Lowell Institute, March, 1907. New York. Putnam's Sons. XXII+280 pp., 73 figs.

1909 Certain ideals of medical education. Journ. Amer. Med. Assoc, vol. 53,

pp. 502-508. The inheritance of ability. Youth's Companion, vol. 83, pp. 471-472.

1910 A laboratory text-book of embryology. 2d edition, revised. Blakiston.

XII+402 pp., 262 figs.

1911 The method of science. Science, vol. 33, pp. 119-131. Nature, vol. 86,

pp. 94-97.

Henry Pickering Bowditch. Science, vol. 33, pp. 598-601.

Notes on the blastodermic vesicle of the opossum. Anat. Record, vol. 5, pp. 295-^00.

Die Entwicklung des Blutes, des Gefasssystems und der Milz. I. Die Entstehung des Angioblastes und die Entwicklung des Blutes. Keibel-Mall, Handbuch d. Entwicklungsgeschichte d. Menschen," Band II. Leipzig. Pp. 483-517. (Also in the English edition, 1912, vol. 2, pp. 498-534).

1912 Antrittsrede. Berliner Akadem. Nachrichten, vol. 7, pp. 31-33. Sci ence, vol. 36, pp. 771-776.

1913 Die Methode der Wissenschaft und andere Reden. (Uebersetzt von Dr.

Joh. Kaufmann.) Jena. Fischer. 205 pp.

Modeme Probleme der Biologie. Vortrage, gehalten an der Universitat Jena in Dezember, 1912. Jena. Fischer. Vl-flll pp., 53 figs.

Die Entwickelung des Todes. Abschiedsrede. Berliner Akad. Nachrichten, vol. 7, pp. 128-134.

A tribute to Joseph Leidy. Science, vol. 37, pp. 809-814.

Modem problems of biology. Lectures delivered at the University of Jena, December, 1912. Phila. Blakiston. IX+124 pp., 53 figs.


BIOGRAPHICAL NOTES AND RESOLUTIONS CONCERNING DR. MINOT

American Association for the Advancement of Science. Minute adopted in memory of Dr. Charles Sedgwick Minot. Science, 1915, vol. 41, p. 59.

American Association of Anatomists. Resolutions on the death of Professor Charles S. Minot. Anat. Record, 1915, vol. 9, pp. 42-43.

American Society of Zoologists. A memorial presented at the Colimibus meeting, Dec. 1915, will be published with the Proceedings, in Science, 1916.

Abbott, Maude E. Bull. Intern. Assoc. Med. Mus., 1915, No. 5, pp. 16-17.

Allen, C. Frank. The Technology Review, 1915, vol. 17, pp. 91-95.

Bremer, John Lewis. Harvard Grad. Mag., 1915, vol. 23, pp. 375-378.

Donaldson, Henry H. Science, 1914, vol. 40, pp. 926-927.

Donaldson, Henry H. Proc. Boston Soc. Nat. Hist., 1915, vol. 35, pp. 79-93.

Eliot, Charles W. Science, 1915, vol. 41, pp. 701-704; also in Proc. Boston Soc. Nat. Hist., 1915, vol. 35, pp. 89-93.

Lewis, Frederic T. Boston Med. and Surg. Journ.,1914, vol. 171, pp. 911-914.

Porter, W. T. Boston Med. and Surg. Joum., 1915, vol. 172, pp. 467-470.


Proceedings Of The American Association Of Anatomists

Thirty-Second Session

At the Osbom Zoological Laboratory of Yale University y New Haven, Conn., December 28, 29 and SO, 1915

Tuesday, December 28, 9.30 a.m.

The thirty-second session of the American Association of Anatomists was called to order by President G. Carl Huber, who appointed the following committees:

CemmiUee on Nominations for 1916: R. R. Bensley, chairman; F.^T. Lewis, J. B. Johnston.

Auditing Committee: C. M. Jackson, chairman; S. Walter Hanson.

The morning session for the reading of papers concluded with an address by the Vice-President, Professor F. T. Lewis, A biographical sketch of Professor Minot.

Wednesday, 12.30 p.m., Association Business Meetjng, President G. Carl Huber, Presiding.

The Secretary reported that the minutes of the Thirty-first Session were printed in full in The Anatomical Record, volume 9, number 1, pages 35 to 143, and asked whether the Association desired to have the minutes read as printed. On motion, seconded and carried, the minutes of the Thirty-first Session were approved by the Association as printed in The Anatomical Record.

Prof. C. M. Jackson reported for the Auditing Committee as follows: The undersigned Auditing Committee has examined the accounts of Dr. Charles R. Stockard, Secretary-Treasurer

165

THB ANATOMICAL BBOOBD* VOL. 10, NO. S

166 AMERICAN ASSOCIATION OF ANATOMISTS

of the American Association of Anatomists and finds the same to be correct with proper vouchers for expenditures and bank balance on December 21 of $264.09. (Signed) C. M. Jackson, S. W. Ranson, N^ Haven, Conn., December 29, 1915. The Treasurer made the following report for the year 1915:

Balance on hand December 23, 1914, when accounts were last

audited $285.85

Receipts from dues 1915 1693.81

Total deposits $1979.66

Expenditures for 1915:

Postage 33.00

Printing and Stationery 37.25

Collection and Exchange 2.32

Expenses of Secretary-Treasurer, St. Louis Meeting 90.00

Wistar Institute for subscriptions, Jour. Anat., and Anat.

Record 1546.00

Return on over paid check 2. 50

Debit unhonored check 4.50

Total expenditures, 1915 1715.57

Balance on hand $264.09

Balance on hand, deposited in the name of the American Association of Anatomy in the Corn Exchange Bank, New York City, December 21, 1915.

On motion the reports of the Auditing Committee and the Treasurer were accepted and adopted.

The Committee on Nominations, through its Chairman, Prof. Ross G. Harrison, placed before the Association the following Dames: For President, Prof. Henry H. Donaldson; for Vice-President, Prof. Clarence M. Jackson; for members on the Executive Committee, terms expiring in 1919, Prof. Eliot R. Clark and Prof. Reuben M. Strong.

On motion, the Secretary was instructed to cast a ballot for the election of the above-named officers.

The Secretary presented the following names recommended by the Executive Committee for election to membership in the American Association of Anatomists:

PROCEEDINGS 167

Leslie B. Arby, Ph.D., Instructor in Anatomy, Northwestern University Medical School, £4^1 Dearborn Street ^ Chicago, Illinois.

Chables Baglet, Jr., M.D., Phipps Institute, Johns Hopkins Hospital, Baltimore, Maryland,

George Alfred Baitsell, Ph.D... Instructor in Biology, Yale University, New Haven, Connecticut.

Charles W. Bonnet, A.B., M.D., Demonstrator in Anatomy, Jefferson Medical College, Philadelphia, Pennsylvania.

John W. Broadnax, Ph.G., M.D., Associate Professor of Anatomy, Medical College of Virginia, Richmond, Virginia.

Charles Brookover, Ph.D., Professor of Anatomy, University of Arkansas, Little Rock, Arkansas.

Louis Casamajor, B.A., M.D., Assistant Professor of Neurology, Columbia University, 4S7 West 59th Stceet, New York City.

Harold Saxon Burr, Ph.B., Ph.D., Instructor in Anatomy, School of Medicine, Yale University, New Haven, Connecticut.

Robert Chabcbbrs, Jr., A.M., Ph.D., Assistant in Anatomy, Cornell University Medical College, New York City.

Charles Manning Child, Ph.D., Associate Professor of Zoology, University of Chfcago, Chicago, Illinois.

Wesley R. Coe, Ph.D., Professor of Biology, Sheffield Scientific School, Ycde University, New Haven, Connecticut.

Edgar Davidson Congdon, Ph.D., Assistant Professor of Anatomy, Leland Stanford University, School of Medicine, SSO Coleridge Avenue, Palo Alto, California.

Wera Danchakoff, M.D., Rockefeller Institute, New York City.

Samuel Randall Detwiler, Ph.D., Assistant in Biology, Yale University, ' New Haven, Connecticut.

D. H. DoLLBT, M.D., Professor of Pathology, University of Missouri, Columbia, Missouri.

Jules Duesberg, M.D., Research Associate, Carnegie Institution of Washington, Johns Hopkins Medical School, Baltimore, Maryland,

J. H. Globus, B.A., Assistant in Anatomy, Cornell University Medical College, New York City.

Francis Wenger He age y, A.B., M.D., Instructor in Anatomy, Columbia University, 4S7 West 59th Street, New York City.

James Peter E^ll, D.Sc, F.R.S., Todrell Professor of Zoology and Comparative Anatomy, University of London, University College, Gower Street, London, W, C, England.

Arthur Hofewell-Smith, L.R.C.P., M.R.C.S., L.D.S., Professor of Dental Histology Histo-Pathology, Comparative Odontology, University of Pennsylvania Denial College, Philadelphia, Pennsylvania.

Arthur Keith, M.D., LL.D., F.R.C.S.,F.R.S., Hunterian Professor of Anatomy, College of Surgeons, London, England,

John D. Kernan, Jr., A.B., M.D., Assistant in Anatomy, Columbia University, 4S7 West 59th Street, New York City.

J. A. Key, B.S., Instructor in Anatomy, Creighton Medical College, Omaha, Nebraska.

168 AMERICAN ASSOCIATION OF ANATOMISTS

Hugh McMillan Kinokrt, AJM., InBtructor in Histology and Embryology,

Cornell University , Ithaca, New York. William Babri Kibkham, Ph.D., Instructor in Biology, Sheffield Scientific School,

Yale University, New Haven, Connecticut, Henbt Laurens, Ph.D., Instructor in Biology, Yale University, New Haven,

Connecticut, Clarence E. McClung, A.M., Ph.D., Professor of Zodlogy, University of Pennsylvania, Philadelphia, Pennsylvania, M. M. Miller, Ph.D., Instructor in Anatomy, Vanderbilt University Medical

School, Nashville, Tennessee, Mae Lichtbnwalner Myers, M.D., Associate Professor of Anatomy and Director of the Laboratories of Histology and Embryology, Woman's Medical

College of Pennsylvania, North College Avenue and Hist Street, Philadelphia,

Pennsylvania. Theophilus S. Painter, Ph.D., Instructor in Biology, Sheffield Scientific School,

Yale University, New Haven, Connecticut. George Papanicolaou, Ph.D., M.D., Assistant in Anatomy, Cornell University

Medical College, New York City, Charles W. M. Potnter, B.S., M.D., Professor of Anatomy, College of Medicine,

University of Nebraska, Omaha, Nebraska. Sidney Sigsfried Schochbt, M.D., Instructor in Anatomy, Dartmouth Medical

School, Hanover, New Hampshire, John W. Scott, A.M., Ph.D., Professor of Zoology, University of Wyoming,

Laramie, Wyoming. M. Deforest Smith, A.B., M.D., Assistant in Neurology, Columbia, University,

JiS7 West S9th Street, New York City, 'Chester A. Stewart, A.M., Assistant in Anatomy, University of Minnesota,

Minneapolis, Minnesota, Alaric Collender Sutton, A.B., Student, Johns Hopkins Medical School,

BaltimorS, Maryland, F. J. Taintor, M.D., Assistant Professor of Anatomy, St, Louis University,

St. Louis, Missouri. Joseph M. ThI^nger, M.D., Assistant in Histology and Embryology, Harvard

Medical School, Boston, Massachusetts. Richard Watkin Watkins, B.S., Assistant in Anatomy, University of Chicago,

Chicago, Illinois. James Crawford Watt, B.A., M.B., Lecturer in Anatomy, University of Toronto,

to Hawthorne Avenue, Toronto, Canada. Theodora Wheeler, A.B., Medical Student, Johns Hopkins Medical School,

Baltimore, Maryland. George Bernays Wislocki, A.B., Student, Johns Hopkins Medical School,

Baltimore, Maryland.

On motion the Secretary was instructed to cast a ballot for all the candidates proposed by the Executive Commjttee. Carried.

Professor Harrison announced to the Association the death of Prof. Moritz Nussbaum which occurred on November 16.

PROCBEDINGS 169

Following this anDoimcement President Huber appointed a Conunittee: R. G. Harrison^ chairman; W. H. Lewis, W. M. Baldwin and Davenport Hooker to draw resolutions expressing the regret of the Association over the loss of Professor Nussbaum, one of its honorary members.

It was moved and voted that the Association express through its Secretary appreciation of the valuable aid the Wistar Institute is rendering the Association and the progress of anatomy in this country by its very generous and efficient publication of the anatomical journals. And further the Wistar Institute lends greatly to the success of the present meeting by its prompt publication and distribution of the abstracts of the communications to be presented.

On motion the offer of The Wistar Institute to supply all members of the Association, for the dues now paid to the joiunals, not only all numbers of The American Journal of Anatomy, and the Anatomical Record, but in addition The Joxu-nal of Comparative Neurology and the Journal of Morphology, was formally accepted.

Thursday, December 30. A Short Business Session Followed the Scientific Session.

The Committee through its Chairman, Professor Harrison, presented the following resolutions on the death of Professor Nussbaum:

Whereas, in the death on November 16, 1915, of Gteheimer Medizinalrat Doktor Moritz Nussbaum, Professor of Biology in the University of Bonn, this Association has lost from its roll of Honorary Members a man of keen intellect, profound learning, and broad sympathies, admired and beloved by all who came in contact with him or his work.

Be it therefore resolved, that we, The American Association of Anatomists, express our very deep sense of the loss of an honored coworker, teacher and friend and.

Be it further resolved, that this resolution be placed upon the permanent records of the Association, and that a copy be sent to his f ainily .

(Signed) Ross G. Harrison, Chairman, Warren H. Lewis, W. M. Baldwin, Davenport Hooker.

170 AMERICAN ASSOCIATION OF ANATOMISTS

The President then appointed Prof. Florence R. Sabin and Prof. J. P. Schaeflfer a committee to formulate resolutions expressing the loss of the Association in the death of Mrs. Susannah Phelps Gage.

The Committee presented the following resolutions:

The American Association of Anatomists, assembled at the Thirtysecond Session at New Haven, express profound sorrow at the death of Susannah Phelps Gage. For many years she has been a constant participant at the meetings of the Association and an active contributor to the Uterature of anatomy.

The members of the Association who came into more personal contact with Mrs. Gage are also mindful of the kind and generous help, advice, and sacrifice of time which she so unstintingly gave.

It is directed that the foregoing Minute be published in the Proceedings of the Thirty-Second Session, and that the Secretary be requested to send a copy to Mrs. Gage's family.

(Signed) Florence R. Sabin,

J. P. SCHABFFER.

Before the adjournment of the last session it was voted: That this Association express through the Secretary our thanks and appreciation to Yale University, the Local Committee and to Professors Harrison and Ferris our hosts at this session, for their generous hospitality and the perfect manner in which the Association has been accommodated and entertained.

Charles R. Stockard,

Secretary of the Thirty;-SeooQd Session of the American Aasociation of Anatonusts.

ABSTRACTS OF PAPERS

PRESENTED AT THE

THIRTY-SECOND SESSION

OF

THE AMERICAN ASSOCIATION OF ANATOMISTS

December 28, 29 and 30, 1915

AT

NEW HAVEN


All papers marked with an asterisk (•) were read by title only


1, Cell changes in the hypophysis of the albino raty after gonadedomy.

William H. F. Addison, University of Pennsylvania.

There is a striking alteration in the structure of the distal glandular portion of the hypophysis of the albino rat, following removal of the testes or of the ovaries. These changes are apparent during the second week after gonadectomy, but progress for several months, until the appearance of this portion of the hypophysis is conspicuously altered. The most evident difference is the development of large, vesicular cells, which have the cjrtoplasm arranged as a peripheral ring, containing in the center a homogeneous colloid-like material.

Alterations in the histological appearance of the hypophysis after removal of the sex gland were first described by Fichera ('05). In Biedl ('13) there is an illustration of the hypophysis of the castrated albino rat, showing, however, only the early changes.

The cells of the distal glandular portion of the hypophysis of the adult albino rat may be grouped in three classes. Already at birth histogenesis has proceeded far enough for these three varieties to be distinguished. In the new-bom rat most of the cells are of the primitive imdifTerentiated type, and from these, here and there, two other cell types (b and c) are seen developing. One (type b) contains distinct granules, which stain more than the other cells with Altmann's acid fuchsin, after neutral formalin zenker fixation (Bensley '11, Am. Jpum. Anat.). These are the oxyntic or acidophile cells. The other type of

171

172 AMERICAN ASSOCIATION OF ANATOMISTS

cell (type c) is distinguished by its larger amount of cytoplasm, which is only finely granular in early life.

At forty-five days of age, the organ has greatly increased in size. The direct descendants of the primitive cells form masses of cells with closely aggregated nuclei and small amount of c3rtoplasm. These cells (type a) form the principal mass of the cell cords between the blood vessels, and often receive the name of 'chief cells.' The acidophile cells (type b) have greatly increased in number and their granules stain more intensely. The large cells (type c) are also more numerous. These two latter varieties appear to be always on or near the margin of the blood streams.

These three types are believed to be the main varieties of cells foimd in the adult, the primitive type giving rise to the chief cells and also to the other two kinds. As age progresses the large cells (type c) often develop in their cjiioplaan one or more smaU vacuoles, and, sometimes a denser spot, which is perhaps connected with the secretory activity of the cell, appears in the cytoplasm. As age increases, also, the granules of the cytoplasm of the type c cells are more apparent and tend to stain somewhat more readily with basic dyes.

After castration (e.g., at thirty days of age) changes are seen at the end of one week and at the end of two weeks these changes become clearly apparent. The acidophiles (type b) remain nearly unchanged in number and size, but the large cells (type c) are definitely enlarged and more granular. After the lapse of several months, however, a much more striking alteration is seen. Some of the large cells (type c) are seen to have increased in size, developing a large vacuole or space in which a clear colloid-like substance is present. The small chief cells (type a) appear to be decreased, probably some of their number growing into the large cells (type c). Several stages are apparent in the growth of the large vacuolated cells. 1) Some of the cells retain the nucleus of spherical shape, the C3rtoplasm granular, and in it a small vacuole and perhaps also a small denser spot. 2) As the vacuole or space, with the colloid-like material becomes larger the nucleus becomes eccentric, the main part of the cell being made up of the colloid-likecontaining space. 3) Finally the cells become ring-shaped in section, the cytoplasm forming the periphery of the cell and containing at some point the flattened nucleus. Nearly the entire cell is filled with the colloid-like material. Often the denser spot remains in the peripheral cytoplasm near the nucleus.

In the normal albino rat sometimes small amoimts of the colloidlike substance are seen in the vascular channels and in the residual lumen of the pouch of Rathke. In the castrated animals, however, the amount of this substance is increased and it is more often found in the blood vessels and in the persistent lumen.

In female animals the reaction in the cells of the hypophysis after removal of the ovaries follows the same general course as does the reaction in the hypophysis of the castrated male animals.

PROCEEDINGS 173

B. On the fate of the ^^Ultimcbranchial bodies" in the pig. J. A. Badbbt SCHER, University of Indiana.*

The considerable amount of work of both a comparative and more specific character that has been done in recent years on the ultimobranchial bodies has led to the interpretation that they do not contribute any structural elements to the mammalian thyroid gland. There is, however, still wanting a detailed histogenetic study through a wide range of developmental stages of well fixed mammalian embryos to give conclusive evidence of the fate of these bodies. The present investigation is concerned with the histogenetic changes that occur in the ultimobranchial elements imtil they are no longer recognizable in the thyroid gland.

The ultimobranchial bodies in early stages are hollow syncytial structures. Before fusion with the thyroid occurs their lumina become . partially obliterated and their walls become partially vacuolar. The thyroid in early stages breaks up into cell cords while in the ultimobranchial bodies this process begins first in 21 to 23 mm. pig embryos or in even later stages. The cell cords when first formed are usually much coarser than those found in the thyroid to which they are fused and apparently are produced in two ways: a) by the invasion of mesenchymal and vascular tissues and b) by the growth of buds from the main stem. The extent to which 'budding' occurs is quite variable in different developmental stages. The posterior part of the ultimobranchial elements usually remains a solid mass longer than their more anterior portion.

The extent to which vacuolation of these bodies occurs varies greatly. In an 18 mm. embryo in which they are not yet fused to the thyroid, the vacuoles are mostly confined to their more central portion. In later stages (up to 125 mm. or thereabouts) after the structure has become partially or entirely broken up into cell cords, the vacuoles are confined mostly to portions of the main 'stem' and to the proximal portion of some of the coarse cell cords attached to it, but not necessarily to the entire structure which, according to recent investigations, is the case in young stages of hmnan embryos.

The structiu'es of the nuclei of the ultimobranchial bodies is uniform 'up to stages 18 mm. in length and cannot be distinguished from the nuclei of the thyroid gland. In the ultimobranchial bodies of a 19 nmi. embryo a few deeply stained nuclei are present. These increase in number and are most numerous in embryos ranging from 21 to 33 mm. in length. In later developmental stages (48 to 125 mm.), in which the ultimobranchial bodies can be distinctly recognized as such, the deeply stained nuclei have almost entirely disappeared. Pyknotic or degenerating nuclei were found in appreciably large numbers in localized areas in the ultimobranchial bodies of 23 and 24 mm. embryo, and in later developmental stages they have almost entirely disappeared. The significance of the deeply stained nuclei is still unknown to me but I am quite certain that they do not represent a general degeneration of the ultimobranchial elements as is held by Simon, Verdun and others.

174 AMERICAN ASSOCIATION OF ANATOMISTS

The nuclei of the ultimobranchial bodies in some stages are quite variable in size but their structure (excepting the darkly stained ones) is the same as of those of the thyroid.

The location of the ultimobranchial bodies in the thyroid is variable. In stages from 19 to 28 nam. in length they are fused to and partially or entirely imbedded in the dorso-lateral margin of the gland. In the later developmental stages examined they may be of greatly unequal length in the same stage, and variable in position in different stages. They may be entirely located in the middle third or two-fourths of the gland or entirely in its caudal half or third. Their usual position in the thyroid is lateral to the median line, more or less deeply imbedded below the dorsal border but may be found imbedded near its lateral border.

In a few stages one or both of the ultimobranchial bodies were found only partially imbedded in the thyroid. Thus, in a 45 mm. embryo their anterior ends remain separated from the thyroid gland. Except- . ing for a small vacuolar area and traces of the lumen in the left one, both have a structure identical with the thyroid gland. In a 60 mm. embryo the ultimobranchial element on the right side is only partially imbedded in the dorso-lateral border of the posterior third of the thyroid and is represented by coarse and loosely arranged cell cords. In a 125 mm. embryo the right ultimobranchial body is fused to the middle two-fourths of the lateral border of the thyroid but not imbedded in it. It is composed of syncytial masses and of large tortuous cystoid or overgrown follicles which do not contain colloid, although colloidcontaining follicles are quite uniformly distributed throughout the thyroid. In a 145 mm. embryo the right ultimobranchial element lies lateral to the medial line of the thyroid along its dorsal surface. Along its entire extent it is partly exposed to the free surface. The portion imbedded in the gland is represented by cell cords in which small follicles containing colloid are quite numerous. These cell cords are identical in structure with those in the thyroid of earlier stages in which colloid is just beginning to form. In the exposed part of tWs structure are cystoid follicles in which colloid is present. Also, along the right lateral border of the posterior two-thirds of the thyroid in a 245 nun. embryo is an area of many cystoid follicles among which are scattered smaller follicles, all of which contain colloid. This area occupies a similar position to the ultimobranchial bodies in some earlier stages and on account of its many cystoid follicles it apparently represents an ultimobranchial element that was only partially imbedded in the thyroid gland.

In late developmental stages no cystoid follicles were found in entirely imbedded ultimobranchial bodies. In the thyroid, follicles containing colloid appear first in a 75 mm. embryo while in the ultimobranchial elements the colloid-containing follicles appear first in a 145 nrni. stage. In a few late developmental stages (including a full term embryo) it was impossible to recognize the ultimobranchial bodies by structural features, but in all (excepting a 175 mm. embryo) areas of small colloid-containing follicles are present. The position of these areas

PROCEEDINGS 175

in the thyroid, in different stages, is variable but they correspond to the variable position of the ultimobranchial bodies in stages in which these bodies can be recognized structurally.

The observations indicate that the now generally accepted view of the fate of the ultimobranchial bodies in all classes of mammals is erroneous. In the pig they apparently contribute to the formation of the structural elements in the thyroid gland.

3, The origin and structure of a fibrous tissue formed in wound healing.

(Illustrated and with demonstrations.) George A. Baitsell,

introduced by R. G. Harrison, Yale University.

In a previous paper (Jour. Exp. Med., vol. 21, pp. 455-479) the author was able to show that in living cultures of adult frog tissues there occurs, in many instances, a transformation of the plasma clot in which the living tissue is imbedded. This transformation results in a consoUdation or fusion of the elements of the fibrin net and a consequent formation from it of a fibrous tissue which is identical in its form and structure and in many of its staining reactions with a regular coUaginous connective tissue. It appeared evident that, whether or not the fibrous tissue directly formed from the fibrin clot remained as a permanent connective tissue, such a reaction must play an important part in wound healing. The present paper gives the results obtained from an extensive series of experiments undertaken with the purpose of studying the action and fate of the fibrin clot formed during wound healing and they may be briefly summarized as follows:

1. In experimental wounds in frog skin made by removing various sized pieces of skin from the animal there is a rapid coagulation of the blood plasma and lymph to form a coagulation tissue which fills the -wound cavity.

2. The observations on living animals show that if the animal remains quiet for a few minutes after an operation the coagulation tissue becomes resistant and is of suflBcient strength to hold the cut edges of the wound in place and to retain its position in the wound cavity. The coagulation tissue is also resistant to the action of water and the animal can be placed in an aquarium a few minutes after an operation has been performed without any injury to the coagulation tissue which serves, at least temporarily, as a connective tissue and as a base for the epithelial cells which rapidly move in from all the cut edges to close the wpimd cavity. Observations have been made on the heaUng of various types of wounds viz., 1) wounds in which only a cut was made in the skm and no tissue was removed, 2) wounds in which pieces of skin of various sizes were removed and 3) wounds in which pieces of skin were removed and either another piece of skin or the stratum compactum obtained by pancreatin digestion of a piece of adult frog skin were implanted. In all these types of wounds the coagulation tissue formed by the clotting of the lymph or blood plasma was an essential feature of the rapid healing which occurred since it filled the wound cavity and held all the cut edges in their proper positions.

176 AMERICAN ASSOCIATION OP ANATOMISTS

3. The study of prepared sections show that, at first, in the coagulation tissue, formed in the wound as a result of the clotting of the blood and lymph, a typical fibrin net is present. Later this fibrin net is transformed into fibrous tissue containing bundles of wavy fibers in which, in many instances, the individual fibrils can be noted. In the course of a few days the entire clot becomes transformed into this new fibrous tissue. This transformation of the clot and the formation of the fibrous tissue is not due to any intracellular action inasmuch as it take place before the connective tissue cells wander into the coagulation tissue. It is a direct transformation of the fibrin clot and is identical with the process which was previously found (loc. cit.) to take place in the fibrin clots in living cultures of adult frog tissues and which has been noted above. The connective tissue cells which wander into the newly formed fibrous tissue from the cut edges do not digest the fibers but move among them and evidently cause a breaking up of the larger bundles of fibers into smaller ones and later we have formed, aS a result, a tissue which is typical in appearance with regular connective tissue. The connective tissue cells are, in many cases, rounded cells when they first appear in the fibrous tissue but later they assume the typical elongated spindle shape of fibroblast cells. This change in shape appears to be due to the action of the cells in stretching along the fibers. The preparations do not show any connection between these spindle shaped cells and the fibers which had already formed, nor is there any evidence of an attempt by them to form new fibers intracellularly.

4. The staining reactions of the new fibrous tissue appear to be identical with the staining reactions of the connective tissue in frog skin. However the newly formed fibrous tissue can be digested in pancreatin and in this reaction it differs from the connective tissue in the skin of the adult frog. On the other hand extensive experiments with pancreatin on embryonic but fully formed connective tissue, obtained from the tail and skin of tadpoles of various ages, show that the pancreatin will digest it just as it does the newly formed fibrous tissue.

5. The experiments do not offer any evidence that the fibrous tissue formed by a direct transformation of the fibrin net is ever replaced by a connective tissue formed by an intracellular action . The newly formed fibrous tissue also appears to be identical in appearance, structure and function with regular coUaginous connective tisue. Digestion with pancreatin will differentiate between the new tissue and adult connective tissue from the skin of the frog but this test fails to differentiate between the new tissue and embryonic connective tissue.

4. A standard of measurement in determining the relative size of the heart,

C. R. Bardeen, University of Wisconsin.

The weight of the heart varies with the weight of the body according to the formula:

B = ah,

where B = weight of the body, h = weight of heart and a is a constant.

PBOCEEDINGS 177

According to Vierordt's table based on 590 cases, a equals about 200 for all ages from one month up to 25 years. While other values have been given by various investigators it is piobable that this figure is approximately correct when the heart is carefully freed from blood before weighing. There are, of course, individual variations but the average is remarkably constant.

The area of the shadow of the heart, when in parallel light rays perpendicular to the dorsal or ventral surface, varies with the weight of the heart according to the formula:

m^ = xh,

where H == the area of the heart shadow; h = weight of the heart and

X is a constant, depending on the shape of the heart. While data on a

large nmnber of cases are lacking observation on foiur hearts has shown

that the average heart shadow was 81.89, the average weight 0.196 k.

so that

lp/2

X = ^ = 3800 ri

The area of the X-ray shadow of the heart when studied by means of the orthodiagraph or in radiographs, after making suitable allowance for the divergene of the X-rays, varies with the weight of the body according to the formula:

B = ?iff3/2 X

where B = weight of body; H area of heart shadow; a the constant

proportion of heart to body weight and a; = a constant depending on the

shape of the heart.

By a study of a tabulation based on several hundred cases it appeared

a 1 that the formula -=jr;^ would best fit the observations. This formula X 20

agrees fairly well with the values for a and x given above and arrived at

by diflferent methods.

If a = 200, then x = 4000 If X = 3800, then a = 190

Plotting a curve, according to the formula 5 = -jV IP^^ and analyzing* the results shows that the heart shadow of a person weighing 50 k. (110 lbs.) should be 100 sq. cm., and that of a child weighing 20 k. (45 lbs.) should be about 55 sq. cm., therefore for weights below 50 k. subtract l\ sq. cm. for each kilo {2\ lbs.) At 10 k. the size of the heart shadow according to this ciurve should be 35 sq. cm. Therefore for each kilo below 20 k. (heart area 55 sq. cm.), down to 10 k. subtract 2 sq. cm. At 66 k. the area of the shadow is approximately 120 sq. dm. Therefore for weights between 50 k. (110 lbs.) and 66 k. (145 lbs.) add 1.25 sq. cm. for each kilo (2| lbs.) For weights above 66 k. add 1.1 sq. cm. for each kilo. At 120 k. (265 lbs.) the area of the heart shadow according to this ciure should be 180 sq. cm.

178 AMERICAN ASSOCIATION OF ANATOMISTS

To test out the value of this curve for which together with other mathematical aid, I am indebted to my colleague, Prof. Max Mason, I have retabulated such available trustworthy data as have been at hand. First Dietlens 261 cases (74 women, 187 men), used in his valuable orthodiagraphic studies of the normal heart, second 123 of Schieflfers 125 cases used in his orthodiagraphic studies of the effects of severe muscular work on the heart and third 61 cases of my own studied by the teloradiographic methods (4 children, 38 normal adults, and 19 healthy athletic students), in all 445 cases. These cases have been plotted individually, then tabulated in averages for each kilogram of weight and finally averaged for each ten kilograms for the sake of simplifying the plotting of the curves.

The curves plotted from the combined data follows closely the theoretical curve from 18 k. (39 lbs.), 50 sq. cm. up to 45 k. (99 lbs.), 94 sq. cm. From here it diverges slightly toward proportionately larger areas than called for by a given weight up to 75 k. (165 lbs.) 136 sq. cm. At this point the average heart area is 5 sq. cm. larger than called for by the theoretical curve 131 sq. cm. Beyond this point the curve diverges in the opposite direction so that at 83 k. (183 lbs.) the average area (132.4 sq. cm.) is 7^ cm. smaller than called for by the theoretical curve (140 sq. cm.). At 94 k. (207 lbs.) the average area is 145 sq. cm., seven less than called for by the theoretical curve (152 sq. cm.).

The divergence of the curve toward the larger areas for weights between 45 k. and 75 k. is undoubtedly due to the inclusion of the relatively large number of Schieflfer's cases which were selected to study the effects of hard muscular exercise on the heart and were shown by Schieflfer to prove that the hearts of those engaged in physically severe occupations or took part in hard bicycle riding are larger than the hearts of average individuals. This is shown by the fact that the curve based on averages of Dietlen's 187 normal men varies not more than the distance which represents a square centimeter from the theoretical curve from 45 k. up to 75 k. Beyond here the curve diverges like the general curve sharply toward a smaller size than called for by the theoretical curve. The average of Dietlen's four cases weighing 80 k. or more is 128 sq. cm. while the theoretical curve calls for 141 sq. cm. for the same average weight.

This divergence is also shown slightly less marked in the averages of my own cases of normal men, the curve of which corresponds closely to the theoretical curve up to 75 k. In case of athletes and of those who follow severe occupations the hearts of individuals of average weight are as a rule considerably larger than called for in the theoretical curve but in such individuals above 75 k. (165 lbs.) in weight the size of the heart shadow approaches closely to that called for by the normal curve. This divergence from an average area much greater than the theoretical normal approximately to the theoretical normal is shown in SchieflPer's cases and in the athletes studied by me. The relatively smaller hearts thus found in heavy individuals can best be accounted for, I believe, by the facts that as a rule individuals who approach 85 k. (187 lbs.)

PBOCEBDINGS 179

or more in weight have relatively more fat and less muscle than those of lighter weight and that the size of the heart is strictly speaking determined rather by the development of the skeletal musculature than by mere body weight, although the latter alone is subject to direct measurement.

This probably also accounts for the fact pointed out by Dietlen that tall individuals of a given weight are apt to have larger hearts than shorter individuals of the same weight. I have not attempted to plot a curve from this point of view but the curve showing the average area of the heart shadow for women probably illustrates the same thing. A woman weighing 50 k. (110 lbs.) as a rule is shorter and fatter than a man of the same weight. The average size of the heart shadow of women averaging 45 k. (99 lbs.) coincides with the theoretical size called for by the curve (92 cm.). But the average heart shadow area of women averaging 55 k. (121 lbs.) in weight is 7 sq. cm. below that called for by the theoretical ciurve (about 100 instead of 107 sq. cm.) and for those averaging 63 k. (138 lbs.) in weight the divergence is still greater (106 sq. cm. instead of 115.5 sq. cm.).

While we have as yet insufficient data thoroughly to test out the curve, especially for individuals below 40 k. (88 lbs.) and above 75 k. (165 lbs.) in weight it gives promise of furnishing a useful standard with which to compare the heart shadow of an individual of a given weight and thus to determine whether or not his heart is of normal size and if not to what extend it deviates from the normal. In carrying out such a study many factors must betaken into account beside those mentioned, such for instance as the shape of the chest, but certainly far more accuracy is now possible than in the standard clinical method of guessing at the amount of cardiac hypertrophy from the relation of the "apex beat" to the nipple line.

5. The devehpment of the hypophysis in turtles, E. A. Baumgartner, Department of Anatomy, Washington University Medical School. In the present work embryos of Chrysemys marginata and Aromochelys odorata were used as the basis of the material for observations on the development of the hypophysis. Chrysemys picta and Pseudemys elegans were the forms chosen for the study of the adult hypophysis.

The first definite outpouching of the hypophysis as observed in 3 mm. embryos consists of a single median evagination of the epithelium of the roof of the mouth. This outpouching is similar in form to that described for other vertebrates and constitutes the so-called Rathke's pouch. In a 5 mm. embryo two additional phases in the development of the hypophysis become evident — the formation of two lateral buds from Rathke's pouch and a thickening of the epithelium anterior to the pouch. These two anlagen, together with the median pouch, constitute three primary structures participating in the formation of the adult organ.

Embryos of 7 mm. Of the three structures, the epithelial thickening has undergone a marked evagination including the roof of the mouth and the base of Rathke's pouch; it is the beginning of the anterior

180 AMERICAN ASSOCIATION OF ANATOMISTS

lobe of the adult. This, together with Rathke's pouch and the lateral buds is partially constricted from the roof in such a manner that ah3rpophysial stalk can now be described joining the floor of the anterior lobe and the roof of the mouth Rathke's pouch and the lateral buds now open in common into the caudo-superior part of the anterior lobe. In larger embryos the free ends of the lateral buds extend forward and dorsaUy and the area of their attachments extends forward with the cranio-caudal development of the anterior lobe.

Embryos of 17 mm. The long diameter of the hypophysis is cephalocaudal. Of the latercd buds, the distal parts present a wing-like form and extend forward beneath the brain floor; the proximal parts, lateral to the anterior lobe being crescentic in section. The caudo-dorsal tip of Rathke's pouch has developed into a flattened mass, the superior lobe. This structure is closely applied to the floor of the infundibulum.

In larger embryos many cord-like growths develop on the surface of the superior lobe. In newborn the lateral lobes show two changes; the wing-Uke parts are united across the median line forming a layer which is closely attached to the floor of the diencephalon cephalad to the infundibulum; the crescentic proximal parts are united by outgrowths of their free edges both dorsally and ventrally around the anterior lobe, so that the latter is partly enveloped by the layer so formed. Dorsad to the anterior lobe these two layers are connected by a small stalk.

Adults. — ^The hypophysis appears as a large ovoid organ ventral to the brain. In sections the parts described above are readily seen. The proximal parts of the early lateral lobes completely surround the middle portion of the anterior lobe in the form of a layer. This is composed of cells arranged in cords which are continuous with similar structures of the anterior lobe. The distal portion of the lateral lobes, imderlying the brain floor, is also arranged in cords which have been invaded by a considerable amount of connective tissue. The anterior lobe consists of cords and short tubules continuous laterally with the cords of the surrounding lateral lobes. The superior lobe, made up of layers of cells surroimds the ventral, lateral and caudal walls of all the branches of the infundibulum and is in contact ventrally with the anterior lobe. The cords and tubules of the anterior lobe are composed of polyhedral cells, some granular and acidophilic, others clear and much darker staining. Occasional colloid-Kke secretions are found in the tubules. The cells of the lateral lobes are small, non-granular polyhedrals, staining darkly. The layers of the superior lobe are composed of tall, colunmar, non-granular cells which take eosin lightly.

Summary. The caudo-dorsal tip of Rathke's pouch develops into the superior lobe of the adult hypophysis. The remainder of Rathke's pouch and a later anterior evagination form the anterior lobe. The lateral buds develop into a thin layer underlying the floor of the diencephalon and a narrow layer surrounding and forming part of the adult anterior lobe. Three parts differing histologically can be distinguished in the adult hypophysis.

PBOCEEDINGS 181

6, Notes on the alimentary canal of the hyper-ontomorph and the Mesoontomorph. Robbbt Bennett Bean, Tulane University of Louisiana.

Epitheliopath or Hyper-ontomorph Mesoiheliopaih or Meso-ontomorph

Stomach small Stomach large

Stomach low Stomach high

Stomach J shaped- Stomach oval

Stomach vertical Stomach diagonal or transverse

Stomach far to left Stomach more to right

Liver small Liver large

Liver low Liver high

Liver vertical Liver transverse or diagonal

Liver far to right Liver more to left

Smfidl intestine small Small intestine large

Small intestine short Small intestine long

Length 15 to 20 ft. or less Length 20 to 25 ft. or more

Colon long Colon relatively short

Colon, low hepatic flexure Colon, high hepatic flexure

Colon, high splenic flexure Colon, low splenic flexure

Colon, transverse, long low loop Colon, transverse, short high loop

The position of the viscera may account in part for the susceptibility of the Hyper-ontomorph to tuberculosis, insanity, pellagra, leprosy, and carcinoma, and the comparative immunity of the Meso-ontomorph to these diseases. The five diseases mentioned seem to be diseases due in some measure, probably primarily, to faulty nutrition.

Data: 1002 patients in hospitals, 317 post-mortem exammations.

7. The origin of the posterior portion of the vena cava inferior in the white rat. (Lantern.) Alexander S. Begg, Harvard Medical School. The changes in the posterior cardinal system of veins in the white rat

can be readily made out, since in this animal the Wolfiian bodies are quite small and certain of the complications met in other mammals are not present. The relation of the developing kidneys to the posterior cardinal veins is immistakable. The kidney Ues at first ventral and later lateral to the corresponding vein, so that at no time does the vein cross the path of its migration. Consequently a peri-ureteric loop is never formed. Moreover, below the diaphragm there is no supracardinal system of veins. The right subcardinal vein anastomoses with the hepatic sinusoids and establishes the vena cava. Below the entrance of the renal veins, the vena cava of the rat is the persistent right posterior cardinal vein. The left posterior cardinal disappears through most of its extent. The liunbar veins drain first into the cardinals and later into the vena cava. In the thorax, there is apparently a supracardinal system which gives rise to the azygos veins. Owing to the relatively slight development 6{ the Wolffian bodies in the rat, its venous system may be much simpler than that in other mammals, notably

THE ANATOMIOAL RECOBD, VOL. 10, NO. 3

182 AMERICAN ASSOCIATION OF ANATOMISTS

than in the pig, where these organs are very large. It is therefore questionable whether conclusions drawn from either the pig or the rat are of general application, but the conditions in the rat, as here described, mayreadily be demonstrated.

8. Endocranial markings of the human occipital bone and their relations

to the adjacent parts of the brain, with special reference to the so^aUed

'vermiform jfossa/ Davidson Black, From the Anatomical

Laboratory, School of Medicine, Western Reserve University.

The study upon which these observations are based began as an

investigation into the relations between the human cerebellum and the

occipital bone in the presence on the latter of the so-called 'vermiform

fossa.' Since October, 1914, I have encountered this anomaly in the

dissecting room four tiines (among some sixty odd subjects examined)

and have been able to prepare endocranial, endodural and encephalic

casts in each case. In making these casts, I have followed the method

used by Symington in his researches upon cranio-cerebral topography

(vide: Edinburgh Med. Jour., Feb., 1915).

In the course of this work a striking diflference was noted in the character of the variations of the endocranial markings on the squama occipitaUs below the sulcus transversus as compared with those of the superior fossa above this line.

In any enquiry into the significance of such differences in the endocranial relations of adjacent parts of the same bone to the underljdng portions of the brain, the phylogenetic relations of the cerebrum and cerebellum to the skull must be taken into consideration.

Endocranial surface of fossa occipitalis superior. EUiot Smith, Le Double and others have called special attention to the peculiar asymmetry of the endocranial markings of this region of the occipital bone and to the definite influence of the occipital poles of the. cerebrum upon these markings. My own series of casts amply bears out the observations of these authors and furnishes' additional evidence of the great variability of the endocranial pattern in this region. Though it is evident that a close correspondence obtains between such juga cerebralia and impressiones digitatae as are present and the fissural pattern of the occipital poles of the cerebrum, yet it should also be noted that all the details of cerebral pattern in this region are not recorded in every case by endocranial m9,rkijigs. The significance of this poinf will be discussed in a subsequent communication. In the present connection the interest in these observations lies in the fact that they demonstrate once more that the markings on the lateral areas of the endocranial surface of the superior occipital fossa are intimately associated with corresponding irregularities on the surface of the caudal poles of the cerebrum.

Such a close association, however, does not obtain between the cerebellum and all adjacent parts of the occipital bone, for the volume of the posterior skull fossa may be appreciably increased by the presence of a well marked 'vermiform fossa,' throughout the greater part of which no portion of the cerebellum comes in contact with the dura

PROCEEDINGS 183

Thus cerebellar growth can not be the direct caused factor in the production of this anomaly.

The relation of the convex surface of the cerebrum to the endodural and endocranial surfaces, except perhaps in the neighborhood of the superior sagittal sinus, is everywhere an intimate one. Small or large subarachnoid clefts (fiumina) are of course present where the cerebral arachnoid bridges across the various fissiures, but it is just such locations, analogous to the cistema magna, that favor the development of endocranifid ridges however slight. These ridges, while they are poorly developed in man and seldom extend deeply in the intergyral spaces, furnish by their presence direct evidence of the action of endocranial growth phenomena of an entirely opposite variety to those causing the formation of an anomalous endocranial fossa overlying the cistema magna.

Endocranial surface of fossa occipitalis inferior — Vermiform fossa. The following series of lantern slides showing endocranial, endodural and encephaUc casts of both the normal and anomalous human occipital region is sufficient to demonstrate conclusively that the so-called * vermiform fossa' occiurs absolutely independently of the inferior vermis. When the fossa is present, a part of it may lodge a small area of the tonsils and biventral lobules of the lateral cerebellar lobes. For the most part, however, it represents a great enlargement of the space between the dura and the brain and thus probably of the cistema magna. I have been unable to demonstrate any considerable enlargement of the cistema magna in the cases here recorded, but that may readily be due to injury to the arachnoid during removal of the brain. After removal the relation of the arachnoid bridging the cistema cerebello-medullaris differed in no discernible way in these specimens from that normally obtaining in this region.

A median cerebellar fossa as a normal and characteristic feature occurs among anthropoids only in Hylobates. At the same time in this form alone of the Simiidae is the fossa excavated for the lodgement of a definite part of the so-called vermis cerebelU.

In contrast to the condition obtaining in the large anthropoids and man, the median cerebellar fossa is a characteristic featiure of the occipital bone elsewhere among the mammalia, and it is excavated in these forms for the lodgement of a median cerebellar protubeiance. This fossa is well developed in insectivores, bats and all rodents and carnivores. It is present in the Sirenia and Cetacea and also in most ungulates, edentates and marsupials. In Echidna it is a well marked feature, and, as in Ornithorhynchus, there is a hiatus in the occipital bone over most of the area corresponding to this region.

Endocranial surface of fossa occipitalis inferior — Lateral areas. Unlike the inferior-vermis, the great lateral lobes of the human cerebelliun lie in contact with the dura of the occipital bone. This contact extends over the lateral areas of the inferior occipital fossa. An indication of intimate association between the lateral cerebellar lobes and the bony wall of this region is seen in the presence of a poorly defined but fairly

184 AMERICAN ASSOCIATION OF ANATOMISTS

constant ridge on each lateral portion of the inferior occipital fossa. This ridge conforms in the recent state to a part of the great horizontal fissure of the cerebellum. Other depressions and ridges may occur in this region, but these are both variable and inconstant and may be due either to direct cerebellar influence or to that of irregular vascular channels.

Mention may here be made of the trigonum vermianum of Schwalbe, but only to point out that the term is a misnomer and the area to which it is applied has a variable relation to the tonsils and a part of the biventral lobules of the lateral cerebellar lobes and never to the vermis. The relations of parts of the lateral cerebellar lobes to the walls of the posterior skull fossa are subject to much variation. This is evident when it is noted that in not a few cases a portion of both tonsils and biventral lobules may project into the foramen magnum. (Vide: Foriep, Anat. Anz., Bd. 19, 1901; Schwalbe, Verhand, d. Anat. Gesellsehaft, Apr., 1902).

The arrangement of the sagittal and transverse sulci and internal occipital protuberance requires no description in this connection.

The occurrence of the median cerebellar fossa in the human occipital bone has been credited both to the presence and to the absence of the ossicle of Kerkring. The fallacy of such arguments, however, has been pointed out by Le Double, who has shown that the ossicle of Kerkring is inconstant in its presence both in carnivores and in rodents, though the fossa in question is constantly present in these forms.

Both Le Double and Poirier et Charpy state that this fossa in man is probably an atavism, but these authors lay no stress upon the important changes in endocranial relations that permit the expression of such a phenomenon in this region

Throughout the mammalian series, neopaUied expansion and cerebellar growth have been the dominant factors in causing increased skull capacity. In most of the lower gyrencephalous mammaUa the superficial convolutional pattern over the convex surface of the cerebrum is accurately recorded by endocranial ridges. These ridges, as I have pointed out above, are in themselves direct evidence of cerebral influence upon endocranial growth whether they accurately conform to the details of cerebral pattern or not.

The areas of the cerebellum in contact with the endocranial surface of the skull in the lower mammalia comprise the floccular lobes and, broadly speaking, that part of the great interfloccular mass lying behind the fissura prima. The postero-median lobe of Bolk varies greatly in its development in different mammals, but in all forms below the large anthropoids and man, it is more or less closely related to the skull. By this cerebellar contact the bone is excavated to form the middle cerebellar fossa.

The great development of the anso-paramedian lobe of Bolk in man and to a lesser degree in the gorilla, chimpanzee and orang, has caused the postero-median lobe of the cerebellum to become completely separated by a deep cistern from the endocranial surface of the occipital bone.

PROCEEDINGS 185

In this cistern between the lateral gerebellar lobes there is usually situated a small phylogenetically recent fold of dura (falx cerebelli), whose endocranial attachment is marked by the internal occipital crest. Thus there is a small median area of the endocranial surface of the occipital bone between the lateral areas of the inferior fossa, in man and the great anthropoids, which is no longer subject to the direct and positive influence of a postero-median cerebellar lobe. In about 4 per cent of human skulls (Le Double) there appears in this region a fossa which is independent of direct cerebellar influence but resembles in all other essential respects the similarly situated median cerebellar fossa characteristically developed in all the lower mammalian familes. The so-called 'vermiform fossa' in man and the great anthropoids may thus be considered to be the result of the action of certain atavistic forces (the mneme of Henri Bergson) which have not yet been completely overcome or neutralized by phylogenetically recent changes in the endocranial relations of the cerebellum.

9. A topographical study of the 13 mm. chick embryo, Edward A. BoTDBN, Harvard Medical School.*

This work has been undertaken as a contribution to the comparative embryology of birds and mammals. The body of the account is a description of the gross anatomy of a chick embryo of five days' incubation (13 mm. length), based on reconstructions designed to duplicate those made by F. T. Lewis in his account of the 12 mm. pig, but supplemented by gross dissections, reconstructions of older and younger stages, wax models and India-ink injections. That portion of the work now presented deals with the comparative study of reconstructions showing the 13 mm. chick and 12 nmi. pig in mid-sagittal section; with dissections of the body cavities of the two embryos in which the more striking differences in the arrangement of the abdominal viscera are exhibited; and with reconstructions of the brain and cerebral nerves of the two embryos. In conclusion, special attention has been given to the interesting relations of the upper spinal, and occipital nerve roots in the hypoglossal complex, the details of which will be described in a later paper.

10. The interrelations of the mesonephroSf kidney j and placenta in different dosses of animals. J. L. Bremer, Harvard Medical School.*

The status of the mesonephros as an excretory organ is questioned by Felix, in the Keibel-Mall Embryology, and by Weber, both of whom find that in man and many other mammals the organ degenerates before the kidney can possibly be considered functional, and that thus no continuity of embryonic excretion is apparently provided for. Rather than accept the idea of this discontinuity, they prefer to consider that excretion is not necessary until the kidney is developed, and assign to the mesonephros perhaps some unknown function, but certainly not that of excretion. Weber was strengthened in this opinion by the condition foimd in the mouse and rat, in which animals the mesonephros is always

186 AMERICAN ASSOCIATION OF ANATOMISTS

rudimentary^ and in the mole, in which the organ is small and degenerates very early. He was puzzled by the fact that in the pig the mesonephros persists in full functioned activity until late fetal life, when the kidney is apparently fully active, but thought that this single case where continuous fetal excretion was possible, but not proved, should not vitiate his opinion gained from so many other animals.

That some other fetal organ might supply the needed excretory function when neither mesonephros nor kidney is capable of activity does not seem to have been considered by these authors, nor have they thought of the possibiUty of differences in the various classes of animals as to whether or to what extent this other organ of excretion should be called on. Yet physiologists have for some time been familiar with the fact that certain substances may pass from fetus to mother by placental exchange, and were only at a loss to understand why this was not foimd to be true in certain classes of animals, as well as in others.

Taking as a standard sign of the possibility of urinary excretion the arrangement of a thin epithelial plate overlying and in close contact with a fetal capillary, such as one finds in the glomeruli of mesonephros or kidney, I have examined the placentae of different classes of mammals, at various ages, and compared the results with the conditions of the mesonephros and kidney in corresponding embryos and fetuses. The following conclusions have been reached.

The WolflSan body or mesonephros is a gland of urinary excretion.

Mammalian embryos may be divided intQ two classes; those which retain functional WolflSan bodies imtil the kidneys are sufficiently developed to excrete urine, as is the case in birds and reptiles, and those in which the WolflSan bodies degenerate before the kidneys reach functional ability. The first class includes the pig, sheep, and cat, the second, the rabbit, guinea pig, man, and rat.

Within reach of these classes individual animals show great differences in the size and presumable excretory ability of the Woffian bodies, without regard to the length of their duration.

The allantois is the receptacle of the urine formed within the body of the embryo; it is present as a reservoir only in those animals with an embryonic excretion, and its size varies with the size of the Wolflfian bodies and with their duration. The urethral opening, though present, is not normally used for the passage of fetal urine.

In those animals without the possibility of a continuous urinary excretion within the embryo, i.e., with an early degeneration of the WolflSan body, the placenta is provided with an apparatus similar to that found in the glomeruli of the WolflSan body or the kidney, thin plates of epithelium overlaying the fetal capillaries. These appear in the placenta at about the time when the WolflSan body commences to degenerate, or in the case of the rat, which never develops mesonephric glomeruli, at about the time of the normal development of the glomeruli in other embryos. These plates continue and increase in number till term. They are apparently of greater extent in animals whose embryos are provided with large WolflSan bodies.

PROCEEDINGS 187

In the placentae of those animals with a continuous embryonic urinary excretion, similar plates are not found, whether the placentae be of the apposed or conjoined type.

From these facts it appears that embryonic and fetal urinary excre* tion takes place wholly through the placenta in the rat, at first through the Wolffian body and later through the placenta in the rabbit, guinea pig, and man, but never through the placenta in the pig, sheep, or cat. A knowledge of these differences should lead to more intelligent experiment on the permeability of the placenta.

11. The development of the vertebral column in the domestic cat, from the

membrarums to the completion of the cartilaginous stage. Alfred J.

Brown, The Anatomical Laboratory, Columbia University.

The study of the development of the vertebral colmnn prior to the stage at which completely cartilaginous vertebrae are present affords a basis for the analysis of the mammalian vertebra into elements, which on the one hand can be homologized with the separate osseous elements of certain extinct reptiles, and on the other may assist in the interpretation of some of the anomalies of the adult spine.

The first differentiation of the mesenchyme about the chorda is initiated by the appearance of condensations in the form of flattened plates which are situated opposite the intermyotomic intervals. They are obKquely placed and inclined caudo-ventrad. Laterad they extend to the mesial border of the myotomes and are pierced at their centres by the notochord. These plates of thickened mesenchyme have paired dorsolateral and ventrolateral extensions which lie in the intermyotomic intervals, the former between the dorsal margins of the myotomes, the latter between the ventral. Between the bases of the two processes on each side the membrana reuniens is but feebly developed. The whole complex corresponds to the "primitive wirbelbogen" of Troriep.- The central portion of this condensation will form the intervertebral disc while the dorso and ventrolateral projections mark the site of development of the future neural arches and costal processes respectively. The condensation develops a vertical ridge on its cephalic and caudal surfaces which tends to divide the compartment between each pair of platelike septa (site of future centnun) into lateral compartments, which in turn are divided into a. pair of cephalo-dorsal compartments and a pair of caudo-ventral compartments by the notochord. The cephalo-dorsal arms of the condensation enlarge cephalad and the caudoventral arms do the same, the latter becoming bifurcated at their extremities, one arm of the bifurcation passing ventral to the interprovertebral artery and the other dorsal to the same.

In the above process the main portions of the vertebra are laid down. The undifferentiated mesenchyme between the condensations will form the major portion of the centrum. The dorsal arms are the basidorsals and become the neural arch. The central portion of the condensation becomes the intervertebral disc and the ventral arms are the basiventrals which eventually will be divided into the costal element or basiventral proper and the intercentral element.

188 AMERICAN ASSOCIATION OF ANATOMISTS

Chondrification, Begins first on the caudal surface of the basi-dorsals. FoUowmg this four centers are found, two on each side of the ventrodorsal partition arranged as follows: the dorsal pair occupy a cephalic position dorsal to the notochord and the ventral a caudal position ventral to the notochord. Each lateral pair fuses rapidly forming a ventro-dorsal bar of cartilage. These fuse ventral and dorsal to the chorda thus forming a ring around that structure. In the early stage thi? ring shows bifurcation cephalo-dorsally and caudo-ventrally thus still indicating its original quadrupartite character. These structures are homologous to the interdorsals and interventrals of reptiles.

The basidorsal loses its connection with the intervertebral disc and become attached to the cephalic portion of the corresponding interdorsal, thus exhibiting a caudal shift.

The common basiventral and intercentral is still membranous. It breaks away from the intervertebral disc at the junction of its mesial limit with the disc. It also exhibits a caudal shift and still membranous lies between the caudo-lateral surface of the interventral and the cephalo-lateral surface of the intervertebral disc, filling up the interval between the caudal surface of the growing cartilage and the intervertebral disc. Chondrification begins in this membrane at two points, one adjacent to the interventral and one in the intermyoiomic interval, lateral to the first. The center adjacent to the interventral fuses quickly with the caudo-lateral portion of the latter and chondrification proceeds caudal gradually filling up the membranous wedge between interventral and intervertebral disc. This portion of the basiventral corresponds to the intercentral of the reptile. The portion <5hondrifying in the intermyotomic interval represents the costal ele ment of the cervical vertebrae and the true rib of the thoracic segment of the column.

The elemeiits present in the form of individual cartilaginous or membranous units at various stages of development are: Two basidorsals = Neural arch.

Two interdorsals \ 1

Two interventrals f \ = Centrum.


s f _ \ two intercentrals ,


Two basiveiitral8= ^ ^^^ ^^^ processes.

The above units fuse with each other except for the costal processes in the thoracic region of the spine which persist as separate elements through the development of joints between the capitular portion and the centrum on the one hand and the tubercular portion and the neural arch on the other.

12. The regeneration of the forebrain of Amblystoma. H. Saxton BuBR, Anatomical Laboratory of the School of Medicine, Yale University. (Introduced by H. B. Ferris).

The following is a report of one of a number of experiments on the regeneration of the brain of Amblystoma. Two series of operations were performed on Amblystoma larvae possessing neither peripheral

PROCEEDINGS 189

nerves nor a circulatory system. In the first of these the right nasal placode and the right telencephalon were completely extirpated. In the second, the right cerebral hemisphere was removed but the right olfactory anlage was returned to its original position.

The results of the first operations were definite. No regeneration of nervous tissue occurred. Theependymal cells surrounding the interventricular foramen, left open by the operation, formed a thin curtain completely closing it. These ependymal cells are under certain conditions potential neuroblasts, into which they will develop upon stimulation from the proper source, in this particular instance the nasal placode. The removal of the olfactory anlage precludes any stimulus, chemical or otherwise, being derived from the mere presence of the placode and in addition prevents the stimulus normally foimd in the ingrowth of the olfactory nerve. It is not surprising, then, that no regeneration occurred.

The operations of the second series were interesting because here the layer of ependymal cells closing the foramen of Monroe was subjected to the stimulus of the ingrowing olfactory nerve fibers, and later to the stimulus of the functioning olfactory organ, since the nasal placode was returned to its proper position after the removal of the imderlying telencephalon. The result was the regeneration from the ependyma of a new hemisphere which in one case, the oldest larva of the series, was complete.

Regeneration of the telencephalon in Amblystoma, then, occurs whenever connection between it and the functional end organ is not prevented.

IS, The functional relation of intercellular substances in the body to certain structures in the egg cell and unicellular of^anisms, Montbose T. Burrows, Department of Pathology, Johns Hopkins University. In an article presented last year before this society the author introduced facts to show that whether an embryonic heart muscle cell in a tissue culture grows and divides, grows to form a sjrncytium, differentiates, contracts rhythmically, or remains inactive depends upon the particular kind of environment into which it is placed. Many of the peculiarities of the environment peculiar to each form of activity were described at that time and evidence was presented to show that these various activities are not the result of an organization contained within the cell but that they are the result of differential changes of the surface tension of these elements. Each form of work and the structure peculiar to each form of work is the result of a specific kind of change. The changes in the surface tension are brought about and maintained by an organization in the environment, external to the cell but influenced by the cell as any phase of any physical chemical system of this kind influence other phases.

These experiments indicate that the cells of the body differ from egg cells and many unicellular organisms in that they do not possess the total organization necessary for their activity but depend for parts

190 AMERICAN ASSOCIATION OF ANATOMISTS

upon structures external to them. In the Kght of this fact it became of interest to compare the structure of the egg with the structure of the organization pecuKar for the growth and the mitotic division of the body cells. It seemed evident that by this method one could determine definitely the true significance of many of the structures peculiar to these cells. What could be applied for the study of the egg cell could be appKed also for the study of many unicellular organisms.

In the present article the author wishes to point out certain analogies between the organization of the environment suitable for the growth of the body cells in the cultiure and certain structures peculiar to the ^g and unicellular organisms as well as the relation between this organization peculiar for the growth of the cells in the culture and the organization peculiar to growing parts of the developing and the mature individual*

14» Microdissedion studies an •cell structures, Robert Chambers, Jr.,

Cornell Medical College, New York City.

(Demonstration of a double Barber pipette holder for the use of two needles and of a substage condenser which produces excellent definition for oil inunersion work at a working focal distance of almost one centimeter.)

The limitations of the microdissection method owing to the injurious effects of puncturing with the needle must be reaUzed and false interpretations are to be guarded against. One must also take into account the effect of surface tension phenomena of the dissection medium which is used as a hanging drop and, of course, the suitability of the medium in preserving normal conditions. The injurious effects of heat are eliminated by the use of a water filter and the various layers of glass through which the Ught must pass before reaching the object frees the light of its injurious rays.

Ivjurious effects of the needle. Tearing of the cell is very generally followed by a swelling with the absorption of water and a change in the chemical reaction of the protoplasm. The cell nucleus is also almost instantly affected by the touch of the needle, a granular precipitate being followed by a condensation of nuclear material into a viscous, highly refractive, gelatinous and amorphous mass.

Experimental data. 1) A morphologically differentiated film or membrane on the surface of the cell has been demonstrated only in free living cells, e.g., eggs. Protozoa. 2) The physical consistency of protoplasm may be summarized as follows — an optically homogeneous, gelatinous substance differing markedly in consistency in different cells and at different times. On extensive tearing or by quick and rapidly repeated thrusts of the needle the protoplasm turns acid in reaction, swells and goes into solution; or becomes vacuolated and granular and coagulates. Normal conditions, however, are apparently maintained if the needle be carefully manipulated and in this way the structure of the cytoplasm of orthopteran germ cells, sea urchin eggs and various somatic cells has been studied. 3) The physical consist PROCEEDINGS 191

ency of morphologically diflferentiated cytoplasmic structures has been studied. 4) The production of a chromatic network and of chromosomes in the hyaline substance of the nucleus has been studied, also the consistency of the spindle-shaped nuclear mass in dividing cells. 5) Localized surface changes in dividing cells were produced by the needle and by various solutions. 6) Localized differences in response to stimuli have been noted in the cjrtoplasm of certain cells. 7) Transmission in protoplasm of the effects of mechanical stimuli has been noted.

16, A study of the reaction of mesenchyme cells in the tadpoWs tail toward injected oil globules. Eliot R. Clabk, University of Missouri. ♦ In their later studies on the mode of development of Ijnnphatics, Huntington and McClure have repeatedly reiterated the view that lymphatics are formed by the transformation of mesenchjrme cells, in the following manner. They hold that fluid accumulates in the tissue spaces, forming small lakelets; that the mesenchyme cells are pressed upon by the fluid collected, and that as a result of the mechanical pressure supposed to be exerted, the mesenchyme cells react by flattening out and forming membranes aroimd the lakelets. Innumerable isolated blisters formed in this manner are held to be the beginnings of the lymphatic endothelium, vessels being formed by the coalescence of neighboring blisters.

The evidence for this view consists almost entirely in the interpretation of certain appearances, seen in cross sections of embryos, which are in many cases open to other interpretations. No facts have been presented which prove conclusively that, if fluid accumulates in the tissue spaces, the mesenchyme cells, pressed upon by such accumulations will form endotheKal membranes, and that membranes so formed will become lymphatic endothelium.

The present investigation was undertaken in order to study, in the living, the reaction of the mesenchyme cells to the pressure of globules of fluid artificially introduced into the tissue spaces, to see whether they react by the formation of membranes, and, in case they do, to study the property of such membranes, particularly their reaction toward blood-vessels and lymphatics.

The studies consisted in the introduction into the transparent fin expansion of the tail of Bull-frog and Fowler's toad larvae, at early stages, of minute globules of paraffin oil, and of watching, in the living larvae, the reaction of the individual mesenchyme cells, blood, and lymphatic capillaries in the neighborhood of the injected globules.

It was found, considerably to our surprise, that the mesenchyme cells showed no sign whatever, of even a tendency to flatten out and form membranes around the globules. Instead, they maintained their identity as branched mesenchyme cells, with their property of slow progression, by the sending out of processes on the one side, and retraction on the other. This was true even of mesenchyme cells against which the injected globules were forced by the injection, as well as those against which the globules pressed, as a result of a slight shifting of

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position of the globule, which occasionally occurred. Obviously, since the mesenchynfie cells failed to form membranes around the globules, it was impossible to study the reaction of blood and lymph vessels to such membranes. Toward the oil globules themselves, however, the blood and lymph capillaries remained quite indifferent — ^apparently entirely unaffected by their presence.

The only observable reaction on the part of the living cells to the injected globules consisted in a more or less intense leucocytosis, which usually subsided in the course of a few days, and which was probably caused by slight grades of infection. In some cases there was almost no leucocytosis. In several instances leucocytes were watched as they moved to the oil globule, flattened out on its surface, and moved away again. It is possible that, if sections were made at the time when leucocytes were flattened out against the globules, one might interpret them as flattened endothelial cells.

It thus appears that the notion that mesenchyme cells are stimulated by the mechanical pressure of globules of fluid to flatten out and form membranes, and that these membranes are the beginnings of lymphatic endothelium, fails to stand the test of experiment, and of direct observation on the living animal.

16. The loose connective tissue, as seat of lympho^ranulopoesis. Wera Danchakoff, Rockefeller Institute, New York City. (Introduced by W. H. Lewis.) The loose connective tissue of an adult hen includes beside the

fibroblasts many kinds of amoeboid cells and small nodes of true

lymphadenoid tissue.

1) The amoeboid cells of the connective tissue are the product of the differentiation of the mesenchymal syncytium in the nodes. After destruction of the lymphoid tissue by X-rays the regeneration of the lymphatic cells takes place at the expense of the mesenchymal syncytium in the nodes.

2) The mesenchymal anlage of the amoeboid cells in the connective tissue and the mesenchymal anlage of the other hasmatopoetic organs are identical. One may interchange the different lines of the differentiation of the mesenchyme. The same mesenchyme, which for example normally forms merely thin connective tissue septa between the muscle bundles, is capable of differentiating into haemoblastic and leucoblastic tissue. This occurs in the embryonic mesenchyme of a chick after grafts of an adult chick embryo. These changes of the mesenchyme being ubiquitous, we have to conclude that the loose mesenchyme of a chick embryo (6 to 10 days) is equivalent in all the regions of its localisation and is polyvalent in its potencies of development.

The once differentiated cells are not capable of interchanging, but the various lines of differentiation of the stem cells may be interchanged. The differentiation of the mesenchymal cells is directed by the force of external conditions whether these conditions consist in the special

PROCEEDINGS 193

physico-chemical milieu of the vessels or in an impulse through an enzyme-like material.

17. The effedts of light on the retina of the turtle and of the lizard. Samuel R. Detwiler, Osborn Zoological Laboratory, Yale University. (Introduced by R. G. Harrison.)

Although the efifects of light on the Reptilian retina have been studied by many investigators, differences of opinion still exist as to the results obtained. According to most investigators (see Garten) no migration of the epithelial pigment takes place, nor do the cones contract. The present investigation was taken up with the hope of finally ascertaining whether or not light had these effects on the retina of reptiles. Coincident with this work it was found that the anatomy of the retinae studied differed in some respects from that of other forms and it was therefore necessary to make an anatomical study which might serve as a basis for physiological experiments.

If the form of the outer segment of the visual elements be taken as the criterion in determining the differences between rods and cones, then it can be said that the retinae of the turtles, Chelopus guttatus, Chelopus insculptus and Chrysemys picta and of the lizard, Sceloporus undulatus, contain no rods. Both double and single cones occur.

Garten's explanation of why the pigment migrates and the cones contract in light necessitates the assumption that, in a retina in which the visual elements are all of one kind, these reactions do not take place. Nevertheless light brings about not only a migration of the retinal pigment but a shortening of the cones in both the turtle and lizard. W)^en the eyes of animals which have been placed in bright sunlight for 6 hours are compared with those of animals kept in darkness for 24 hours it is found that light causes a forward migration of the pigment in the turtle (Chrysemys picta) on the average of 3.6/i and in the lizard (Sceloporus undulatus) of 3.1/i. In addition to this effect on the pigment it is found that light causes a contraction of the cones to the extent of 2.3/x (based on a series of 10 measurements) in the turtle retina as well as a flattening of the pigment epithelial cells of 2.7/i.

Not only are these changes brought about in normal eyes, but also upon eyes the optic nerves of which have previously been severed. In such cases the pigment in the light eyes is 2.bii nearer the external limiting membrane than in the light eye with the nerve intact, but this does not represent a greater relative amount of migration. The pigment in the dark eye in which the optic nerve is cut is 4/x nearer the external limiting membrane than in a dark eye with the nerve intact. A series of measurements show that the migration caused by light in eyes with the optic nerves cut is on the average of 2/i in contrast to a migration of 3.6m in normal light eyes.

In addition to these effects, light is abo found to reduce the amount of chromatin and Nissl substance in the ganglion cells so that they stain less darkly and more diffusely. It also decreases sUghtly the ability of the outer nuclei to take on stain, but, while it does cause

194 AMERICAN ASSOCIATION OP ANATOMISTS

them to lengthen slightly, it has no effect on the form and volume of the ganglion cells.

Attempts to see whether the pigment could be induced to undergo a greater migration by means of stimuli other than light were carried out by stimulating with currents, both interrupted and constant. Stimulation of the enucleated bulbus with an interrupted current of moderate strength brings about a position of the pigment approximately equal to that produced by Ught i.e., a migration of about 3.6m.

Constant currents of 18 M.A. passed through the eye either in a centrifugal or centripetal direction for a period of 15 minutes cause a migration of approximately 2/i more than that produced by light. It also results in a considerable elongation of the cone myoid.

18. The development of function in the Purhinje cell of the dog and its relation to growth. (Lantern.) Davtd H. Dolley, Laboratory of Pathology, University of Missouri. (Introduced by E. R. Clark.) Postdivisional growth and the development of function proceed simultaneously in the Purkinje cell of the dog's cerebellum. An elemental analysis has become possible, first, because function from its definite morphology can be investigated as a process independent of growth, and second, very exact limits for the strictly developmental progress of both can now be set. Viewing growth independently, it is the resting cell type, uninvolved in function, through which the line of growth proper is accurately to be traced. As to the limits, the beginning is from a cell just out of the divisional phase, so embryonic that the cytoplasm is minimal. The terminal definitive period is set by the attainment of the nucleus-plasm norm standard for the species uncjer the law of its species identity formulated by Dolley (Jour. Conxp. Neur., 1914, vol. 24, 445).

Considering first growth separately, as expressed by nucleus-plasma coeflScients, measurements of fourteen puppies of various ages show a steady progress with minor variations from coeflBcients of approximately to 0.4 in the fetus at term to the adult norm of IIH- at five to six weeks.

It is not in the slightest conflict that there remains the potentiality of further growth for the infantile animal after six weeks. What has become constant is the nucleus-plasma relation. This post-developmental growth can be demonstrated to be identical with the growth which results from functional usage, the functional hypertrophy. There is an increase in the absolute size of nucleus and of plasma, but the same quantitative ratio holds between them in conformity to the law of species identity. It follows that the substances remain the same, and post-developmental growth, whether it is an undergrowth, an overgrowth, or maintains its status quo, is quantitative.

Considering next the phenomena of function separately, its morphologic changes become measureable at 10 days, though recognizable in immature form as early as 6 days. The conclusions of this paper, based on volumetric data, go no further back than the 10 day period. M eas PROCEEDINGS 195

urements of resting and functioning cells were made at 10 dajrs, 17 days, and 27 days, and compared with the adult in respect to volumes and nucleus-plasma coeflScients. There is complete objective homology between the curves of activity at all periods, and therefore the mechanism of infantile function is identical with that of the adult. Further, it is shown by plotting the data that there are definite quantitative levels, and that the changes of activity for any period are quantitatively based on the nucleus-plasma figure which subtends the resting cell b^ longing to that period. However, the limitations of average measurements of consecutive stages are too great to permit of an independent induction of the quantitative nature of infantile function and growth, though the indication of this is strong.

That adult function is quantitative is sufficiently established. That adult growth and overgrowth are quantitative is true if the law of species identity holds. The premise that was lacking in extending the quantitative principle to the developing cell is whether the substances of that cell, those involved in growth and those involved in function, are identical with the adult materials. In default of chemical knowledge, one has to turn to the mathematics of chemistry. Constant proportions for corresponding stages must indicate a constant chemistry. If there is the same proportion between resting cell infant and functioning cell infant as between resting cell adult and functioning cell adult, and if, as would follow, there is a common ratio of infant to adult for all phases at any period, it could only mean the same proportion of combination and nucleus-plasma interchange, and on a chemical basis, the same substances. Differences between infant and adult would be reduced to one of quantity for both function and growth.

In search of such exact proportions, resource was again had to the method of wax reconstructions of individual cells from one micron sections in serial. This gives the dud possibilities of determining the relative mass of plasma and nucleus by weighing the models and of applying the prismoid formulas for volume estimation, a mutual check. Two series of 12 adult functioning cells and of 15 infant, ranging from Stage 6 to Stage 9 (DoUey, Jour. Med. Research, vol. 22, 1910) were made and the data plotted in comparison with those of two series of 5 resting cells each from the same adult and infant. There was an identity of proportion lacking 0.24 of 1 per cent for the wax data and 1.5 per cent for the data of prismoid formulas, and the results were within the limits of the mathematical probable error.

The conclusion is indicated that back to the 10 day period the substances of the infantile cell are identical with the adult. The sub- ' stances being identical, the quantitative principle may be independently induced as the basis of both developmental function and growth in common with the adult. Analyzed separately, the residuum of stable intrinsic substances which provide for growth and recuperation may be distinguished from the superficial transitory substances devoted to immediate energy liberation, the chromatin and nucleolar substance which disappear with work and return with rest. Each group must

196 AMERICAN ASSOCIATION OF ANATOMISTS

have a constant ration to the whole, that is, a constant chemical constitution, otherwise the sum total of constancy would not be maintained.

What then is the relation of these two processes, each embodying a conmion principle, to each other? The nerve cell is the embodiment of a single attribute, irritability. In this it conforms to the law of the specific energies of protoplasm. All stimuli produce only a quantitative reaction. Its growth, therefore, must be conceived to relate only to that attribute. The substances which primarily grow are themselves preparatory and solely devoted to function. In the chromatin may be seen an end product of nucleus-plasma interchange and synthetic upbuilding destined to be transformed into the kinetic energy of function. Recuperation after the exhaustion of chromatin is as much a growth as is the infantile development of chromatin. Function then fundamentally rests upon the residual more stable substances which provide the passing material for each cycle of activity. Function further brings in no new element. It involves the same substances in a quantitative increase or decrease which are in metaboUc equilibrium in the resting cell.

The phenomena of growth and function in the nerve cell are therefore concurrent, coinciding phenomena. For function it needs only to be shown that it is propogative, that the reaction of function is to excite the upbuilding of more materials for more function, and the whole growth phenomenon becomes merged in function, a property of function. It is an established principle of pathology that such is the inevitable effect of continued function, witness the functional hypertrophy.

Developmental growth, therefore, is a functional growth.

If growth of the nerve cell after division ceases is a functional growth, reversely, without function — that is, in the absence of stimuli — there should be no growth. This points the way to the experimental test of the conception.

19» The size of the rnedidlated axons of the Purkinje cerebellar neurons in

the albino rat. Elizabeth H. Dunn, Marine Biological Laboratory,

Woods Hole.*

Golgi material shows us that the axons of the Purkinje cells of the cerebellar cortex are efferent, but has not determined the relation of these axons to the medullated fibers of the cerebellar cortex.

While working over, for another purpose, considerable brain material of the albino rat, I found that in some of the double stained material

the relation of the medullated sheath to the Purkinje neuron could be determined. The medullated sheath was laid on very close to the neuron body, and the medullated fibers thus formed were among the largest found in the cerebellar cortex. The medullated process appears, therefore, to be correlated with the size of the neuron body which is the largest appearing in the cerebellar cortex.

See 66, Herbert M. Evans, page 264.

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£0. On the reversal of laterality in the limbs of Amblystoma embryos, Ross G. Harrison, Osborn Zoological Laboratory, Yale University. For the purpose of investigating certain factors concerned in the development of paired limbs a series of experiments have been made upon Amblystoma embryos. That portion of the body wall from which the fore limb normally develops was excised before differentiation had begun, and the disc-shaped piece thus obtained and consisting of ectoderm and mesoderm, was transplanted to another embryo. The results were as follows:

1. A limb bud from one side of the body implanted in the place of a bud previously removed on the opposite side, develops in a large proportion of cases (over one-third) into a limb of the side to which it has been transplanted, i.e., its laterality is reversed. The limb thus grown is in most cases perfect in form and function, though it is usually somewhat retarded in development as compared with the normal nontransplanted bud. Perfect regulation of the transplanted part with respect to the organism as a whole is thus brought about. In other cases (almost one-third) duplicate limbs arise, the twins being of opposite laterality. Observation of many of the cases in which single normal limbs develop shows that in early stages the bud tends to grow out as of the side to which it originally belonged, i.e., not reversed; then a reduplicating bud appears and this soon gains the upper hand, ultimately becoming the limb with reversed lateraUty, while the original is resorbed or in some cases reduced to a small spur which may remain permanently attached to the fully developed limb. In a few cases a single limb of original laterality develops though none of these have been found perfect. In the remaining cases the limbs are either rudimentary or the transplanted tissue is entirely resorbed.

2. If the Umb bud from one side of the body is transplanted to the opposite side and placed not in normal position but on the side of the body between the fore and hind limb, the results are very different. A much larger proportion (over half) develop very imperfectly or are absorbed, about one-quarter develop with their original laterality and some reduplicated appendages may arise. In but one case has the laterality of the single limb been reversed.

3. In cases where the limb bud is implanted in normal location on the same side but turned upside down, about one-third of them develop into normal Imbs in normal position. About one-sixth give rise to twin limbs and the remainder are mostly imperfect or are resorbed. Observation of the development in individual cases shows that the single limbs gradually acquire their normal orientation by rotation.

4. Limb buds placed upside down on the same side of the body between the fore and hind Umb develop in one-half of the cases into limbs of opposite laterality. In no case has a single limb of the original laterality been produced and in only one case a pair.

5. Experiments in which the limb bud was transplanted to the same side of the body in normal position all resulted in normal development, showing that the results of the previous experiments are due to the

THE ANATOMICAL RRCORD, VOL. 10, NO. 3

198 AMERICAN ASSOCIATION OF ANATOMISTS

abnormal relations of the transplanted rudiment with respect to its surromidings, and not to the operation as such.

£1. Changes in the chemical composition of the entire body of the albino rat during the life cycle. S. Hatai, The Wistar Institute of Anatomy. The entire body of the albino rat was analysed at birth and at 7, 15, 22, 28, 35, 42 and 294 days and the variations according to age were determined for water and soUds, and from the solids for the following chemical components: protein, fat, organic extractives and salts. The stomach contents of the young rats fed by the mother's milk exclusively were analysed also. Analysis of the stomach contents suggests that the milk of the albino rat is highly concentrated and rich in fat.

(1) The percentage growth of the soUds reaches nearly a maximum (30 per cent) while the young are still nourished almost entirely by the mother's milk (end of third week). At the end of 42 weeks the additional growth in soUds is only sUghtly over four per cent.

(2) The components of the soUds show the following age variations: Protein. The protein content is highest at birth and diminishes gradually until the end of the lactation period after which it rises again sUghtly.

Fat. The fat content rises rapidly during the lactation period after which it diminishes steadily.

Organic extractives. The organic extractives decrease rapidly towards the end of the lactation period, after which there is another steady increase. In general the variation in the organic extractives is similar to the variation in the protein.

Salts. The salts show a slight progressive reduction during the lactation period, after which they increase steadily.

(3) When the chemical compositions of several mammals are compared with one another it appears that the bodies of these mammals during growth pass through identical phases of chemical alteration, so far as the water, protein, fat and ash contents are concerned, and that the percentage of water is an indicator of the corresponding phases of the chemical alteration in different species, while neither the calendar age nor body weight of the animals can be used for the same purpose.

S^. Some results from reversing a portion of the spinal cord end for end in frog embryos. Davenport Hooker, Anatomical Laboratory of the Yale University School of Medicine.

A piece of the spinal cord, together with the skin covering it and the dorsal portions of the myotomes, was excised from frog embryos in the closed neural tube stage, turned end for end and grafted into the space from which it had been removed. The cephalic end of the cut passed through the extreme caudad end of the medulla. The pieces were about one mm. long. In the majority of cases the reversed piece healed readily in situ. Twenty-four to forty-eight hours after operation, the head region cephalad to the cut responded to light mechanical stimulation of the body caudad to the grafted piece. On the second to the fourth day after operation, the embryos moved spontaneously.

PROCEEDINGS 199

The portion of the dorsal fin that was reversed with the piece of the spinal cord continued to exhibit its original polarity. This became more marked as the embryo increased in size. With this exception, many of the operated embryos were entirely normal in appearance. They grew well, reacted to stimuli in the normal manner and behaved as normals.

Microscopic examination of the reversed cord shows that in the majority of cases the fusion of the cord ends had successfully taken place at both ends. The original caudal end of the reversed piece, now directed cephalically, has fused with the medulla. The canaUs centralis has in every case widened so that the transition from the large cavity of the medulla to the narrower canal of the cord is a gradual one. The reversed portion of the medulla, now lying at the caudal end of the reversed piece, has not disappeared. In all but two cases, it is present as a distinct enlargement of the canalis centralis. This enlargement, like the medulla itself, has an extremely thin roof.

In a few cases, the notochord was injured in the operation. In one or two of these embryos, its end became turned up between the cut ends of the cord. While this presents a mechanical obstacle to the healing of the cord that at times effectually prevents the process, it is perfectly possible for the cord to regenerate around it. This is accomplished by the outgrowth of nerve fibers from both ends of the cord and the prolongation of the canalis centraUs either to one side of the obstacle or in such a manner as to envelop it.

A very interesting problem is raised by some of those embryos in which the cord ends did not fuse. Even in those cases where the lack of fusion was apparent at both ends of the reversed piece, the embryo was capable of fairly well coordinated movement during the beginning of the development of the swinuning reflexes. The question raised here, as also by the embryos with completely severed spinal cord described in the Journal of Comparative Neurology for October, is whether the first swimming movements in the frog embryo are in reality dependent on a fully developed, though primitive, reflex system as has been found to be true for Amblystoma by Herrick and Coghill. It seems at least possible that the coordination in the swinuning movement during the early stages of its development is materially assisted by the drag on the skin over the myotomes produced by the side to side swaying of the head. Muscle tissue that is beginning its differentiation is peculiarly susceptible to such stimuli. If this be true, the coordination is mechanical and not nervous in nature. It is, further, lost very soon as is the response to this type of stimulus by developing muscle tissue.

2S. On the growth of the albino rat 05 affected by environment and by feeding

varioits dVrCtless glands (thyroid, thymus, hypophysis, and pineal) . E. R.

HosKiNS, The Institute of Anatomy of the University of Minnesota.

In this investigation were used 59 female and 73 male albino rats from

29 different litters. One or two of each Utter were kept for controls,

and the others distributed as nearly as possible among four other

200 AMERICAN ASSOCIATION OF ANATOMISTS

groups, the two sexes of course being kept separately. Each group received on alternate days thyroid, thymus, hypophysis, or pineal substances in different amounts, and the control animals were given equal weights of muscle. Both dried and fresh glands and muscle were fed, but the same results were obtained in either case. All the animals were given whole wheat (Graham) bread soaked in whole milk as their regular diet. The total number of rats fed, the number autopsied in each case, and the amount of glandular substances fed is shown in the following tables:

TABLE 1 — NUMBER

Total fed A u topsied

Group Males Females Males Females

Thyroid 13 9 11 9

Thymus 12 11 9 11

Hypophysis 17 11 13 11

Pineal 11 12 10 12

Muscle (controls) 20 16 16 16

Total 73 59 59 59

132 118

TABLE 2 — ^DOSAGE

Gland Dry Fresh

Thyroid 10 mgm. to 200 mgm. 40 mgm. to 800 mgm.

Thymus 15 mgm. to 300 mgm. 70 mgm. to 1400 mgm.

Hypophysis 5 mgm. to 100 mgm. 25 mgm. to 500 mgm.

Pineal 7.5 mgm. to 150 mgm. 40 mgm. to 800 mgm.

Muscle 8 mgm. to 160 mgm. 25 mgm. to 500 mgm.

Feeding was begun at 3 weeks of age and continued for varying lengths of time until the rats were from 10 weeks to 8 months of age. Each of the rats was treated individually, weighed frequently, and carefully autopsied at the termination of the experiment. The older or ' summerborn' control rats grow at about the same rate as described by some previous workers along this line, but most of the younger or 'winterborn ' were considerably larger at every age beginning with 3 weeks. These averaged at 3 weeks 32.6 gms. (males) and 29.6 gms. (females) ; at 6 weeks 84.0 gms. (males) and 80.0 gms. (females); at 10 weeks 168.5 gms. (males) and 129.6 gms. (females) ; at 13 weeks 197.4 gms. (males) and 145.3 gms. (females). The figures given are for an average of only 15 rats of each sex in the control group, but since the gland feeding in the other groups had little or no effect on the growth rate of the body as a whole, these figures represent indirectly the approximate average growth of 46 males and 43 females.

The rapid growth of these animals as compared with the published norms for other albino rats selected at random may be due to one or more of the following causes. They were the offspring of a mixture of two different strain?, they were kept in a well ventilated room in

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large, clean, airy cages, they received three tunes a day an abundance of wholesome food, and once a day clean water. The temperature of the room was carefully regulated. Of these factors the diet is probably the most important. A growth norm should be established for earh group of experimental animals.

Measurement of the tails of 59 male and 59 female rats at autopsy showed that, as has been noted, males have relatively shorter tails than do females. The tail-body ratio in rapidly growing rats averaging 13 weeks of age was about 85 per cent for females and about 80 per cent for males. The tail. of these rats was relatively shorter than that of previous norms.

The relative weights of the parts and organs of the control rats agree in most cases fairly well with previously published data.

Th3rroid subf^tance fed to rats had Uttle if any effect at all on the growth rate of the whole body as compared with the control rats.

Autopsy showed that there was a slight loss of fat from the skin and the body cavity but the weight of this was made up by the overgrowth in some of the organs of the thyroid-fed animals, as compared with the controls. The heart of the th3rroid-fed animaJs when compared with the controls showed an increase in relative weight of from 30 to 50 per cent in all rats of both sexes except in the case of a few males which received very small doses. The liver was increased in relative weight over 20 per cent in all but the few males just mentioned; the spleen is also more than 25 per cent heavier in the thyroid-fed rats, but this organ is extremely variable. The relative weight of the suprarenals was increased by from 17 to 45 per cent in the females and over 50 per cent in the males, excepting the few which received small doses. The kidneys were increased to about the same extent as the Uver.

Other organs showed a constant hypertrophy to a lesser extent among the th3rroid-f ed animals, but the difference in these might possibly be accounted for by chance variations. These parts are the alimentary canal, testes and hypophysis (male). A still smaller increase in weight is shown by the skeleton, pineal body, brain and eyeballs. The variation in this last group of organs might well be due to normal variability especially as the number of data is not large.

In none of the groups of rats fed with thymus, hypophysis (whole), or pineal were constant differences in relative weight of the body or of the organs or parts demonstrable, except such as possibly can be explained imder normal variabiUty. These will be discussed in a later paper.

£4 • The renal tiibules of birds^ G . C arl Huber, University of Michigan . The author, after completing a study of the development of the mammalian renal tubule, in which the Born reconstruction method was freely used, projected a study on the morphology of the renal tubules of vertebrates. The renal tubules of the frog (mesonephros) and of several reptiles, (Chrysemys marg., Alligator miss., and a lizard) were readily reconstructed. For the time the reconstruction of the renal tubules of adult mammals and birds, owing to the long medullary loop, seemed

202 AMERICAN ASSOCIATION OF ANATOMISTS

beyond the reach of possibility. A method for completely isolating the renal tubules of mammals by teasing was later developed, by means of which it was possible to isolate complete renal tubules of an adult mammal (rabbit) and to determine the chafacteristic epithelium of their different segments. After further interruption of study of the renal tubules of birds was undertaken, as here reported. The bird's kidney studied was that of the chicken (roosters, two to four years old) ; the method of maceration and teasing, that developed by the author; injection of 75 per cent hydrochloric acid through the arterial supply, teating under water with the aid of a steroscopic binocular. Many complete renal tubules of the bird's kidney attached to portions of the duct system have been isolated.

The kidney of the bird is of special interest in that it presents a transition stage between the simple renal tubules without medullary loop as observed in reptilia and the more complex tubule with long medullary loop having distinctive epithelium as observed in mammalia. A bird's renal tubule of the mammalian type possesses a relatively large renal corpuscle and glomerulus, a relatively long proximal convoluted portion, a proximal limb of the medullary loop with flattened epithelium, the loop itself and the distal limb of the medullary 'oop with short cubic epithelium and a relatively long, much-coiled distal convoluted portion. The bird's renal tubules of the simplest type — ^reptilian type — present relatively small renal corpuscles and glomeruli, with the tubular portion arranged in the form of a compressed W; the proximal V, near the glomerulus, presenting the epithelium of the proximal convoluted portion, the distal V, joining the duct, being lined by short cubic epitheUum. Intermediate types, ranging between the mammalian and the reptiUan types of tubule, are observed.

25. The significance of different and distinctive types of bronchial architecture mthin the same order of mammals, Geo. S. Huntington, Columbia University.

The detailed study of the bronchial system of mammalia reveals within ordinal and generic groups individual forms which dilBFerentiate themselves sharply in the architecture of the bronchial tree from the prevalent bronchial type f oimd in all other members of the group to which the aberrant forms are zoologically assigned. In some instances the departure in bronchial organization typical for the remaining members of a taxonomic group may fairly be interpretated as a progressive adaptation to changed environmental conditions affecting respiration. Thus, among rodents, in the subfamily Hydromyinae, Xeromys, a purely terrestrial form, exhibits the dominant asymmetrical bronchial type, common to most members of the order, with the eparterial bronchus confined to the right side. In its close relative, Hydromys, strictly aquatic inhabit, a symmetrical bilateral eparterial district has developed. The same condition is encountered in the aquatic rodent, MyopotamuSy in which the left eparterial bronchus appears in association with the equally well developed left infra-cardiac bronchus.

PROCEEDINGS 203

These isolated instances of left eparterial bronchial extension in some aquatic rodents seem to place themselves in line with the corresponding puhnonary organization found in its full development in many of the pinnipede carnivores and some cetaceans.

The adaptation to aquatic life calls for more or less prolonged periods of suspension of respiration during submersion. Accessory modifications of the vascular system render possible the storing of CO2 in the blood, until the same can be exchanged in large quantities and rapidly when respiration is resinned. The huge hepatic caval sinuses of Phoca and the enormous plexiform network of the abdominal and urogenital veins in Macrorhinus are examples of these secondary and associated vascular modifications. Under such conditions the process of respiration becomes exceedingly active and rapid. The lung responds to this heightened physiological demand by the greatest possible unfolding of the respiratory area. The ultimate respiratory expansion in any mammalian lung is in direct ratio to the distances separating the origins of the primary derivatives from the stembronchus and to the calibre of the tubes. The physiological significance of the eparterial bronchial development is the attainment of the greatest possible interval between the origins of the first and of the succeeding tiers of primary bronchi from the stembronchus, with the resulting greater unfolding of the respiratory area in the cranial divisions of the lung. The latter, in lungs with fully developed eparterial districts, show increase in size, more complex lobar organization, freer mobility in expansion and in adaptation to the topographical conditions of the thorax and its contents. They become distinctly more eflScient organs.

The same significance attaches to the right tracheal eparterial bronchus found imiformly throughout the Artiodactyls, and to the bilateral eparterial development of the Perissodactyls and Proboscideans. Here increase in pulmonary development is apparently called for by massive bulk, great muscular development, and, in many forms, by the necessity for rapid and long continued locomotion, all factors calling for increased tissue combustion and active respiratory exchange.

Respiratory metabolism throughout the vertebrates appears reflected in pulmonary structure primarily in reference to the rapidity with which the gas exchange is to be effected. All more highly organized complex lungs indicate either a high rate of tissue combustion at a fairly constant ratio (Ungulates), or a rapid metabolism called for at definite intervals (aquatic adaptations), with intermissions, during which respiration is suspended. The lung appears to acquire morphologically the development corresponding to the highest degree of eflSciency which can be functionally demanded in any given form under all conditions.

The organ is, so to speak, overpowered for the work it ordinarily has to perform, but is hence capable of responding to unusual demands.

While many of the aberrant lung types among mammalia may thus be analysed as adaptations to specialized environmental factors, others do not fit themselves into this interpretation, and call for special consideration.

204 AMERICAN ASSOCIATION OP ANATOMISTS

A distinctive bronchial type, in which all primary derivatives arise from a distal tracheal enlargement (bulla) and unfold as bilaterally sjrmmetrical hyparterial bronchi, is found among the rodents, in some Hysiricomorphs (Hystriz cristata) and in one member of the carnivore family of the Mustelidae, Taxidea americana. In all the other Mustelidae, whose p^lmonary organization is known, and at least in some of the Hystricomorphs, the dominant mammahan asymmetrical bronchial tree obtains.

Both animals possessing the aberrant pulmonaiy pattern fairly conform to the nearest associated tyF>es in the carnivore and rodent groups to which they are respectively assigned, in respect to weight, body-build, habitat, mode and speed of locomotion, hibernatioA, etc., all external factors which might be regarded as influencing pulmonary development. Likewise in food habits, dentition and alimentary canal they agree with their zoological colleagues — Taxidea has an alimentary canal which, in respect to the structure of the stomach, the relative length of the individual intestinal segments, the simple ileo-colic jimction without caecum, can hardly be distinguished from that of Meles. Hystriz conforms in every particular of its alimentary canal to the rodent type.

We are hence confronted by the important question as to the value of pulmonary organization in determining phyletic relationship. In spite of the nearly congruent structure of all parts subject to the external influences of environment and food, Taxidea, in its pulmonary ground plan, is not a typical Mustelid badger, nor, judged by the same evidence, does Hystrix agree with all other rodent porcupines.

To take the case of Taxidea for closer examination, the morphological facts may be analysed in one of four ways:

1. My friend. Prof. William K. Gregory, of the American Museum of Natural History, who has devoted especial attention to the phylogenetic history of the MusteUdae, derives the extant members of the group from the lower Oligocene Plesictis. Accepting Plesictis as the common ancestor of the modem MusteUdae, it is possible to assume that the Plesictis-lung was constructed on the bilateral symmetrical hyparterial type, retained today in only one of the living descendants, Taxidea, while in all other recent Mustelidae, as far as known, environmental changes have so altered the constitution of the germplasm as to produce the changed pulmonary type with right eparterial development.

It is probable that the influence of changed environment upon the developing germ cells, and through them on evolution, has, if anything, been underestimated in the past, as notably by Weismann. But in the case in hand, the above outUned hypothesis is untenable, because all our available evidence proves the mammalian lung to be extremely sensitive in its structural response to environmental demands. Witness the above cited instances of pulmonary adaptation in isolated members of rodent groups to aquatic life furnished by Hydromys and Myopotavius. Now any changed environment capable of producing

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chromomeric alteration in the developing germ cells of the Plesictis descendants must have operated equally and evenly on all to produce the prevalent Mustelid type. On no possible grounds could Taxidea be exempted, when in all other environmental adaptations it conforms to the other members of the group. The lung of Taxidea is unquestionably physiologicaUy as efficient as are the lungs of the other Mustelidae. Its occurrence in this form, which in general body structure, alimentation, locomotion, habitat, etc., agrees with its other taxonomic colleagues, alone proves this. Otherwise Taxidea would either have become extinct or its lung would have adapted itself structurally to the new demand. The perpetuation side by side of the Taxidea lung and of the prevalent pulmonary type of the other Mustelidae disproves the premises on which the first hypothesis is based.

2. It might on the other hand be reasoned that the Plesictis lung had already developed the dominant d6xtral eparterial distribution, transmitted imchanged to all of the modem descendants, with the single exception, as far as known, of Taxidea. In Taxidea pulmonary reduction occurred, resulting in the loss of the right eparterial lung segment and the acquisition of the tracheal bulla.

Aside from the fact that no environmental changes are known, capable of inducing this reduction and confined in their operation to Taxidea to the exclusion of the other MusteUdae, the above supposition assumes morphogenetic processes, which on close examination we find nowhere in the mammalian series. I know of no single instance of even a probable reduction of a formerly developed pulmonary element in a mammal. In fact the phylogenetically acquired metameric reduction of the trunk cavity in manunalia would of itself negative this assumption. The inclusion of the cardiac lobe in the phrenico-mediastinal angle of the right lower lobe in man and some other manamalia is merely in consequence of the closure of the pericardio-phrenic space consequent upon the fixation of pericardium to diaphragm. The morphological character of the lobe is retained in the cardiac branch of the right stembronchus.

D'Hardiviller's discovery of an ephemeral left eparterial bronchial vesicle in* the embryo of the rabbit, if confirmed, points far more directly to an attempt on part of the left lung to acquire additional respiratory territory, than to the evanescent appearance during ontogeny of a pulmonary element lost in the phylogenetic evolution of the mammalian lung. This is confirmed by the occurrence of adult bronchial variants. I think we are justified, on the basis of aU of our actual facts, in concluding that any manmialian lung which has once acquired a focal point of entodermal bronchial proliferation, retains that point in its structural organization of the lung, even against apparently imfavorable local and topographical modifications, as e.g., of the thoracic space.

Neither of the above hypotheses 1 and 2 answer in any way the problems raised by the aberrant pulmonary type of Taxidea and Hystrix. Neither offer more than agnostic circumlocution, a pseudoscientific way of leaving them unexplained.

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3. The assumption that the lungs in question are the result of variation by mutation in one of the phylogenetic hues, transmitted by inheritance to the modern descendants, is the only one compatible with the acceptance of a monophyletic derivation of the aberrant types. It labors under the enormous handicap that mutations of this degree, affecting exclusively a single organ system, are heretofore imknown in manmiaUan organogeny.

4. Hystrix and Tcmdeaj conforming in skeletal and alimentary characters to the respective rodent and carnivore types with which they are zoologically grouped, yet dififerfrom them radically in their pulmonary organization. This difference is not one of degree, not merely an exaggeration or reduction of a pulmonary character common to all manamaJia. It is fundamental, inherent in the genetic groundplan of the organ, and it manifests itself in the earliest ontogenetic differentiation of the endodermal lung tube. 'Its development implies a histonal selection differing from that governing the genesis of the dominant manamalian type. Comparison of the lung of Taxidea with that of Meles, or Mephitis or (xuloy or of any one of the Mustelidae, whose pulmonary organization is known, render the conclusion inevitable, that the same germplasm, or the same determiners of the chromomeres, cannot be held responsible for the production of these divergent results in adult structure. The modem zoological groups of the carnivore Mustelidae and the rodent Hystricomorphs contain, therefore, today on the evidence of their lung structure, each a form which departs so fundamentally in this regard from its respective colleagues, as to create at least a grave doubt concerning their germinal kinship. These considerations raise the question as to a diphyletic origin for certain of the modem zoological groups. It is possible that all the extemal congruent characters uniting two given types are the results of a high degree of convergence and adaptation to identical conditions of habitat, locomotion, alimentation, dermal protection and other (environmental factors.

^6. An interpretation of connective tissue and neurogliar fibrillae. Raphael Isaacs, from the Anatomical Laboratory of the University of Cincinnati. (Introduced by H. McE. Knower.)* Observations on Connective Tissue and Neurogliar Fibrillae. The study of living connective tissue in cover glass and hanging drop preparations, checked up by experiments with gelatin, albumin and fibrin, and compared with fixed tissue, shows the intercellular substance to be homogeneous, that is, without a formed network in the ground substance in the living condition. The methods and materials used, confirm many points dscribed by different investigators and suggest interpretations of others. The connective tissue fibrillae, described as exoplasmic fibrillae by Mall and others, as well as the corresponding fibrillae of neurogliar tissue, do not appear in the Uving intercellular connective tissue colloid, as they arenot visible in the living tissue, nor can they be demonstrated by stains or optical methods. They can be produced in fresh tissue under the microscope through any agency

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which will cause the material, which is distributed in the intercellular spaces, to shrink up. This shrinkage produces fibrillae with spaces, formerly occupied by the ground substance, between them. The possibility of washing out interfibrillar substance is eliminated by this method. The pattern and delicacy varies with different fixatives and resembles the pattern of the fibrin coagulmn in the blood vessels. The freedom of movement of the cells and the appearance of fibrillae across gaps in separated pieces of tissue which had been placed in contact and fixed in this position, further supports the view of the origin of the fibrillae by the coagulation of the ground substance. The fact that the white fibers and elastic fibers of flie tissue, when first formed, have a jellylike consistency, and not the consistency of a transformed network of preexisting fibrillae, is additional evidence. The fibers, studied stage by stage, are shown to become progressively stronger and denser by the concentration of their material through increased deposit of substance. The movement of the connective tissue cells probably effects the distribution of the material through chemical or other action and causes the fibrillated structiu-e of the adult fibers.

Forces acting on the jelly-like embryonic connective tissue affect the cells and colloid alike, but the cells have the power to readjust themselves to the new condition, thus producing permanent structures in reaction to pulls and pushes.

The colloid is continuous between the cells and is more jelly-like than either lymph or blood. No free-flowing intercellular fluid could be demonstrated.

The spindle-shaped type of connective tissue cell appears to be the most stable form, the stellate cells often reverting to this shape when freed from the surrounding pressures. The cells he free in the colloid in the younger embryos and can be easily separated mechanically. This observation is in accord with the findings of Clark, Ferguson and Stockard, that the mesenchyme cells in embryos exercise the power of active locomotion.

By means of experiment with fresh tissues, it is found that the fibrillae of fixed tissue span cut surfaces which had been allowed to touch. This would suggest that if pressure were exerted on an embryo, (the contraction on fixation,) early lymphatic aniagen, which have been described as irregular spaces in the mesenchyme, and which are at first not lined by typical endothelium, could be closed in places by these 'fixation adhesions.' As the weaker colloid of younger embryos leaves less precipitate than that of the older stages, we can assume that regions of less concentration in the older embryos will exhibit more deUcate fibrillae. As such regions have been described (Kampmeier) it is probable that we have pathways physiologically determined by the establishment of regions of less concentrations. Into these, capillaries may grow by following the lines of least resistance. This would seem possible from the fact that young capillaries, both blood and Ijonph, show no condensation of connective tissue cells around them, as do the more solid organs as the salivary glands, thyroid and thymus, which have pushed into the more dense tissue.

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27. Effects of inanition upon the structure of the thyroid and parathyroid glands of the albino rat. (Lantern slides.) C. M. Jackson, Institute of Anatomy, University of Minnesota.

The material for this study was derived from numerous young rats held at constant body weight by underfeeding for various periods, and from adult rats subjected to acute and chronic inanition.

In the younger rats, the histological changes are varied as follows. The nuclei of the follicular epithelial cells in many cases undergo chromatololysis (various stages of karyolysis), and are sometimes swollen. Hyperchromatosis, however, is more frequent. Some stage of karyopycnosis is usually present. In earlier stages the nucleus may be nearly normal in size and structure, excepting a pale homogeneous coloration of the nuclear background. In more advanced stages the nucleus diminishes in size, with deepened coloration, forming a dense homogeneous mass (typical pycnosis). In extreme cases the nucleus becomes fragmented (karyorrhexis). Mitosis is never found.

The cytoplasm of the follicular epitheUimi is usually reduced in amount, but may show no marked change in structure (simple atrophy). Sometimes the cytoplasm is not reduced in volume, but in this case it always appears rarefied, with decrease of the normal granulation and usually with marked vacuolization. In advanced stages, the cytoplasm may disintegrate, forming irregular, deeply -stfdning masses of varied appearance.

The intrafollicular colloid may show no abnormal changes. Advanced stages of degeneration in the follicular epithelium, however, are accompanied by dissolution and distintegration of the colloid. The colloid is often replacd by desquamated epithelial cells in various stages of degeneration, and the entire follicle may collapse into an irregular mass.

The interfollicular connective tissue (stroma) usually shows no marked change in structure, but in some cases the ground substance is greatly increased in amount, giving an edemic appearance. On this account, a gland may show little change in gross weight, although there has been a marked atrophy of the parenchyma.

In the adult rats subjected to acute and chronic inanition, the changes in the structure of the thyroid gland are likewise varied, but in general appear similar to those found in the younger rat^s. The interpretation of the changes in the older rats is more diflScult on account of the frequent occurrence in the normal (control) rats of degenerative changes similar to those found in advanced stages of inanition.

The parathyroid gland is in general more resistant to inanition. The effects upon its epithelium are somewhat similar to those described for the thyroid, though usually less marked. The nuclei may remain nearly normal in size and structure, though usually slightly smaller and exhibiting various stages of karyolysis or (more frequently) karyopycnosis. Mitosis ceases. The cytoplasm may be either reduced in amount (sometimes deeply staining) or may remain normal in volume,

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with marked vacuolization. The stroma may remain normal in amomit or may be increased in volmne by infiltration of gromid substance. Variations also occur normally in the structiu-e of the parathyroid (through less marked than in the thyroid), so that here likewise caution must be observed in drawing conclusions as to the effects of experiments.

£8. Notes on the neuromeres of the brain and spinal cord, (Lantern.)

Franklin P. Johnson, University of Missouri.

While studying a human embryo of 23-24 segments (a description of which is now ready for publication), it was discovered that not only does the rhombencephalon possess neuromeres, but the whole medullary tube as well is definitely marked by a series of alternating constricted and expanded portions. The fact that the medullary tube possesses neuromeres has apparently been overlooked in the numerous descriptions of young human embryos. Minot (Human Embryology^ 1892) however, noted their presence in mammalian embryos, and recently Watt (Carnegie Contributions to Embryology, vol. ii, 1915) observed them in twin human embryos of 17-19 segments. I have since found them to be constant in all other young human and pig embryos which I have examined. The present work was undertaken to determine the similarities which exist between the neuromeres of the brain and spinal cord, the relations of the nerves to the neuromeres, and the relations of the neuromeres to the body segments. The following points have thus far been revealed:

1. The neuromeres of the spinal cord are intersegniental in position, that is, the sweUing of the neuromere is placed opposite the intersomitic spaces. This fact was noted by McClure (Jour. Morph., vol. iv) in embryos of amphibians, reptiles, and birds, and by Minot (Human Embryology) in mammalian embryos. The same is true of the neuromeres of the rhombencephalon in the region occupied by the occipital somites.

2. Each neuromere of the spinal cord gives rise to the ventral root and receives the dorsal root of a spinal nerve. Likewise the rhombic neuromeres, with the exception of the first and possibly the fourth neuromeres, give rise to and receive the motor and sensory roots of the cerebral nerves.

3. The relations existing between the neuromeres and cerebral nerves are as follows:

The first rhombic neuromere in the embryos I have thus far studied does not give rise to any nerve. It is possible, however, that the trochlear nerve is a later outgrowth from this neuromere.

The second and third neuromeres both contribute to the efferent root of the trigeminal nerve. Both receive the afferent fibers from the ganglion of the same.

The fourth neuromere gives rise to the efferent and receives the afferent fibers of the facial nerve. In addition it receives the afferent fibers of the acustic nerve.

The fifth neuromere gives rise to the abducent nerve, although this

210 AMERICAN ASSOCIATION OF ANATOMISTS

ft

nerve appears somewhat later than most of the others. In addition it receives a small number of the afferent fibers of the acustic and facial nerves. These fibers, however, enter the brain on the fourth neuromere and pass immediately in the brain wall to the fifth neuromere.

The sixth and seventh neuromeres give rise to the efferent and receive the afferent fibers of the glossopharyngeal and vagus nerves respectively.

The eighth and ninth neuromeres give rise to the efferent roots of the hypoglossal nerve. The eighth receives the afferent fibers from the root ganglia of the accessory nerve; the ninth, a few fibers from the root gangUa of the accessory nerve together with a few from Foriep's ganglion.

The succeeding neuromeres belong to the spinal nerves, the first cervical nerve to the tenth neuromere, the second to the eleventh, etc.

The lateral roots of the accessory nerve proceed from the eighth to the thirteenth neuromeres inclusively.

4. Since the first cervical nerve belongs to the tenth neuromere, the preceding nine belong to the rhombencephalon. The lower limit of the rhombencephalon may, therefore, be definitely defined in young stages as the constriction between the. ninth and tenth neuromeres. This lies opposite the last pair of occipital somites.

5. The lumen of the central nervous system likewise shows expanded and constricted portions corresponding to the neuromeres and the depressions between them. This is well known concerning those of the rhombencephalon and easily demonstrable in case of those of the medullary tube. .

29. A comparative microscopic study of cardiac and skeletal musde of

Limulus, H. E. Jordan, University of Virginia.

Limulus heart muscle in stained sections is practically identical with vertebrate heart muscle; it has a coarser texture, but it is syncytial in structure and contains a conspicuous telophragma or Z stripe, the Q and J stripes being generally indistinct. Skeletal (spine) muscle of Limulus, Hke that of vertebrates, consists of finer and more robust multinucleated fibers; but from the viewpoint of its myofibril arrangement and its cross striations it resembles more closely the cardiac type of muscle. Among the cross striations, the Z stripe is the most conspicuous. Moreover, the skeletal muscle, like cardiac muscle generally, lacks a mesophragma or M membrane. In both types of muscle, in the contracted condition, only contraction bands are visible; these are coarser darker discs at the levels of the telophragmata. In view of its detailed similarity to cardiac muscle, including the promiscuous location of the nuclei either centrally or peripherally in the fiber, the skeletal muscle of Limulus must be regarded as a less highly differentiate type of striped muscle.

The close structural similarity between cardiac and skeletal muscle in Limulus extends to still other details: The apparent fibrillar unit of structure, the myofibrilla or sarcostyle, resolves on higher magnification and under certain artificial conditions into still finer fibrils, to

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the limit of visibility. This observation supports Heidenhain's *Teilkorper Theorie' ('histomere' or ' prctomere' theory) enunciated as a general histologic principle, and based largely upon the study of striped muscle development in trout (Arch. mikr. Anat. 83: 4, 1913.)

A sarcolenmia is present in both cases, distinguishable from the closely investing endomysium by a different staining reaction to acid fuchsin. Meek (Joum. Morph. 20: 3, 1909) reported the absence of a sarcolenmia in Limulus cardiac muscle. He conceives of this musculature as a double syncytium composed of trabecule 'individualized by connective tissue sheaths.* But with Van Gieson's stain, successfully applied, a delicate inner layer remains unstained, whereas the outer portion stains a light red color. When the application of the stain is unduly prolonged, the sarcolemma also stains red, but so do also the ground membranes, the nuclear membranes, and the peripheral sarcoplasm. Moreover, the muscle nuclei are larger, more vesicular, and more regularly oval than those of the intertrabecular endomysium.

This material furnishes precise data also with respect to the nature of the telophragma or ground membrane, and its relation to the sarcolemma and the nuclear wall. The Z stripe results from a continuous membrane extending completely across the fiber; to it are firmly attached bundles of the ultimate fibrillae, forming the so-called sarcostyles. At the point where fibrilla and membrane meet the latter appears to swell and presents the form of a spherical granule; this circumstance gives to the Z membrane a granular appearance. Peripherally the membrane unites with the sarcolenmia, the latter frequently showing a uniform festooning. Centrally the membrane joins with the nuclear wall; the latter frequently, when in shnmken condition, presents a series of spinous projections, corresponding in number and spacing to the number and spacing of the corresponding Z membranes.

The continuity of the telophragma with the nuclear wall across the perinuclear sarcoplasm presents conclusive evidence against the interpretation of striped muscle structure in terms of 'muscle cells' and extracellular myofibrillae, as first suggested by Apathy and recently again urged by Baldwin. (Zeits. f. Allgem. Physiologie, Bd. 14, 1912).

Nuclear multiplication in growing Limulus muscle is amitotic; as many as eight or even more nuclei may lie in series in a single sarcoplasmic area. Not a single mitotic figure occurs; but all stages in amitosis can be found.

In cross-section the myofibrillae of both types of muscle are seen to be arranged in radially placed lamellae; in the skeletal muscle these lamellae undergo peripheral radial and central tangential splitting, recalling an early ontogenetic phase in striped muscle histogenesis of certain teleosts, e.g., trout.

In an earlier study (Jordan and Steele, Am. Jour. Anat. 13: 2, 1912) I came to the conclusion, therein confirming Meek (1909), that intercalated discs are lacking in the Limulus heart. Reinvestigation with a different staining technic (iron-haematoxylin, after alcohol-nitric acid fixation) reveals a few discs of the simple 'comb' type. On the basis of

212 AMERICAN ASSOCIATION OF ANATOMISTS

my interpretation of intercalated discs in tenns of irreversible contraction bands, following strain incident to prolonged rhythmic contraction,, intercalated discs would be expected to occm* in Limulus heart muscle, since the latter beats rhjiihmically and has a structure essentially like that of vertebrate myocardium. In view of the slowness of the heart beat (about 20 to 32 beats per minute— Carlson; Patten), only a small number of discs of simple construction were to be expected. This expectation is actually realized; the intercalated discs are of the 'comb' type, and generally divide contracted from relaxed portions of the fiber, as is frequently the case in mammalian heart muscle.

The position of the intercalated disc at the lev^l of the ground membrane, the firm union of the fibrillae to the membrane, and the resolution of the fibrillae into finer fibrils as seen in Limulus cardiac muscle throw light upon the origin of the serrated type of intercalated discs almost exclusively present in hypertrophied mammalian hearts, and to some extent in apparently normal mammalian hearts. What was difficult of interpretation hitherto was the character of the connecting substance between successive crests of the 'saw-toothed' discs (Anat. Rec. 6: 9, 1912). Hypertrophy is essentially a matter of fibril increase; moreover, in hypertrophied heart muscle the usual tensions among the fibrils are conceivably readily disturbed throwing adjacent fibrils out of functional synchrony and even placing them under directly opposed stresses. Granting the latter possibility, serrated types of discs are readily derived from simple straight *comb' types by a splitting of the original sarcostyle, the resulting fibrils of which, after more or less complete separation at the level of the original 'comb' disc, are drawn in opposite directions, or for imequal distances in the same direction, the ground membrane meanwhile retaining its connection with the displaced division products of the disc, thus producing a serrated condition.

With respect to the mode of conduction of the impulse to heart beat the Limulus myocardium answers the requirements of the neurogenic theory (Carlson) ; vertebrate cardiac muscle apparently acts in accordance with the myogenic theory. But the two musculatiu-es (Limulus and vertebrate) agree in theij* rhythmic functional activity, their sjoicytial structure, the presence of intercalated discs, and the presence and relationships of cross-stripes and sarcolemma. The difiference in the method of impluse conduction inheres most likely in the absence in the Limulus heart of anything analagous to the longitudinal coordinating muscle bands, the atrioventricular conducting bundle of His, of mammals and certain vertebrates. Longitudinal muscle fibers are practically absent in the Limulus heart. As a consequence the only remaining mechanism which could mediate conduction of the impulse for the 'apparently instantaneous' (Carlson, Am. Jour. Phys., vol. 12, 1904) contraction of the whole series of the nine annular segments of the essentially metameric heart, is the longitudinal nerve cord.

The structurally dififerent constituents of the Limulus sarcostyle are the Z membranes and the Q and J discs. Q and J most probably

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differ only in the matter of a relatively greater abundance ofcertain darker staining^ materials (the so-called 'anisotropic granules') in the former. In the wing muscle of the fly Meigs, (Zeits. f . Allgem. Physiologie, 8: 1, 1908) recognizes only Q> Z and M; he regards J as the optical effect of a greater refractive index on the part of Z as compared with its enveloping substance, Q. That this interpretation cannot properly apply to Limulus muscle seems indicated by the relatively great width of J as compared with the Z membrane, and the different degree of stainibiUty of Q and J. Moreover, there is not the slightest indication of an M membrane, either in the cardiac or the skeletal muscle. Thulin (Archiv. mikr. Anat., 86: 3, 1915) records the absence of an M membrane also in the wing muscle of Coleoptera, and in the analogous (pectoral) muscles of birds and bats. An M membrane apparently only occurs in certain more speciaUzed types of striped muscles; it is probably never present as a true membrane in cardiac muscle.

SO. The development of the occipital region of the domestic cat with an

interpretaiion of the paracondyloid process. John D. Kernan, Jr.

From the Anatomical Laboratory, Columbia University.

Investigators are by no means agreed as to the elements entering into the composition of the basal part of the occipital region. Weiss (1) working with rat embryos found that the basioccipital was formed from hypochordal elements exclusively, without body elements. Noordenbos (2) working with Talpa, and Gaupp (3) working with opossum and rabbit embryos confirmed the findings of Weiss. On the other hand, Froriep (4), with calf embryos showed that in this animal the basioccipital is formed through chondrification of perichordal tissue dorsal to the hypochordal arches. Thus, according to him, there is formed a body to the occipital vertebra; which later fuses with the bodies of the unsegmented vertebrae which form about the chorda in a more cranial position. The united bodies form the basioccipital to which are subsequently joined the independently arising arches. The hypochordal arches winch appear in the early embryos are absorbed without chondrifying.

In view of these contrary findings, the conditions found in early cat embryos are of interest. In an embryo of 9 mm. foiu- membranous arches can be made out in the occipital region. These show a row of dense masses laterally, joined ventrad to the chorda by hypochordal bars. These hypochordral bars are in contact with the chordal «heath, and form with it a row of perichordal crosses such as is foundin other areas of the vertebral column. Of the four membranous arches which can be made out, the two cranial are indistinct and have a tendency to fiise.

In an embryo of 10 mm. the tissue of the lateral masses has advanced far toward chondrification, and they have begun to fuse longitudinally, forming two precartilaginous bars between which Ues the chorda free of cartilage. These bars may very well be compared to the parachordal processes of lower vertebrates.

THK ANATOMICAL RECORD, VOL. 10, NO. 3

214 AMERICAN ASSOCIATION OF ANATOMISTS

In an embryo of 11 mm., the lateral bars of cartilage have begun to unite across the median line ventral to the chorda. There is thjis formed a cartilaginous trough in which lies the chorda, with no cartilage dorsal to it.

It must be noted that throughout the formation of the basioccipital as thus far traced no perichordal cartilage forms between the primitive arches, where the bodies form in other regions of the vertebral column.

In an embryo of 12.5 mm., for the first time, cartilage dorsal to the chorda is present. This is in immediate connection with the perichordal sheath, in an area craniad to the occipital region. At no stage does the chorda receive a dorsal cartilaginous covering in the occipital region.

Comparison of this mode of formation of the basioccipital with that of the other vertebra shows it to be the same as that by which the atlas originates, that is by chondrification of two lateral masses, which subsequently are joined by chondrification along the line of the hypochordal bar. The basioccipital of the cat then may be said to be made up of vertebrae of the atlas type. This view has already been suggested by the authors quoted, most clearly by Gaupp.

The paracondyloid process is ordinarily interpreted as being constituted by the transverse and costal processes of the oc dp'tal vertebra. It forms the ventral tip of a lateral expansion of the occipital wing, known as the 'lamina alaris' (Voit) (5). The lateral border of tins lamina extends along the caudal margin of the otic capsule and fuses with it, thus forming the commissura occipito-capsularis.

Examination of a series of cat embryos from 15 to 35 mm., shows that the lamina alaris is connected with the occipital wing craniad and dorsad to the hypoglossal foramen. This would indicate that the lamina alaris and its free ventral tip, the processus paracondyloideus, belong, not to the occipital vertebra, but to the unsegmented vertebrae craniad thereto. An examination of a reconstruction of a 20 mm. human embryo and the sections of the same gives evidence for this interpretation. In the sections it is seen to consist of two parts as regards its cartilage, a lateral and a mesal. The mesal part is a lateral protrusion of the occipital wing. The lateral part is a bar of cartilage parallel to the mesal ridge, and in close contact with it, but showing a separation of its cartilage at certain levels. It is entirely independent at the tip. Then for a distance dorsal to the tip it is in close contact with the mesal part, but separated from it by a layer of perichondrium. Dorsal to this area of close contact, there is for a distance a fairly wide separation, then an area of complete fusion. Still more dorsally there is again an area of separation, then complete fusion, which is not again interrupted. From the tip of the paracondyloid process there extends mesad and craniad a thin process of cartilage which reaches the basioccipital dorsal and laterad to the hypoglossal foramen. This was found by Macklin (6) in his 40 mm. embryo, and bounded laterally and oranially a foramen which he termed paracondyloid foramen. It corresponds in position to a ridge of bone which in the adult passes from the jugular process

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mesad and craniad to the basioccipital. It represents as Macklin suggests a costal process, not that of the occipital vertebra, but of on^ of the unsegmented vertebrae, probably the second, since its connection to the basioccipital and occipital wing are laterad and dorsad to the hypoglossal foramen.

The paracondyloid process then is the free extremity of the lamina alaris, and this has been shown to be a bar of cartilage uniting at intervals with the parallel ridge of the occipital. The outer bar represents fused costal elements, and may be termed the costal bar. The points of union correspond with the transverse processes, the areas of separation indicating the intervals between the processes of successive vertebrae. Three such processes can be enumerated. That of the occipital vertebra is small and shows no independence of the costal elements. The second vertebra shows an independent costal element which is drawn out caudad and forms the paracondyloid process. The most cranial vertebra helping to form the basioccipital shows no free costal element. The costal bar is piolonged upward on the occipital wing as a ridge which represents the inferior nuchal line of the adult bone. The caudal prolongation of the costal bar, the paracondyloid process, overhangs laterally the costal and transverse processs of the occipital vertebra. These subsequently imite with it, though the division can still be traced in this embryo by the arrangement of the cells in the sections.

(1) Weiss, A. Die Entwi6keluiig der Wirbelsfiule der weissen Ratte, besonders

der vordersten Halswirbel. Zeitschs. f. wissenschaftl. Zool., Bd. Ixix, Heft 4, 1901.

(2) NooRDENBOs, W. Weber die Entwickelung des Chondrocranium der Sftu gethiere. Petnis Camper. Deel., iii, 1905.

(3) Gaupp, E. Zur Entwickelungs geschichte und vergleichenden Morphologie

des Schadels von Echidna aculeata var. typica.

(4) Froriep, a. Zur Entwickelungs geschichte der Wirbels&ule, insbesondere

des atlas und Epistropheus und der Occipital region. Arch, f . Anat.

and Physiol. Anat., abth. 1886. (6) VoiT, M. Das Primordial cranium des Kaninchens. Anat. Heft, v. 38, 1909. (6) Macklin, C. C. The skull of a human fetus of 40 mm. American Jour, of

Anat.. vol. 16, no. 3, 1914.

31. On the relation of mitochondria to zymogen granules, J. Albert

Key. Creighton Medical College, from the Department of Anatomy

of the University of Chicago. (Introduced by R. R. Bensley.)*

The material used for this study was the pancreas of the toad. A

number of toads were injected with pancreatic secretion or pilocarpine

and the pancreas was fixed at various periods after injection. In some

cases the injection was repeated at regular intervals for several days.

The mitochondrial methods of Bensley, Altman, Benda, Regaud, and

Kolster were used. But the best preparations were obtained by fixing in

Bensley^s acetic-osmic-bichromate mixture and staining with acid fuchsin

216 AMERICAN ASSOCIATION OF ANATOMISTS

and dififerentiating with picric acid. For zjrmogen granules the pancreas was fixed in Bensley's formalin-zenker solution and stained with neutral gentian or neutral safTranin.

In the normal resting pancreas of the toad the zjonogen granules crowd the portion of the cell between the nucleus and the lumen and may extend beyond the nucleus into the basal zone. The mitochondria are chiefly ifound in the basal zone but may extend around the nucleus into the mass of zymogen granules. When the gland is subjected to prolonged stimulation the large zymogen granules are discharged and the central zone of the cell is occupied by very minute granules having the same staining reactions as the zymogen granules. The mitochondria on the contrary are not exhausted even by prolonged stimulation, but in many cases seem to increase in length and extend to the central zone of the cell.

In both the resting and in the stimulated pancreas the mitochondria curve around the nucleus and wind through the cytoplasm with their long axes roughly parallel to that of the cell. They vary greatly in length and in diameter. Branching forms are fairly common, as are also the knobs and spindle like swellings on the filaments which have been interpreted as evidence that the mitochondria break up to form the zymogen granules. However these swellings do not stain with neutral gentian, neutral saflfranin, or neutral red. Nor are they well fixed in formalin-Zenker material. By combining his neutral gentian and acid fuchsin methods Bensley is able to dififerentiate the mitochondria and the zjonogen granules in the same proportion. I was unable by this method to detect any difference between the swellings and the rest of the filaments. Hence I concluded that they do not contain zymogen granules. Likewise the absence of reciprocal changes in the amount of mitochondria with variations in the content of zymogen granules leads me to believe that the zymogen granules are not formed directly by the mitochondria.

32. Studies in peripheral nerve regeneration, Edwin G. Kirk and

Dean D. Lewis, Morris Institute of Medical Research of Michael

Reese Hospital, Chicago.

Technique, Segments of the dogs' ischiadic nerve varying in length from 1 to 3 cm. were excised, fascia lata from the same dog being used to construct a tube leading from proximal to distal stump, the hiatus not being otherwise closed. Thus, regeneration following trauma may be studied without interference from various external mechanical factors. Also the comparative behavior of proximal and distal stump is more easily determined than when the ends are approximated. Present material includes 36 nerves, of which 14 are complete serial sections.

Observations. In addition to confirming the work of Hanson in almost all details, the following points were noted. The empty fascial tube early fills (first to seventy-second hour) with serum containing a few mononuclear migratory cells into which rapidly grow from the proximal stump, protoplasmic bands containing numerous neurilemmal nuclei, these being direct outgrowths of the neurilemmal sheathes. These

PROCEEDINGS 217

begin invading the serum the first two days and from that time on continuously proliferate down the tube. These protoplasmic bands are not tubular, but solid strands of cjrtoplasm, anastomosing frequently as a network. They constitute channels or beds along which the non-medullated fibers from*the proximal stump rapidly grow, — so rapidly that often the protoplasmic bands precede the non-medullated fibers by not more than the length of one or two neurilemmablasts. The non-medullated fibers (as previously shown by others) originate a) by terminal division of the original bundles of non-medullated fibers of the proximal stump; b) by subneurilemmal division of medullated axones of the proximal stump. A gap of 1 cm. is bridged by numerous protoplasmic bands and their contained non-medullated fibers within 5 to 6 weeks — and correspondingly for larger gaps. (Above observations on Cajal-Ranson technique).

About the sixth week many of the fibers bridging the gap acquire a myelin sheathe in their upper part, i.e., that nearest the proximal stump but yet unmistakably belonging to the regenerated part. (Mallory's phosphotungstic technique.) Many of these non-medullated fibers are exceedingly minute, not more than one-tenth the diameter of a fully developed medullated fiber. At the seventh week the upper 3 or 4 mm. of the regenerated segment contains many medullated axones. At the ninth week many of the fibers bridging a gap of 12 mm. have acquired a medullation throughout this extent and even some distance into the old neurilemmal sheathes of the distal stump. All eflicient regeneration of nerve fibers is from the proximal axone. All medullated nerve fibers regenerate through the gap as non-medullated ones which later acquire medullation. All medullation begins proximally and proceeds distally.

Interpretation, The myelin is laid down in situ, whether by activity of axone or by neurilemma we have not determined — but undoubtedly it appears only in those parts of the new fiber which have reached an age of 6 to 6§ weeks, hence it appears proximally first. But many of the regenerated fibers which have undoubtedly attained this age do not yet possess myelin sheathes. Presumably some of these are derivatives of the original non-meduUated fibers.* [*For earlier series see Kirk and Lewis. Jour. Am. Med. Assoc. 1915. Vol. LXV. p. 486-491.]

SS. The germ oell cycle in the mouse, W. B. Kirkham, Osbom Zoological Laboratory, Yale University, introduced by R. G. Harrison. The material for this research is (1) a complete series of embryos, at about 24 hour intervals, from the fertilization of the egg to the time of birth, and (2) a complete series of gonads of both sexes, likewise representing 24 hour intervals, and covering the period from birth to 50 days old.

The primordial germ cells have not been found in embryos younger than 11 days, but from that time on their development has been traced without a break. They are characterized by having large, round nuclei, and clearly defined cell membranes, while the adjacent epithelial cells have smaller, oval nuclei, and indistinct membranes.

218 ' AMERICAN ASSOCIATION OF ANATOMISTS

Mouse embryos of 11 days show the primordial germ cells scattered through a much larger number of epitheUal cells, without .sexual diflferentiation. At 13 days testes can be distinguished from ovaries by the evidence in the form of tubule formation. In male embryos of 15. days it is evident that the primordial germ cells form the cores, while epithelial cells line the inner walls of the tubules, and this arrangement assumes great significance in view of the fact that older male embryos show degeneration of the primordial germ cells, and a simultaneous increase in size, together with a rounding up of outline, on the part of the nuclei of the peripheral epithelial cells, which can now be called spermatogonia. Some of the primordial germ cells in male gonads divide after tilbules are formed, but all appear sooner or later to degenerate, and about 8 days after birth none are longer visible.

The development of spermatozoa from the spermatogonia goes on at about the following rate, variations of 2 or 3 days being allowable in each case to cover individual differences : the first primary spermatocytes appear on the thirteenth day after birth; first secondary spermatocytes on the twenty-third day; first spermatids twenty-sixth day; and first spermatozoa, attached still to Sertoli cells, on the thirtieth day. Not until the thirty-eighth day after birth have spermatozoa been found in the seminal vesicles, and then in only small numbers.

No new evidence has been found as to the origin of the Sertoli cells, nor can any definite statement be made as to the time of their first appearance in the testes. In testes of 30-day old males Sertoli cells are present, and they can easily be found in any older specimens.

The female gonads during the embryonic period show, in contrast to those of the male, no degenerating cells, but instead a constant increase in size and in number (by mitosis) of the primordial germ cells. At the time of birth the largest primordial germ cells already possess a single enveloping layer of epithelial cells, and can properly be termed oogonia.

After birth the primordial germ cells of the female, or oogonia, increase still further in size, but no increase in number takes place. Many layers of epithelial cells arrange themselves around the largest oogonia, lacunae appear inside the follicles thus formed, and it would seem on the seventeenth day after birth as though ovulation was iabout to occur. However, instead of follicles rupturing at this early age, from about the sevent'Centh to the fortieth day the oogonia which have grown to full size degenerate within their follicles, the first indication being the chromatolysis of the follicle cells, which may or may not be followed by the maturation or fragmentation of the egg. In no instance has a normal maturation spindle or polar body been seen in this material.

The stimulus which causes ovulation first comes at about 40 days after birth, as the earUest ruptured follicles in this material are in ovaries from a 40-day old female, but a female of 50 days was found to contain enabryos which correspond with the usual development at 12 days, while another female whose age was 48 days possessed ovaries with no ruptured folUcles.

PROCEEDINGS 219

In brief this investigation shows that the sexual cycle in the mouse is completed in about 60 days, but that the male and female germ cells come from different cell lines, the oogonia being direct descendants of primordial germ cells, while the spermatogonia arise, not from primordial germ cells, but from what appear to be epithelial cells.

34' The prolonged gestation period in nursing mice. W. B. Kirkham,

Osbom Zoological Laboratory, Yale University, introduced by R. G.

Harrison.

, This is an embryological investigation of the discovery published by Prof. J. F. Daniel in 1910 that among mice, nursing females if again fertilized give birth to a new litter only after a longer gestation period than that of non-nursing females. He further stated that the delay in parturition was equivalent to approximately one day for each young mouse nursed.

Two series of embryos have been studied, one taken from nursing, the other from non-nursing females. Both series are spaced at 24 hour intervals, and cover the period from the fertilization of the eggs to the time of birth.

The differential characteristics of. each day's development having been determined in the series from non-nursing females, a comparison with the muring series shows the following facts:

(a) Ovulation and fertilization, when they occiu*, occur at the same time relative to parturition, — namely the following night, — ^in both nursing and non-nursing females, although muring females are much less liable to ovulate.

(b) Segmentation of the fertilized eggs and their passage down the Fallopian tubes take place at the same rate in both nursing and nonnursing females, resulting in blastulae in the lumen of the uterus on the fourth day.

(c) Implantation occurs in non-nm^ing females during the fourth day, while in nursing females implantation does not take place until the close of the twelfth day. This delay would appear to be connected with the fact that up to this time the nursing young are obta ning all or most of their nourishment from the females.

(d) The stages following implantation show progressive development in non-nm^ing females, but in nursing animals the time elapsed since fertilization bears no fixed relation to the stage of development of the embryos, nor does this appear to be associated with the number nursing nor with the total number of embryos. It can be stated, however, that almost every set of embryos examined is behind the stage of development it should have reached if growth and differentiation at the rate of embryos in non-nursing animals were to bring about birth at the date set by Daniers formula, a day's delay for each one nursing.

The prolongation of the gestation period in nursing mice is therefore largely due to delayed implantation.

220 AMERICAN ASSOCIATION OF ANATOMISTS

85. On the Mesenterium commune of hum^n embryos, (Lantern.) Frederic T. Lewis, Harvard Medical School and James W. Papez, Atlanta Medical College. (Presented by Professor Papez ) In order to obtain a clearer insight into the factors involved in the primary rotation of the intestinal loop, a series of models has been made, showing the mesentery of human embiyos in situ. Although a satisfactory mechanical explanation of the intestinal rotation has not been found, and the work is still incomplete, the models are perhaps of sufficient interest to justify their description. Since in the adult the mesentery is usually displayed by cutting away the intestinal tube along its attached margin, the same method was followed in preparing the models.

In the youngest specimen — ^the Bremer embryo — ^the mesentery is straight and uninterrupted from the anterior end of the mediastinum to the posterior end of the mesorectum, being comparable with the mesentery of a fish. The large pair of vitelline veins and the several small pairs . of vitelline arteries, together With the pneumatoenteric recesses, are essentially bilaterally symmetrical. (The recesses in this embryo have been previously described before this Association, in connection with a demonstration that tracheo-oesophageal fistula is due to a division of the fore-gut along their dorsal margins — Lewis, 1912.)

A sUght but distinct bending of the mesentery is seen in the 4.9 mm. Begg embryo. The mesogastrium deviates toward the left, and the smaU remnant of the left pneumato-enteric recess has been displaced forward so that it does not appear in the model. The left vitelline vein enters the mesentery a little lower than the right. The mesentery is slightly drawn out toward the yolk-stalk, with its mesenteric arteries still symmetrically placed on either side. There is possibly a slight suggestion of intestinal rotation in the normal direction.

In a 7.5 nmi. specimen, the mesogastrium bulges conspicuously to the left; the left vitelline vein courses upward into the right, making a straight channel, without, however, deflecting the duodenum to any appreciable extent. The prolongation of the mesentery toward the yolk-stalk has increased, and the vitelline artery on its left side has disappeared. The intestinal loop shows a sUght but distinct indication of sagging downward on the right side of the right vitelline artery, and intestinal rotation has therefore begun.

In a 9.4 nmi. embryo, remarkable for the precocious disappearance of that part of the vitelline veins which leaves the mesentery and crosses the abdominal cavity, the intestinal rotation is well established. The stomach swings boldly to the left and it appears that the intestine is carried to the right by a compensatory bend of the elongating epithelial tube. We are inclined at present to regard the gastric and intestinal bends together as forming an 'S,' comparable with those secondary S-shaped bends which soon develop in the intestinal mesentery itself. Accordingly we ascribe the rotation of the intestinal loop to the growth of its epithelial portion, and not to the influence of adjacent blood vessels.

PROCEEDINGS 221

Although the loop of intestine appears to drop over the taut mesenteric artery, as if the position of that vessel on the right of the intestine were an essential factor in rotation, this possibility has been eliminated. For in one of the embryos modelled, there is a normal disposition of the mesentery, although the artery anomalously persists on the left side. Professor Frazer and Dr. Robbins, in the last issue of the Journal of Anatomy and Physiology (October, 1915) suggest that the persistence of the left umbilical vein and its shifting to the median line are the cause of the intestinal rotation. This is a very ingenious suggestion, illustrated by an interesting schematic figure. If it is correct the rare cases of reversed torsion should show an umbilical fissure on the right side of the gall bladder. In Strehl's case of reversed torsion, unfortunately nothing is said of the liver, but such an abnormality, had it existed, could hardly have escaped attention. We are inclined, therefore, to reject the tempting suggestion, but propose to give it more careful consideration in continuing this work.

See 64 P. E. Linbback, page 262.

S6, Anomaly in the circle of Willis, due to absence of the right intenml

carotid artery, Lawson G. Lowrey, Harvard Medical School. No. 59

Danvers State Hospital. Hospital Papers.

Anomalies in the formation of the circle of willis are, in my experience, relatively conunon, but are usually found in the posterior communicating arteries or in the origin of the posterior cerebral arteries. The case here reported is the only one known to me in which the internal carotid artery was missing. The possibility of this anomaly is not mentioned in any of the standard text-books of anatomy, and a hasty search through the available literature has revealed no other reported case.

The condition was found in a woman 75 years of age autopsied by me at this hospital. No other vascular anomalies were found. The right carotid tnmk arose as usual from the innominate, and measured 4 nmi. in diameter. The left arose from the aortic arch, and measured 7 mm. in diameter. Both vessels were traced to the level of the palate and no anomaly of distribution was noted beyond the absence of any trace of the internal carotid branch of the right trunk. No bony canal was found in the petrous portion of the right temporal bone. The left vertebral artery was of unusual size.

The basilar artery divided into two branches of unequal size. The larger, right branch passed forward in the usual position of the posterior communicating artery and turned laterally into the Sylvian fossa, forming the middle cerebral artery. From the upper surface., the posterior cerebral artery passed backward. Both anterior cerebral arteries arose from the left internal carotid which was otherwise normal in its distribution. The right anterior cerebral, arising from the left internal carotid artery, was joined by a very fine twig from the right middle cerebral artery. The right ophthalmic artery arose from the anomalous middle cerebral.

222 AMERICAN ASSOCIATION OF ANATOMISTS

AbseDce of the internal carotid seems chiefly interesting because of the great rarity of the anomaly and because of the functionally eflicient rearrangement of vessels. It means, of course, either complete atrophy or agenesia of the third aortic arch and all of the distal portion of the primitive dorsal aorta on the right side.

37. Experimental confirmation of ike view that lymphatic endothelium

arises in loco from intraembryonic mesenchymal cells and that it is

not derived from the endothelium of the veins. Charles F. W.

McClure. Princeton University.

Does the endothelium of the lymphatic system arise, at any time or place, in a discontinuous manner and independently of that of the veins? As we shall see the determination of this question constitutes a solution of the l3rmphatic problem."

The above question forms the opening sentence of a recent paper by the writer,* a question which he is now able to answer in an affirmative manner on evidence obtained by experimental means.

The investigations of Loeb,* and later those of Stockard,' have shown that teleost embryos when subjected to the influence of certain chemical agents often develop without a circulation. Stockard has shown in particular that the development of the blood-vascular syBtem of these embryos may be arrested in such a manner that the arteries and veins are represented by a series of independent and discontinuous anlagen and that the endothelium of these vascular anlagen is formed in loco from mesenchymal cells. These experiments of Stockard, together with those of Hahn,^ Miller and McWhorter,* Reagan,^ Werber J and Reagan and Thorington,* substantiate completely the observations of Rtickert and Mollier,* Felix,*® Schulte" and others regarding the local origin of intraembryonic haemal endothelium from mesenchymal cells. The 'ingrowth' or 'angioblast' theory of His, hitherto so vigorously exploited by a certain group of American anatomists is now, therefore, merely of historic interest.

It has occurred to the writer if the development of the blood-vascular system can be arrested by experimental means at an early on

Anat. Rec, vol. 9, no. 7, 1915. « Plttger's Archiv, Bd. 64, 1893, and Pop. Sci. M<)nthly, vol. 80, 1912. ' Proc. Amer. Ass. Anat., Anat. Rec, vol. 9, no. 1, 1915 and Amer. Jour. Anat., vol. 18, no. 2, 1915.

Archiv Entwickmech., Bd. 27, 1909. • Anat. Rec, vol. 8, 1914.

Anat. Rec, vol. 9, no. 4, 1915 and Anat. Rec, vol. 10, no. 8, 1915. ' Anat. Rec, vol. 9, no. 7, 1915.

• Anat. Rec, vol. 10, no. 8, 1915.

• Hertwig's Entwickelungslehre d. Wirbeltiere, Bd. 1, erste Teil, aweite Mlfte, 1906.

" Anat. Hefte, Bd. 8, 1897.

^^ Memoirs of the Wistar Institute of Anatomy and Biology, na 3, 1914.

PROCEEDINGS 223

togenetic stage, that the lymphatic system might be influenced in a similar manner. If one could find an undoubted instance of a chemically treated embryo in which a circulation has not been established and in which the arteries and veins, as well as the lymphatics, were foiuid to consist of a series of discontinuous and independent anlagen, it would seem to be positive proof that the endothelium of the lymphatics, like that of the arteries and veins, has been formed in loco from mesenchyme and that it had not been derived from the endothelium of the veins. As far as the subocular lymph sacs of a teleost embryo are concerned, the writer now has such experimental evidence at hand.

In the spring of 1915 the writer subjected the early cleavage stages of the Chub Sucker (Erimyzon sucetta oblongus, Mitchill) to the influence of weak solutions of KCN" and in many cases the embryos developed without a circulation. As described by Stockard for Fundulus, these embryos developed a string-like heart, in some cases solid and without a lumen, and possessed a greatly expanded pericardial chamber filled with fluid. Free erythrocytes could often be seen floating in the pericardial chamber and in some cases the pressure of the fluid became so great that the walls of the pericardial chamber burst. Reconstructions of a number of these oedematous embryos with solid string-like hearts were made, after the method of Bom, of all the definite endothelial-lined vascular anlagen that could be observed under a high magnification, and I can verify the recent observation of Stockard" that the aorta and cardinal veins of the teleost embryo, as I have found to be the case for the main lymphatics," do not develop from a plexus. Most of the reconstructions made by the writer were confined to the region contiguous to and anterior to the cardino-Cuvierian junction. In one of the embryos reconstructed (Princeton Embryological Collection, 1020) a continuous sjrstem of vascular lumina for the aorta and cardinal veins had not yet been established in the region anterior to the cardino-Cuvierian junction nor, as a matter of fact, in the more caudally situated portion of the body. In the region anterior to the cardino-Cuvierian junction, the lumina of the aorta and its branches and those of the precardinal veins were represented by a series of independent and discontinuous endothelial-lined vascular spaces, the precardinal veins being represented by eleven and the aorta and its branches by eight of these vascular spaces. Near the caudal end of each eye and just anterior to the independent anlage of the hyoidean artery ^ which wa^ present on both sides of the embryOy an entirely independent endotheliallined subocular lymph sac was found to he present. The relatively large size, and the location of this sac near the caudal end of the eye and just

" 1 cc. of a 1 per cent solution of KCN in 450 cc. of water. The embryo especially referred to in this paper was treated with KCN on April 26 for 18 hours and killed on May 13.

" Amer. Jour. Anat., vol. 18, no. 3, 1915.

" Memoirs of the Wistar Institute of Anatomy and Biology, no. 4, 1915.

224 AMERICAN ASSOCIATION OF ANATOMISTS

anterior to the hyoidean artery, leaves no doubt regarding its character. The most anterior of the independent and discontinuous anlagen of the precardinal veins which were present in this embryo were found opposite the cranial end of the otocyst, a point which Ues a considerable distance from the anlagen of the subocular lymph sacs. As.no veins or endothelial-lined independent anlagen of the veins are found in the vicinity of these sacs, their endothelium, like that of the independent anlagen of the arteries and veins, must have been formed in loco from mesenchyme.

The study of a number of reconstructions of chemically treated embryos leads me to believe that the aorta is the first of the main bloodvascular channels to establish a continuous lumen and that the continuous lumina of the veins is first estabUshed near the caudal end of the otocyst and in the vicinity of the cardino-Cuvierian junction.

Those investigators who have followed the development of vascular channels solely by the aid of injections seem to have overlooked the fact that they are not studying the genesis of the endothelium, but rather the topographical arrangement of the vessels after a circulation has been established. Otherwise it would not be possible to inject them. After the main arterial and venous channels have been formed and capillary plexuses established, the picture naturally becomes more complicated. It is from this stage of development, however, that the injectionist necessarily draws his conclusions.

It has already been pointed out by the writer^^ that the failure of the independent anlagen (lymph vesicles) of the main lymphatic channels to undergo concrescence and form continuous vessels which connect with the veins, would result in an oedematous condition of the embryonic body. It is evident that the oedematous condition presented by these chemically treated teleost embryos must be due to some analogous cause. During the early stages of ontogeny and before a haemal circulation has been formed, there must necessarily be a gradual increase in the amount of fluid which collects in the tissue spaces of the embryo. In the course of normal development the process of concrescence between the independent anlagen of the haemal vessels adjusts itself to the demands of fluid production so that a circulation is established and an oedematous condition of the body does not occur. When, however, as shown by experiment, the normal process of concrescence between the independent and discontinuous anlagen of the main haemal vessels has been experimentally delayed, without a corresponding arrest of fluid production, a circulation is not established, and an oedematous condition of the body naturally prevails.

»* Anat. Rec, vol. 9, no. 4. 1915

PROCEEDINGS 225

38. Bimideate and mvUinudeate cells in tissiLe cultures. C. C. Macklin, Department of Anatomy, Johns Hopkins Medical School.

The problem of the origin and fate of the binucleate and multinucleate cells found in cultures from embryonic chick tissue, growing in vitro in modified saline solution, was investigated by the method of continuous observation of the living cell, supplemented by a study of these cells in fixed preparations of tissue cultures.

It has been concluded from this research that such nuclei arise by direct division. Nuclear amitosis consists apparently in a constriction of the elongated nucleus, rather than in the formation within the nucleus of an equatorial plate, which subsequently splits. In this process of nuclear division it is believed that the centrosphere and mitochondria may play a part. It is fmther beUeved that multinucleate cells arise by a repetition of this process.

The fate of the binucleate cell has been similarly investigated, and in no case has it been found that the parts became separated to form the nuclei of mononucleate cells. On the contrary the only change which took place in such cells occurred when they were imdergoing mitosis. In such a case the changes in the binucleate cell were essentially the same as those characteristic of mitosis in the mononucleate cell. There was a disappearance of the nuclear membrane with a fusion of the karyoplasm of the two nucelar moieties and the formation of a single amphiaster. In this way there finally resulted two apparently normal daughter nuclei, contained within separate cells.

A double-nucleated cell was followed for almost twelve hoiu^ continuously, and the entire process of mitosis, with fusion of the two nuclear parts, was seen to occur in it, finally culminating in two new separate cells. Fusing nuclei imdergoing mitosis were foimd in fixed preparations.

Nuclei in tissue cultures have been foxmd in which the spireme has formed while the nucleus was bent upon itself, apparently undergoing amitotic division. This superposition of mitosis upon amitosis, combined with the failure of the latter process ever to result in the formation of separate new cells, would seem to indicate that amitosis, in the material examined, is a change in form of the nucleus rather than a method of cell proliferation.

39. A plea for the more critical study of the intimate structure of the cerebral cortex as furnishing the probable anatomical basis of mental development. E. LiNDON Mellus, Brookline, Mass.

In connection with the paper charts will be shown of various areas of the cortex x ca. 1200.

It has been objected that to go over the whole cortex in this way would require so long a time as to make it doubtful of accomplishment, but it is contended that such charts give so good an idea of the intimate structure as to justify the work. Other surveys discussed and compared.

The lamination of the various areas is discussed, and the reason

226 AMERICAN ASSOCIATION OF ANATOMISTS

given for adopting Meynert's scheme of five layers as the division most likely to be ultimately accepted.

The suggestion is made that as a cortical cell develops it becomes invested with more and more protoplasm, that many of the cortical cells are still largely imdeveloped, and are perhaps incapable of function. But little is really known today of the function c)f the innumerable cells in the various layers. The giant cells of the so-called motor area are the only cells of which anything definite can be asserted. Various areas are known as the areas of special sense, but the facts jrielded by pathology and animal experimentation are misty, and often openly contradicted by good authorities.

Ifi, A stvdy of the hypophysis of the pig} M. M. Miller. Department of Anatomy, Vanderbilt University Medical School. This paper is limited to the embryology (origin and development), the morphological significance, and the histological structure of the hypophysis cerebri of the pig.

Material and Methods, The major part of the material used in this study may be foimd in the Harvard Embryological Collection. It consists of pig embryos, ranging in size from 3.1 mm. to 45 mm. The hypophyses of new-born and full grown pigs were also used.

Wax reconstructions were made of many of the younger specimens. High power studies of sections stained with Biondi's stain and other granular stains furnished the histological observations.

Embryology. In the younger specimens the oral plate was intact and the anterior end of the notochord was attached to the phar5mgeal wall. The hypophyseal angle was present. As the development proceeds, Rathke's pouch appears as an evagination from the pre-oral epithelium (ectoderm), the notochord pulls away from the phar3rnx carrying with it a mass of cells (entoderm) which at first lies in relation to the apex of Rathke's pouch. Later these cells enter into the structure of the pharyngeal wall of the pouch, gradually shifting their position from the apex of the pouch to the constricted neck (stalk) of the hypophyseal sac. This change in position is brought about by the relatively more rapid growth of the wall of this pouch nearest the brain tube. The notochord retains its connection to the pharyngeal wall of Rathke's pouch imtil after the 15 nmi. stage. The infundibulum first appears in the 10 mm. stage. After the hypophyseal sac becomes separated from the pharynx, it imdergoes a rotation anteriorly and superiorly which is brought about by the growth of the infundibulum and surrounding structures. Consequently the mass of cells which was in relation to the notochord and hypophyseal stalk is carried forwards and upwards. The lateral cords grow aroimd and encapsulate this mass of cells imtil in the new-born it is situated within the glandular portion, anterior to the ventricle, and forms the medulla of the anterior lobe.

Accepted by the Harvard Medical School is a thesis that partly fulfilled the requirements for the degree of Doctor of Philosophy.

PROCEEDINGS 227

These cells are considered to be of entodermal origin (1) on account of their close relation to the notochord, (2) the difference in the arrangement of these cells from those of the cortex of the anterior lobe, which developed from the lateral cords, (3) and the microscopical structure of the medullary part of the anterior lobe of the functioning hypophysis.

Morphology. The observations on the developing hypjophysis of the pig tend to support Kupffer's idea that the relationship between the mouth, intestine, and brain (infundibulum) is significant of an ancestral communication between the neiu'al tube and intestine. The perforation in the superior free stump of the oral plate, observed in the younger embryos studied, is indicative of a commimication between the intestine and Rathke's pouch. This is considered as the remains of the old mouth (Palaestoma) as described by Kiipffer in his schematic ancestral vertebrate. The small mass of entodermal cells which are attached to the anterior end of the notochord is comparable to the subneural gland as described in Ascidians.

Histology. If the hypophysis is considered as having a three fold origin, the histological structure becomes relatively simple.

The glandular portion (anterior and intermediate lobes) is composed of two distinctly different types of cells, those which have a marked aflSnity for acid stains and stain deeply (chromophiles), and those which stain less intensely and are decolorized by the ordinary staining methods (chromophobes).

The chromophiles form the greater part of the medulla of the anterior lobe. They vary greatly in appearance, depending upon the stage of the functional activity. These cells are arranged in rather definite acini.

The chromophobes predominate in the cortical portion of 4^he anterior lobe and the intermediate lobe. These two lobes are continuous with each other around the ventricle. These cells do not exhibit the regular arrangement found in the medullary part of the anterior lobe. The spindle-shaped supporting cells and colloidal vesicles are other interesting structures in the intermediate lobe.

The posterior lobe (infundibulum) consists mainly of tissue of a fibroglia character and a small amount of white fibrous connective tissue.

Condnsions. Sufficient evidence is presented, in the above investigation of the hypophysis cerebri of the pig, to warrant the following conclusions:

1. That the hypophysis in the pig develops in a different way from that heretofore described in any of the manmialian types.

2. That the entoderm, as well as the ectoderm, enters into this structure. This is in accordance with the development of the hypophysis in birds and lower forms.

3. That this ectoderm comes from two sources, the brain wall and the oral cavity; and that the entoderm is derived from the fore-gut.

4. That the notochord takes an important part in the development of the hypophysis. First, it plays a mechanical r61e in pulling out the diverticulum of the entoderm over the hypophyseal angle; and secondly,

228 AMERICAN ASSOCIATION OF ANATOMISTS

its anterior extremity is in intimate relation with the entoderm from the fore-gut which enters into the structm-e of the hypophysis.

5. That the anterior lobe contains two distinct portions, a medullary part which is of entodermal origin, and a cortical layer which is developed along with the intermediate lobe from the ectoderm which formed Rathke's pouch.

6. That the results here obtained strengthen the theory that the glandular portion of the hypophysis is phylogenetically homologous with the sub-cerebral gland of Ascidians.

7. That the explanation of the development of the hypophysis here presented explains the presence of two distinct types of cells found in the anterior lobe of the hypophysis'.

41- Artificial parthenogenesis in Cumingia. Margaret Morris, Os born Zoological Laboratory, Yale University. (Introduced by

R. G. Harrison.)

The eggs of the mollusc Cumingia telUonoides can be made to develop parthenogenetically by the use of heat followed by hypertonic sea-water. Some of the eggs so treated form polar bodies in a normal manner, but others pass at once to a two-cell stage without imdergoing maturation. Experiments were made which show that the eggs in which maturation has been suppressed may develop to fairly normal cellular larvae, while those which have formed polar bodies do not do so.

It is seen from a study of preserved material that in some eggs the chromosomes of the first maturation spindle divide normally giving rise to two daughter nuclei which are both retained in the egg, which has, therefore, the full amount of chromatin. The cytological study of the later stages of these eggs has not yet been completed, but it seems certain that they give rise to the swimming larvae.

42, Histological changes in testes following vasectomy. Burton D.

Myers, Indiana, University School of Medicine.*

Sterilization is suggested as one of the methods to be used in checking increase in the number of defectives. Indiana has a law providing for steriUzation under certain circumstances, and other states have attempted such legislation. Therefore, it becomes desirable to know what changes occur in the testis after cutting or hgating the ductus deferens.

Though I have been in touch with the proper ofiicials in Indiana for several years with the hope of being able to report on changes in testes of some of the six himdred individuals on whom a vasectomy was performed, no opportunity has as yet arisen for such an examination.

My report is, therefore, confined to the histological changes occurring in the testes of a series of white rats following ligation of the ductus deferens on each side.^ The operated rats were permitted to live ninety

About two years ago I expressed to Dr. Henry H. Donaldson my intention to do the work here reported. He offered to furnish material as he had a series of white rats with double ligation of vas, for another problem. I desire to express my appreciation of this assistance.

PROCEEDINGS . 229

to one hundred and twenty days after the operation, an equivalent of seven and one-half to ten years for man. The testes were fixed in Zenker's and Flenaming's fluid. Several stains were used, the best result being secured with iron hematoxylin.

Before reporting the results of this operation permit me to recall the fact that in addition to the seminiferous tubules with their spermatogenetic cells, the products of which are eliminated through the ductus deferens, the testicle has also an internal secretion. The cells responsible for this internal secretion, interstitial cells,' as the name implies, lie in the intervals between the seminiferous tubules. Though these cells were first described in 1850 it was not imtil 1897 that a discussion of their function was imdertaken, a discussion which later investigation failed to verify. The experimental work of Steinach 1910, 1912, and 1913, established almost beyond question the fimction of these interstitial cells as controlling sex impulse and secondary sex characteristics.

On sectioning the series of testes of the operated rats, I find that the testes of those animals which had been permitted to Uve ninety days after operation show a high degree of disintegration of spermatogenetic cells. The interstitial cells on the contrary are entirely unaffected. In the animals in which 120 days had elapsed between operation and date when killed, most seminiferous tubules are entirely free, as mere connected tissue capsules containing a slight amount of debris. In some seminiferous tubules a few spermatogonia can still be discerned at the periphery, beneath the capsule, all other spermatogenetic elements have imdergone complete degeneration and absorption. The interstitial cells on the contrary show no degenerative changes. Their number seems to be increased. This is not, however, the case, the appearance of an increase being due in part to the fact that they, instead of the seminiferous tubules, are now the conspicuous elements of each microscopic field; and in part to the fact that they are crowded somewhat more closely together than normal as a result of a slight shrinkage following the disintegration of the seminiferous tubules.

Therefore, as a result of vasectomy, or was ligation, we are able to say with much certainty that the steriUzation of the individual is accomplished without in any way disturbing the internal secretion of the testicle. The psychic life and secondary sex characteristics are therefore entirely imaffected. There is nothing about the operation that will make the individual in any way a freak. He will not acquire a falsetto voice, nor will he lose any secondary sex characteristic. These conclusions are verified by observation of men upon whom this operation has been performed.

The result indeed is exactly comparable to that which follows a high urethritis with occlusion of the lumen of the ductus deferens. Therefore, why should not the state provide deliberately for a condition many acquire as the result of disease?


THE ANATOMICAL RECORD, VOL. 10, NO. 3

230 , AMERICAN ASSOCIATION OF ANATOMISTS

4S, Growth and distribiUum of the milk-ducts and development of the nipple in the albino rat from birth to ten weeks of age. (Lantern slides.) J. S. Myers, Institute of Anatomy, University of Minnesota, Minneapolis. (Presented by C. M. Jackson.)

Observations made on 100 rats show the number of glands varies between 10 and 13, the normal number being 12 (6 pairs). Only one supernumerary gland was observed. The second thoracic glands are those most often absent.

Only one primary duct is present in each gland. This duct after reaching the tela subcutanea turns at right angles and pursues a course parallel to the surface of the skin. The majority of the branches of ducts belonging to the first thoracic gland lie cephalad to the nipple, while those from the second and third thoracic glands he latero-cephalad to the nipple. In case of the abdominal and first inguinal glands the greater number of ducts he latero-caudad to the nipples, while in the last inguinal the ducts he caudad to the nipples. The dichotomous method of branching frequently occurs, especially in tha proximal branches.

Reconstructions and cleared preparations show that anastomoses sometimes occur between the ducts of a single gland. It is uncertain whether anastomoses occur between the ducts of different glands.

End-buds are present on a large number of terminal ducts at all stages studied. No true alveoli were observed. Large numbers of lateral buds are present on the sides of all ducts distal to the primary duct during the earUer stages. Such lateral buds later develop into branches of the ducts.

Considerable individual variation in the development of the glands was noticed. Not only do the corresponding glands of opposite sides differ in their degree of development, but also the glands of one individual may be better developed than those of another even seyeral days older.

The characteristic distribution and ramification of the ducts apparently depend upon the space available for their growth.

The growth and branching of the ducts goes on at an unusually rapid rate about the ninth week, probably corresponding to the age of puberty.'

A distinct lumen is present at birth in all ducts distal to the intraepidermal portion of the primary duct. At the end of the second week the lumen extends to the surface of the nipple.

The periphery of the nipple area is marked by a hood-like epithelial ingrowth. The nipples make the most rapid growth during the second week, toward the end of which time they are very conspicuous.

44' Neuromeres and metameres. H. V. Neal, Tufts College.*

The neuromeres of vertebrate embryos have been regarded as important evidence of the primitive metamerism of the body and as decisive criteria of the number of metameres in the head region. The hindbrain neuromeres or ^rhombomeres* appear to be metameric struc PROC^JEDINGS 231

tures, since they correspond numerically with the mesodermic somites, since they seem directly connected with the metameric visceral arches, and since as local thickenings of the medulla they are inexplicable as results of mechanical bending.

Graeper ('13), however, by the use of improved neurological methods upK>n mammaUan embryos finds the relations of the rhombomeres to the visceral arches not so clearly metameric as has been assmned. Using the same methods upon Squalus embryos the writer has been able to obtain results essentially similar to those of Graeper. These are summarized in the present paper.

In embryos of Squalus the motor nidulus of the Trigeminus lies in the second and third rhombomere; that of the facialis nerve extends through four neuromeres, viz., the foiuiih, fifth, sixth and seventh. The nidulus of the glossopharyngeus lies in the sixth and seventh rhombomeres, and that of the vagus extends from the seventh into the unsegmented portion of the medulla posterior to the seventh.

Of the somatic motor nerves, the oculomotorius has its nidulus in the midbrain. That of the trochlearis Ues primarily wholly in the first (cerebellar) rhombomere. In later stages, however, some of the trochlearis fibers come from the midbrain and the niduli of the oculomotorius and trochlearis overlap each other. While, according to Graeper, the nidulus of the abducens nerve in mammal embryos lies in the fifth rhombomere, in Squalus it extends through the sixth, and the nerve receives some fibers from neuroblasts in the fifth and seventh rhombomeres. The hypoglossus has an extended nidulus in the unsegmented region posterior to the seventh rhombomere.

Primarily the somatic motor niduli lie dorsal and lateral to the splanchnic motor niduli just as in mammal embryos (Graeper). As a consequence of this relation the fibers of splanchnic motor and somatic motor nerves cross within the wall of the medulla. These relations are seen with special clearness in the region of the roots of the vagus and of the hypoglossus nerves. The distinction commonly made between lateral and median niduU, therefore, is based upon secondary and not upon primary relationships.

It is difficult to reconcile the present relations of the rhombomeres to motor nerves with any scheme of primitive metamerism. The writer ('14) has attempted in an earUer paper to interpret the present relations of the somatic motor nerves to the neuromeres upon the assumption that their relations were primarily metameric. But such an interpretation is more difficult in the case of the splanchnic motor nerves. Assuming, however, that a single rhombomere was originally connected with a single visceral arch by a splanchnic motor nerve, it is possible to explain the present relations of the trigeminal nerve to two rhombomeres by assuming the disappearance of a gill-cleft anterior to the spiracular cleft. This assumption has often been made and there is good reason to believe that the lost pair of gill-pouches is represented today by the club-shaped gland of Amphioxus.

A more complex problem is presented by the relations of rhombo 232 AMERICAN ASSOCIATIC^ OF ANATOMISTS

meres four to seven. All four of them send fibers into the hyoid arch, while at the same time one of them — the seventh — sends fibers into three visceral arches, viz., the second, third, and fourth. The diflSculty of solving the problem of these confused metameric relations is lessened of course by our knowledge that nerves become connected with their terminal organs by a process of free outgrowth, consequently permitting the possibility of changed metameric relations imder changing conditions. The growth and enlargement of the ear might affect nerve relations in the region of these four neuromeres, but such a change will not explain the growth of post-otic fibers into the pre-otic region. In Amphioxus the nerves of the four metameres homologous with those to which rhombomeres four to seven belong form a plexus on the velum. Consequently there is reason to believe that in the ancestors of Craniotes metameric relations in this region of the body were modified even before the ear made its appearance.

However, the fact remains that the nerve relations of the rhombomeres — the neuromeres considered of the least doubtful metameric value — are difficult to reconcile with the assumption of a primitive metamerism. Consequently, as criteria of the primitive metamerism of the vertebrate head they do not appear as reliable as the mesodermic somites. Indeed, in the light of the facts presented in this paper it would seem that the strongest proof of the metameric value of neuromeres consists, as maintained by the writer ('96, '98), in their numerical correspondence with the mesodermic somites.

1^6, Some phases of cell mechanics. Theophilus S. Painter, Osbom Zoological Laboratory, Yale University. (Introduced by R. G. Harrison.)

In a series of studies an attempt is being made to analyze some of the forces at play within the cell, along the general lines begun by Boveri and by WSson. The material used has been the sea urchin egg and the method of approach has been: first, to produce monasters in the eggs and then to follow, in the living and in the sectioned egg, the changes through which the single aster goes. .Second, to treat the eggs with narcotics such as will inhibit the cytoplasmic radiations and then to follow the centrosomes, chromosomes, etc., in their behavior. Several points of general interest will be touched upon here.

1. Under certain conditions, "spiral asters" appear in the monaster eggs. The conclusion reached after a detailed study of eggs of this type is, that the bending of the aster fibers is brought about by movements in the cytoplasm lying outside of the centrosphere. This interpretation gives a satisfactory explanation for the spiral asters observed by various workers, in Nemerteans, Annelids, Molluscs, and in Vertebrates. A second conclusion, drawn from the study of spiral asters is, that the shifting of the spindle observed in eggs, especially during polar body formation, is due to movements in the cytoplasm.

2. Observations on the living monaster eggs has shown that when the aster retreats to one side of the egg (as in normal divisions) there

PROCEEDINGS 233

is a flow of the superficial protoplasm towards the opposite side of the egg. Thus, in Arbacia, nearly all of the pigment is swept to one pole, the side opposite the aster. Biitschli attributed great importance to this movement in cell division.

3. When eggs are treated with chemicals which suppress the cytoplasmic radiations, a spindle is formed within the boundary of the nucleus, the chromosomes divide and the centrosomes separate. Cytoplasmic division, however, does not follow. Thus it is clear that the cy1;oplasmic radiations do not pull the centrosomes apart, as has been thought by many workers. They are probably concerned with cytoplasmic cleavage.

Ji6. The use of a graph in teaching embryology. A. G. Pohlman, St.

Louis University.

It may be that this method of graphing results is by no means a new thing in the teaching of embryology. The general idea is an up-anddown method of noting the data obtained through the study of serial section. The student is supplied with two pig series of 12 and 16 mm. The first cut in paraffin at 25 and the second cut cleared (Gage-Fish method) in celloidin cut at 50. The student dots every fourth section in the firat series and every other one in the second; giving 100 micron intervals. Next the ordinary cross section paper is taken and a line drawn across the page to represent the first dot in the first slide; allow one space for each dot on the slide and then rule through the next line to show dot one on slide 2, etc. These lines are two mm. apart and therefore the relative positions of organs and parts may be studied x 20. The following structures are represented by up-and-down lines indicating their position in the series: the extent of brain; spinal cord; spinal ganglia; the olfactory area; the eye and lacrimal duct; the hypophysis; the ganglia of the Vth.; Vllth, Vlllth, IXth and Xth nerves; the extent of the membranous labyrinth; the chorda; the esophagus; the stomach; the foldings of the small and large intestine and the anal region; the umbilical and sup. mesenteric arteries; the dorsal aorta and branchial arches on the left; the aortic-pulmonary and auriculo-ventricular valves; the foramen ovale and the pulmonary artery; the trachea and bronchi; the extent of the two lungs; the thymus and th3n'eoid glands; the heart; liver; bile duct including gall; the pancreas; spleen; the extent of the Wolffian body and duct; the kidney and ureter; the bladder and allantois; the sex glands; adrenal.

I have found this method of indicating that the various parts are located in certain sections of a series and that they extend through a given nmnber of sections is easily grasped by the average student and more easily checked by the instructor. When the graph of a given part has been O. K'ed it may readily be transferred to a final graph. I show herewith the graphs of an 8, a 12 and a 16 mm. pig. The great disadvantage in the method is that the usual way of giving the embryology course should be reversed and that the text book must be used almost entirely as a collateral reading. What the student learns is a general developmental picture rather than a series of isolated facts.

234 AMERICAN ASSOCIATION OF ANATOMISTS

It is perfectly possible to do a rough profile reconstruction by projecting selected sections x 20 on the graph paper but this is neither desirable nor expedient.

47. An experimental study of bone growth in the dog, J. L. Pottorf, Anatomical Department of the University of Missouri. (Introduced by E. R. Clark.)*

The object of this investigation was to analyze the extent to which bone growth is regulated by the mechanical factors of stress and strain. The results of the two experiments which have been carried out thus far appear to be sufficiently definite to be worth reporting.

The first experiment consisted in the binding up of the left leg of a puppy about three weeks old. The weight of the fore-part of the body was thereby thrown on the right fore-leg and the left leg was freed of all stress and strain except that resulting from the muscle pull. The animal was allowed to Uve twenty days. Marked differences were found in the two humeri; particularly in the thickness of the compacta, and the size of the epiphyses. The bones were split longitudinally in approximately the same plane. The thickness of the anterior side of the humerus from the imbound leg is, on the average, about 50 per cent greater than that of the bound leg. The epiphyseal ossifications of the imbound leg are noticeably larger than those of the bound leg. The two humeri are of about the same length. A second experiment was carried out as follows: Two ten day old puppies from the same Utter were used. One was killed at the beginning of the experiment, and served as a control. On the second, an operation was performed, with, as nearly aseptic technic as possible, in which all the nerves of the right brachial plexus were cut, in order to eliminate all muscle pull from the fore leg, and the leg was put up in a loose sling. The animal was killed after 20 days. During this time the weight increased from 3.75 poimds to 8.5 pounds. Some of the more striking results are as follows: The increase in length of the entire humerus was nearly as great on the operated as on the imoperated side. Thus, while the length of the humerus in the control animal was 5.08 cm., the length of the humerus on the operated and imoperated sides were 6.2 and 6.3 cm. respectively.

In thickness, however, there was a marked difference in the two humeri. The bones were split longitudinally in as nearly the same plane as possible, and measurements made of the thickness of the compacta at corresponding points of both the anterior and posterior sides of the shaft. In the humerus from the operated side, the thickness of both the anterior and the posterior sides was 0.5 mm. ; in the humerus from the imoperated leg, the thickness of the anterior side measured 2.0 mm., of the posterior side, 0.83 mm. Thus the thickness of the bone on the imoperated side exceeded that on the operated side by from 66 to 300 per cent. (It is interesting to note that, in the humerus from the control animal, the thickness of the shaft is greater even

PROCEEDINGS 235

than that of the humerus from the mioperated leg. There is evidently, then, a reduction, normally, in the thickness of the humerus during this period of growth.)

In addition to the greater thickness of the shaft attained by the humerus from the imoperated leg, this bone is larger in several particulars; its has a larger diameter: 0.8 mm. as compared with 0.7 mm. (antero-post. diameter at about the middle of the shaft) ; the epiphyseal cartilages are wider, and the epiphjrseal ossification centers are slightly larger. It is of interest, however, that the epiphyseal ossification in the head of the humerus from the operated leg is much larger than that of the control hmnerus (11 x 5 mm., as compared with 8x3 mm. in longitudinal section), and that, whereas in the longitudinal section of the control no ossification center is visible in the lower end of the bone, there is present, in the lower end of the humerus from the operated leg, an epiphyseal ossification measuring, in section, 0.46 x 0.26 mm.

There are also distinct differences in the shape of the two bones, both of the ossified part and of the cartilaginous part.

These experiments, particularly the second, indicate that a bone which has been relieved of the stress and strain due to the force exerted by the weight of the body and by the action of the muscles, will increase in length at practically the same rate as a bone subjected to the action of these forces; that epiphyseal ossification centers will increase in size, but that in the thickness of the compacta, such a bone will lag far behind another subject to such forces. It is planned to continue the experiments to an analysis of bone ^owth in older puppies, and over longer periods of time.

48, Experimental studies on the origin of irUraemhryonic endothelium and of blood cells. Franklin Pearce Reagan, Princeton University.

(a) Cytological and experimental studies on the origin of intraembryonic endothelium — a study of mechanically produced meroplasts. Franklin P. Reagan.

(b) The origin of endothelium and of erythrocjrtes in hybrid teleosts. F. P. Reagan and James M. Thorington.

(c) The origin of erythrocytes in chemically treated, and in cardiectomized teleost embryos, and in embryos developed at low temperatures. F. p. Reagan.

(a, b, and c). A series of experimental observations lending support to the following propositions:

1. That the embryonic body is capable of furnishing its own endothelium, which develops in loco, in and from the mesenchyme.*

2. That the embryonic body is capable of developing blood-cells which also develop locally.*

3. That pre-endothelial mesoderm (mesenchyme) may be either of direct entodermal origin or from mesenchyme proliferated from visceral or parietal mesoderm, or from coelomic mesothelium.*

236 AMERICAN ASSOCIATION OF ANATOMISTS

4. That endothelium may be obtained in meroplasts in which its origin could not have been from coelomic mesothelium as such.

5. That mesenchjrme cells in any region where they are foimd in bodies of teleost embryos which have developed without circulation may produce erythrocytes whether the circulation has been prevented by certain methods of chemical treatment, by hybridization, by refrigeration, by removal of the heart, or by mechanical or chemical destroyal of the heart anlage prior to heart pulsation; that the anteriorly located erythrocytes of such embryos may or may not be surrounded by endothelium; that the anterior mesenchyme may be (and perhaps generally is) haematopoetic.f

6. That endotheUum, as seen in the tail of the Uving teleost embryo, is formed partly by the active migration of single mesenchyme cells; that such endothelium once formed may grow by its own proliferation.* (This observation has been made by a number of previous observers.)

7. That erythrocytes may develop locally from* the mesenchyme in any part of the yolk-sac of teleost embryos lacking circulation and that their origm on the yolk-sac is not restricted to the posterior and ventral yolk-surface, t

8. That the hearts of teleost embryos which have always been devoid of circulation may contain erythrocytes.!*

9. That cardiac endothelium may transform into erythrocytes.!*

10. That myocardial anlagen may transform into eryiJirocytes.f*

11. That mesothehum may transform into erythrocytes.!* (Statements followed by an asterisk are at variance with the Angio blast theory of His. Those followed by a cross are opposed to the principal results of Stockard on which he has based his recent support of the so-called Polyphyletic Theory. More complete but preliminary accounts of these observations can be found in the Anatomical Record, vol. 9, no. 4, and in the Anatomical Record, December 1915).

Blood cells and endotheUum can be traced back to a very widely distributed mesenchyme, the cells of which are so much alike that it is at present beyond human power to detect differences among them or to determine further their individual lineages. To claim that a cell of this apparently indifferent cell-complex is destined to this or that fate is to argue from authority which one does not possess. To wait until after a cell has differentiated and then claim that the realized outcome was its predetermined fate is to beg the question. Concerning the existence of preformed but unobservable difficulties in such apparently indifferent stages, endless and futile discussions might be precipitated.

The phenomenon itself that different sorts of cells emerge from this apparently indifferent complex need neither be elaborated into a Polyphyletic theory nor into any other theory; it is an undeniable fact that may be verified with the greatest of ease. Certainly no recent observer need claim credit for the discovery of the phenomenon of differentiation. A claim that the divergence of cell-types from an apparently indifferent cell group supports one theory or another is of interest only as evi PROCEEDINGS 237

dence of the extent to which its sponsor has missed the fundamental problem involved. The monophyletic view allows and even depends in part on the divergence of mJike cells from apparently Uke cells which have a wide distribution. If now this is consistent with the polyphyletic view where is the 'conflict on this point, and why all this phyleticism? The monophyletic view does not preclude the possibility of actual differences at a time before we can observe them; it merely postulates in case of vascular tissues a stage in which mesenchyme cells are truly indifferent, and in which an undifferentiated mesenchyme cell may give rise to diverse sorts of progeny.

Some of us believe, however, that there is, k priori, more to favor the monophyletic origin of a 'diffuse anlage' than there is reason to beUeve its origin to be polyphyletic. Conversely it may be stated that those who believe in the 'specificity' of vascular tissues (in .the narrower sense of the word 'specificity') generally, though not always, realize that their doctrines can stand only when supported by a demonstration of the correctness of the following three postulates — in fact such believers do their utmost to defend these postulates. First, that the particular vascular derivative shall have a restricted locus oif origin — a limited anlage. Second, no other tissue shall ever be foimd producing the tissue of which the 'anlage' is the 'specific' source. Third, the derived type shall never be found producing other types of tissue. It is in testing the vahie of these three postulates that we can analyze to a certain extent the developmental processes which operate during the entire 'indifferent' stage. In any event we can produce nothing more than purely d priori evidence for one view or the other.

The differentiation of divergent types of tissue from apparently indifferent tissue-complexes has been known since cells were discovered and was reasoned about for centuries previous. A 'discovery' that actual but undemonstrable differences exist preformed is at present of little value in that it can neither be proved nor disproved, so far as mesenchymal transformations are concerned.

49. The development of the biliary system in animals lacking a gall-bladder in post-embryonic life. (Lantern slides.) Richard E. Scammon, Institute of Anatomy, University of Minnesota. The gall-bladder is well known to be consistently absent in a number of species of vertebrates. In others, as in man, congenital absence of the gall-bladder is a more or less rare anomaly. The history of the biliary system has been investigated in three species in which the gall bladder is lacking in post-embryonic life. These are the rat, the pigeon and Petromyzon. In Petromyzon a complete biliary apparatus is formed. According to Nestler and to Bujor this later undergoes regression both of the epithelial walls and the surrounding layer of supporting tissue. In the pigeon a cystic anlage is formed and completely dffierentiated but increases little in size with the growth of the liver. The gall bladder is carried out on the distal end of the posterior hepatic duct as in other birds. Its regression occurs late in embryonic life and

238 AMERICAN ASSOCIATION OP ANATOMISTS

may take place in several ways. In some cases there is apparently a partial inclusion of the gall bladder in the posterior hepatic duct. In others the bladder may remain, at least temporarily, as a small sac attached to the posterior hepatic duct and lying within its muscular wall. More commonly the bladder becomes detached from the cystic duct, remains for a period as an isolated cyst and finally disintegrates entirely. ^

The rat has not been completely investigated. In later stages (above 5 mm. in length) there is no trace of a gall bladder. In these stages the hepatic ducts are of large caUber. Helly found no distinct cystic anlage in younger embryos.

50. The embryology and anatomy of the nasofrontal region in man, (Lantern sUdes.j J. Parsons Schaeffer. The Daniel Baugh Institute of Anatomy and Biology of the Jeflferson Medical College. Owing to contradictory or but general statements concerning the anatomy of the ductus nasofrontalis and the ostimn ifrontale, the writer deemed it important to make a more detailed study of the embryology and gross anatomy of the nasofrontal region.

The nasofrontal region is embryologically an outgrowth from the ventral and cephaUc end of the middle nasal meatus and is operculated by the middle nasal concha. The mucosa of this portion of the middle nasal meatus is, therefore, the proton of the frontal recess (early in evidence) and derivatives therefrom. The frontal recess in turn is the anlage of the frontal sinus and some of the ventral ethmoidal cells. For sometime the lateral wall of the frontal recess is even and unbroken and gives no evidence of the later configuration and complexity. In a four-month embryo the lateral nasal plate of cartilage is thickened at certain points along the lateral wall of the frontal recess. These thickenings are the smlagen of accessory frontal folds or conchae of the middle nasal meatus. In the late fetus one finds a variable nmnber of frontal conchae with a corresponding number of furrows or pits. The number of frontal folds differentiated varies from a complete absence to three or four. The reduced number is doubtless due to lack of differentiation rather than to an earUer fusion of neighboring parts. The frontal pits or furrows vary accordingly. The uncinate process and the folds composing the ethmoidal bulla should likewise be considered as accessory conchae of the middle nasal meatus (analogues and homologues of the frontal conchae) and the ethmoidal infundibulum and the suprabullar furrow as accessory meatuses or furrows (analogues and homologues of the frontal furrows).

The accessory furrows are forerunners of paranasal sinuses. The frontal furrows or pits early evaginate and form ventral ethmoidal cells. It is a well known fact that the frontal sinus develops variously by a direct extension of the frontal recess or from one or more of the ventral ethmoidal cells which have their point of origin in frontal furrows. In many instances the frontal sinus is, embryologically speaking, a ventral ethmoidal cell which has grown sufficiently far into the frontal

PROCEEDINGS 239

region to be topographically a frontal sinus. In a term fetus one is scarcely justified to hazard an opinion as to the specific point in the frontal recess from which the frontal sinus will ultimately develop.

The uncinate process and the folds composing the ethmoidal bulla are often in direct continuity with one or more of the frontal folds or conchae. The relations of these structures vary. Likewise, the ethmoidal inf undibulmn and one of the frontal furrows or pits are usually in the same axis. It must here be emphasized, however, that it is rare and very unusual for the ethmoidal infundibulmn to be directly continuous with a frontal furrow. The latter anatomic fact is significant when one recalls the careless statement repeatedly made that **the ethmoidal infundibulmn is continued upwards as the nasofrontal duct into the frontal sinus."

When the frontal sinus develops from a frontal furrow not in the same axis as the ethmoidal infundibulmn the relation of the nasofrontal duct to the ethomidal infundibulmn is less intimate than in those cases in which the frontal sinus develops from a frontal furrow which is in the same axis as the ethmoidal infundibulmn. Even if th^ latter condition does obtain, the relation is only intimate and not direct. The same argument holds when the frontal sinus develops by a direct extension of the frontal recess.

If the frontal sinus represents a direct extension of the frontal recess, there is in most instances no nasofrontal duct. The frontal sinus in these cases extends well into the ventral and cephaUc part of the middle nasal meatus, and a very large' proximal ostium frontale represents the pK>int in the frontal recess from which the sinus developed. The ostium frontale -in these cases is usually in the same axis as the inf undibulum ethmoidale. In the vast majority of cases the infimdibulum ethmoidale ends blindly lateral to the frontal recess in a dilated cephaUc extremity (infundibular air cell). The latter may encroach buUous-like on the frontal sinus.

When the frontal sinus develops from one or other of the frontal furrows or pits, a varied anatomy of the nasofrontal connections may be expected. In some cases there is a narrow serpentine-like channel (a true nasofrontal duct) of varied length and dimensions communicating between the frontal sinus and the frontal recess. In such cases there are really two frontal ostia, one at the distal end of the nasofrontal duct and the other at the proximal end of the duct, i.e., in the frontal recess.

One or more ventral ethmoidal cells may encroach upon the nasofrontal duct and cause it to deviate from its primary axis. These ethmoidal cells often encroach on the frontal sinus. There are also many frontal sinuses which obviously develop from frontal furrows in which no true nasofrontal duct is present. The frontal sinus in these cases extends in a roomy manner towards the frontal recess of the middle meatus. The proximal ostium frontale in the frontal recess indicates the position of the embryologic frontal fiurow or pit. Varied relations of the proximal ostimn exist with the infundibulum ethmoidale, — rarely a direct relation, however.

240 AMERICAN ASSOCIATION OF ANATOMISTS

When the frontal sinus appears in duplicate or triplicate obviously two or more ventral ethmoidal cells (from frontal furrows) or the frontal recess and one or more ventral ethmoidal cells developed sufficiently far into the frontal region to be topographically frontal sinuses. With this condition we may again expect varied nasofrontal connections. Each frontal sinus may have a nasofrontal duct. Some or all of the sinuses may present a proximal ostimn frontale only in direct relation with the frontal recess.

Rarely the nasofrontal duct is in direct continuity with the ventral and cephaUc extremity of the ethmoidal infundibulmn. There are two possible explanations for this anatomic condition. Either the ethmoidal infundibulmn and the frontal furrow from which the frontal sinus developed were early in direct continuity or the bridge of tissue intervening between the two channels was resorbed at a subsequent time. The latter explanation seems the more probable. Indeed, in many of these cases a superficial bridge of tissue persists, connecting the uncinate process and the ethmoidal bulla, beneath and lateral to which one occasionally finds the nasofrontal duct and the ethmoidal infundibulmn in direct continuity. This tends to support the beUef that there is a subsequent resorption of the intervening mucous membranes rather than a continuity of channels at a very early time.

Some of the ventral ethmoidal cells developing from frontal furrows or pits remain topographically ethmoid in position. Often, however they encroach on the frontal sinus. Then again it is difficult to classify some; that is, whether they should be called frontal sinuses or ventral ethmoid cells.

This study leads us to conclude that the ethmoidal infundibulmn is rarely in direct continuity with the nasofrontal duct or in the absence of the latter, with the proximal frontal ostium. In all cases the frontal sinus communicates with the frontal recess, the communication being established either by a true nasofrontal duct or merely by an ostium placed directly in the caudal portion of the frontal sinus. Furthermore the ethmoidal infundibulum usually develops ventrally and cephalically, lateral to the frontal recess and dilates into a ventral ethmoidal cell (infundibular cell). The nasafrontal duct and the ethmoid infundibulum may or may not be in the same axis, depending from which frontal furrow the frontal sinus developed. It is, therefore, obvious that the relation between the ethmoidal infundibulum and the nasal portion of the frontal sinus is very intimate but not direct. In many instances contiguity and not continuity is the relation. However, from a practical standpoint, the relations are so intimate in at least 50 per cent of the cases that any drainage from the frontal sinus, naturally finds its way into the ethmoidal infundibulum, while in the remaining cases the relations are less intimate and drainage from the frontal sinus finds its way more directly into the middle nasal meatus, medial to the uncinate process. The only exception to this statement is when the ethmoidal infundibulum is directly continuous with the

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nasofrontal duct or in the absence of the latter directly with the frontal sinus. It must again be stated that the exception is rare.

This paper is illustrated by lantern slides of reconstructions and of actual dissections. The detailed paper, with figures, is ready for publication and will appear elsewhere.

51, A suggestion as to the process of ovulation and ovarian cyst formation. S. S. ScHOCHET. Department of Anatomy and Histology, Dartmouth Medical School. (Introduced by Dr. F. P. Lord.) The study here reported has been carried out in a large part in the Anatomical Laboratories, Tulane University, at the suggestion of Dr. Irving Hardesty.

It is generally conceded that pressure atrophy of the ovarian stroma is the chief means by which the extrusion of the ovum becomes possible, as a result of the compression of the blood capillaries between it and the surface of the ovary. (John G. Clark, Johns Hopkins Bulletin 1899). Attention has been directed to the determination of the r61e played by the hquor foUiculi.

A series of experiments indicates that the hquor foUicuU possesses a digestive enzyme that can be demonstrated by dialysis and other tests. Controls and comparative tests were made with other fluids of the body and indiflferent fluids. The results obtained may be tabulated as follows:

1. Abderhalden^s dialyzaiion reaction

Ligament

Ovarian Tissse Muscle (White Fibrous) Fibrin

Liquor FoUicuU + + + + + + ++ + + +

Cystic Fluid + + + +

Amniotic Fluid — _ — —

Normal Saline — — — —

2. Grutzner^s fibrin test

Liquor FoUicuU + + +

Ananiotic Fluid

It was noted that there was an increase of stroma (proliferation) instead of an atrophy of the theca of the Graafian follicle. This is not a new fact, but a fact in many instances not sufiiciently emphasized.

I beUeve that I am justified in offering as a tentative interpretation the suggestion that the ruptiu-e of the Graafian follicle is due in part to the digestion of the theca by the Uquor foUicuU. At present I am not in a position to state whether it be (1) a specific ens5Tiie: (2) a concentration of proteolytic substances due to the endosmosis into the antnmi of the folUcle from the blood current: c. a proteolytic substance as a result of the Uquefaction of the folUcular cells. If it be a specific enzyme it is suggestive of a typical antibody production.

The paper is illustrated by diagrams and a review of the theories of ovulation.

242 AMERICAN ASSOCIATION OF ANATOMISTS

52, The fusion of the bilateral anlagen of the heart and the formation of the bulbo-ventricular loop in embryos of the cat. H. von W. Schulte. Anatomical Laboratory of Columbia University.

•• Differences in the details of the fusion between the myoepicardial mantels in mammals have been shown to depend upon the primary position of the mantels in the parietal cavities (Stahl and Carius). They have termed the strip of viscera mesoderm intervening between the mantel and the coelomic angle, the retrocardiac plate, that between the mantel and the. lateral limit of the parietal cavity, the precardiac plate. From the former the dorsal mesocardium is formed, from the latter the ventral. The type of fusion is determined by the relative breadths of the pre- and retro-cardiac plates, in particular the proportion existing between the latter and the transverse diameter of the foregut. If the foregut is wise and its retrocardiac plate relatively narrow, the bilateral anlages will be separated by a wise interval when the foregut is closed and will only secondarily come into opposition; the formation of the ventral mesocardium will antecede that of the dorsal (Cimiculus) . If the converse proportions obtain — foregut narrow, retrocardiac plate broad, the dorsal mesocardium will be formed before the ventral (Cavia).

The cat conforms in general to the tjrpe of the rabbit, although the bilateral mantels are not as widely separated after closure of the forgut as in the shematic figure of Stahl and Carius (1889. Fig. 15). Further the retrocardiac plate is not of uniform breadth in its whole extent but narrows cephalad, so that here the anlages are nearer together and their fusion is accelerated as compared to more caudal portions. The ventral mesocardium is ephermeral and after its disappearance, the myoepicardial mantels are joined by a triangular middle field or plate of mesoderm derived from the precardiac plates, which broadens caudad as the cardiac tubes diverge. The approach of the mantels is accompanied by a contraction of the middle plate, and their ultimate fusion is accomplished by its agency. In these stages it is transformed to an ectal convexity in the line of union, while

• entally it is defined by longitudinal ridges from the mantels of the two sides. Ultimately the ridges are reduced and the site of the middle field is marked only by a moderate thickening of the myocardium, which marks the primitive axial line of the heart during the formation of the loop. Between the middle plate and the foregut from the deep surface of the former there is active production of mesenchyme, in which endotheUal vesicles are formed and subsequently play a r61e in the fusion of the endothelial tubes of the heart.

That the heart tubes become bent prior to fusion was observed by His in a rabbit embryo, and has since been frequently represented (e.g., in the chick Duval, 1889, fig. 88; in the cat, Martin, 1910, fig. 72). The angulation in question is incident at the caudal end of the heart tube and marks its junction with the omphalomesentric veins; it is bilateral and symmetrical. There is in embryos of the cat (8-14 somites) a second bend on each side at the juncture of the bulbus with the ventricle. Primitively these bulbo-ventricular furrows are symmetrical, and with the abrupt change of diameter from the narrow bulbus to the broad ventricle, give this region of the heart much the appearance of a neck rising between two shoulders (12 somites). The bend of the heart is effected by the deepening of the left and the gradual obUteration of the right bulbo-ventricular furrow. It is in its innition as though the left shoulder were raised, the right dropped (14 somites). The left furrow and shoulder are no longer distinguishable in external view at 16 somites, though entally the fundus of the left bulbo-ventricle furrow is still perceptible as a low ridge; the right furrow deepens and persists as the definitive bulbo-ventricular fissure.

The endotheUal tube in these stages conforms in general shape to the myoepicardial mantel. The junction of the bulbus with the ventricle is greatly constricted and sharply bent around the fundus of the bulbo-ventricular fissure on each side. In consequence, the endothehal tubes are early brought into apposition in their bulbar segments. In the ventricular region they diverge and again approach one another at the caudal bend at the junction of the heart tube and the omphalo-mesentric vein. Fusion is accompUshed by the incorporation of the mesenchyme and endothelial vesicles of the middle field into the endotheUal tubes of the uniting cardiac anlages. It is first effected caudad in the region of the sinus venosus; almost at the same time, however, it begins also at the bulbo-ventricular isthmus (14 somites). The ventricles still show remnants of the septum in embryos of 19-20 somites. The definitive bulbus is derived chiefly from the right endotheUal anlage, the auricle chiefly from the left, for in both of these situations one tube enlarges, the other diminishes in size, eventuaUy becoming interrupted in its continuity, and is only partially utilized in the formation of the single cardiac tube.

See 65, Kathebins J. Scott, page 263.

53. On the osteology of the fishes of Bermuda. R. W. Shufeldt, Washington, D. C*

Several years ago, while Uving in the city of New York, Dr. Charles H. Townsend, Director of the New York Aquarium, invited my attention to the fact that many of the Uving fishes, constantly being transported from the Islands of Bermuda for exhibition purposes at the Aquarixun, died, either during transportation in the tanks in which they were kept, or succmnbed to the effects of the trip shortly after their arrival. It was pointed out to me that this material was in excess of what was required for the series of alcohoUc specimens, or for the use of the schools and other laboratories in the State.

The suggestion was made by me that a series of skeletons of these interesting forms be prepared, and subsequently figured and described, if i>ossible. This idea appealed to Doctor Townsend; he offered me the use of the laboratory in the Aquarium building, and the services of those of the staff who could assist me in the work. During the two years that followed, I dtjvoted from two to three days a week to the skeletonizing of the various specimens of fishes that came into my

244 AMERICAN ASSOCIATION OF ANATOMISTS

hands from the aforesaid sources; so that, at the end of that time, when I returned to Washington to continue my researches in biology and other branches, I found I had prepared for study and description over sixty skeletons of the representative species of the ichthyfauna of the Bermuda Islands. These included such interesting forms as all the varieties of the Parrot fishes {ScaruSy Pseudoscarus, and their allies), the Moras {MuroRna)^ the Groupers, Red Hind, Trunkfishes, Porcupine fish, various species of Angel fishes, Pompano, Hog-fish, Turbot, and others too numerous to mention.

My first labor upon these was to prepare a series of photographic negatives of them. This work has now been carried to a point where I find in my collection a nearly complete array of these, and from them I have made many valuable photographs to illustrate my forthcoming contributions in this field of research.

The very distinguished ichthyologist, the late Dr. Theodore Gill, examined, shortly before his death, not only my set of photographs illustrating the skulls, skeletons, and other parts of these Bermuda fishes, but he was so good as to go over some of the material itself, remarking at the time that my studies of these forms should certainly be completed and published. Some of this is quite ready for publication at this writing, and much else of it can easily be so prepared. There is very little being done at the present time in the skeletal structure of fishes; and as a matter of fact there is but scant literature on this important department of biology.

Very well do I remember the interest Doctor Gill evinced as he examined some of my preparations; and upon several occasions he remarked, as he held one or another of them in his hand: "Here is a point that should be carefully worked, as we have little or nothing upon the osteology of this species."

it would be quite out of the question for me to fully abstract my work in this direction as far as it has been carried up to date, for that would soon overrun the legitimate space limits. However, I may say that, in view of the fact that so little — so very little comparatively — is now being done in anatomical structure of fishes in this country, contributions on the subject should be quite welcome, not to say useful. It is my hope to complete my work on this material in due course; when it is sufficiently advanced to commence publication, and not subjected to subsequent interruptions, I further hope to have some of it appear in the Anatomical Record, or, perchance, in some of the various other publications of The Wistar Institute.

64' A disguised type of smooth muscle cell. R. A. Spaeth. Osborn Zoological Laboratory, Yale University. (Introduced by R. G. Harrison).*

The melanophores of the lower vertebrates — fish, amphibia and reptiles — are described by most authors as modified connective tissue cells. The evidence for such a conception rests chiefly upon a m^orphological basis. Leydig showed that no sharp morphological distinc PROCEEDINGS 245

tion exists between impigmented connected tissue cells, inactive, pigmented connective tissue cells and chromatophores (melanophores) containing actively migrating pigment granules.

Morpholgical characteristics alone are not, however, suflScient finally to classify any type of cell. Little evidence is to be adduced from the embryological history of the melanophore concerning its ultimate nature, since both connective tissue cells and melanophores develop from wandering mesenchyme cells. However, a consideration of the physiological responses of the melanophore shows that it bears a far closer resemblance to certain types of Smooth muscle cells, e.g., those of the sphincter pupillae, the digestive tract, and the radial elements of the cephalopod chromatophores, than to any type of conaective tissue cell. Thus, for example, chemical, electrical and thermal stimuli which produce contractions in these three types of smooth muscle also bring about contractions in the melanophore. Appropriate treatment with PaCU initiates a slow pulsation in the melanophore. A study of the rate of rythmic contraction shows it to be of the same order as the spontaneous contractions of excised portions of the digestive tract.

The work of Chun has shown conclusively that the chromatophores of cephalopods develop from single smooth muscle cells by a complicated metamorphosis. Thus the establishing of the vertebrate melanophore as a modified smooth muscle cell — a view previously suggested by Steinach, Franz and Hofifman and formerly questioned by me, is of particular significance, since it reduces the color-changing mechanisms of both vertebrates and invertebrates to a common basis, i.e., a specialized type of smooth muscle cell.

In the melanophore, the motor fimction of the smooth muscle cell is lost and there is developed a modified motility in the migration of pigment granules, which may serve as a means of protecting the animal. An analogous case in that of the electric organ in fish where a striated muscle cell loses its motor function completely, and the action current, normally of relative insignificance, becomes a physiological end in itself and a formidable means of protection.

55. Growth of the body and of the various organs of young albino rats upon refeeding after inanition for various periods. C. A. Stewart, Institute of Anatomy, University of Minnesota. (Introduced by C. M. Jackson.)*

Eight Utters, including 45 rats, were used, of which 9 were controls. Of the experimented rats, 3 were repeatedly starved and refed for short intervals. The others were refed for various periods of maintenance (from age of 3 weeks to age of 4, 6, 10, and 12 weeks).

The average daily loss in weight did not increase in the rats repeatedly starved, and no effect upon the ultimate body weight was noted.

The growth in body weight of the rats refed after maintenance for various periods averaged considerably higher for some time than the normal for (younger) controls of the same body weight. Thus the stunted rats on refeeding were able to overtake the full-fed controls before the end of the normal growth period.

TBB ANATOMICAL RECORD, yOL. 10, NO. 3

246 AMERICAN ASSOCIATION OF ANATOMISTS

As to body proportions, the relative weights of the head, trunk and extremities remain practically normal during the various periods of refeeding after maintenance.

Of the sjrstems, the musculatiu'e and 'remainder' continue practically normal (for corresponding body weight) during the various periods of refeeding. The integument rapidly increases and the skeleton decreases in relative weight, so that both reach their normal proportions within the first two weeks of refeeding after maintenance from three to ten weeks of age.

The viscera which lose weight duriilfe maintenance, — thymus, spleen, thyroid, lungs and ovaries, — ^likewise apparently regain their normal relative weight within two weeks. The thjTnus (and possibly the limgs and spleen) are even above normal at four weeks of refeeding, but all are foimd nearly normal in rats refed to the adult stage.

The viscera whose weight remains nearly constant during maintenance, — ^brain, heart, kidnejrs, Uver and epididymi, — ^in general present approximately normal proportions during the process of refeeding; although the heart appears slightly above normal, and the epididymi somewhat below.

The viscera whose weight increases during maintenance, — eyeballs, spinal cord, alimentary canal, hypophysis, testes and suprarenals, — have in general decreased in relative weight so as to approach the normal within the first four weeks of refeeding.

66. Experimental modification of the chromatin within the germ cells of

one generation and the resulting hereditary transmission of degeneracy

and deformities. (Lantern.) Charles R . Stockard, Cornell Medical

School, New York City.

The results of an experiment now in progress for more than five years permits to some extent an analysis of the influence on the offspring of alcohohzing either one or both parents and the manner of hereditary' transmission of the induced effects to subsequent generations.

The experiments have demonstrated on two different stocks of normal guinea-pigs that the parental germ cells may be so modified by chemical treatments that they are rendered incapable of giving rise to a perfectly normal offspring. This incapacity is probably due to modifications of the chromatin, or carriers of the hereditary quaUties, within the germ cells since the great-grandchildren, the Fs generation, from the treated animals are usually more decidedly affected and injured than the immediate offspring (Fi) of the alcoholized animals.

This then becomes a study of the behavior of diseased or pathological chromatin in heredity. Chromatin rendered pathological more than four years ago is stiU living and has now been passed on to the Fs generation from the alcohoGzed great-grandparents. The F> animals are almost without exception incapable of reproduction and are in many ways subnormal and degenerate.

Studies of abnormal here(Sty may possibly furnish a means of analyzing the normal methods of action by which the minute carriers

PROCEEDINGS 247

of hereditary qualities contained within the fertilized egg are capable of causing the complex developmental and structural changes to reoccur from generation to generation in so wonderfully consistent a manner. Just as the knowledge furnished by studies of experimentally modified embryonic development has supplied valuable data towards a clearer imderstanding of the normal processes and changes which occur in the developing embryo.

The treatment of adult guinea-pigs by an inhalation method with daily doses of alcohol through several years produced little if any noticeable effect upon the organs and tissues of the animals' body. The direct action of alcohol fumes tends to injiu'e the mucosa of the respiratory tract and to render the cornea of the eye dull or opaque. These changes, however, do not inconvenience the animals in any perceptible way, and they remain strong and hardy and live as long and actively as the untreated guinea-pigs. In spite of their healthy appearance the injurious influence of the alcohol inhalation is very decidedly shown by the quality of oflFspring to which the treated guineapigs give rise and the descendants of these oflFspring are even worse than the Fi generation when compared with the different generations of control animals produced under identical cage and food conditions.

The males seem to be more injured by the treatment than the females taking as an index of injury the quaUty of their oflfspring and descendants. Stating it diflferently the spermatocjrtes or spermatozoa are more sensitive to the changed chemical condition of the tissues than are the female germ cells.

There is a larger proportion of degenerate, paralytic and grossly deformed individuals descended from the alcoholized males than from tJie alcoholized females.

The records of 667 oflfspring produced by 566 matings of animals of various types have been tabulated to show the kinds of litters of young produced and their ability to survive. One hundred and sixty-fom* matings of alchoUzed animals, in which either the father, mother, or both were alcoholic gave 64, or almost 40 per cent negative results, or early abortions, while only 25 per cent of the control matings failed to give full term litters. Of the 100 full term litters from alcoholic parents 18 per cent contained stiU-bom young, and only 60 per cent of all the matings resulted in living Utters, and 46 per cent of the individuals in the litters of hving yoimg died very soon after birth. In contrast to this record 72 per cent of the 86 control matings gave living litters and 84 per cent of the yoimg in these Utters survived as normal healthy animals.

The mating records of the descendants of the alcoholized guineapigs although they themselves were not treated with alcohol compares in some respects even more unfavorably with the control records than did the above records of the directly alcohoUzed animals.

Of 194 matings of Fi animals in various combinations 55 have resulted in negative results, or early abortions, 18 still-bom litters of 41 young occurred, and 17 per cent of these still-bom young were deformed.

248 AMERICAN ASSOCIATION OF ANATOMISTS

One hundred and twenty-one living litters contained 199 young, but 94 of these died within a few days and almost 15 per cent of them were deformed, while 105 survived and 7 of these showed eye deformities. Among 115 full-term control young of the same stock not one has been deformed.

The records of the matings of Fj animals are still worse, higher mortality and more pronounced deformities. While the few Fs individuals which have survived are generally weak and in many instances appear to be quite sterile even though paired with vigorous prolific normal mates.

The structural defects seem to be confined chiefly to the central nervous system and special sense organs. Many of the young animals show gross tremors, paralysis agitans, or the hind legs, fore legs or both legs of one side may be paralyzed. Eye defects are very conmion such as opaque cornea; opaque lens, various degrees of monophthalmica asymmetrica and finally several cases of complete anophthalmia have occurred the entire eye ball, optic nerve and optic chiasma being absent.

The quality of the individuals from the same parentage varies inversely with the size of the litters in which they are produced. Animals bom one in a litter are rather strong even though derived from very bad alcoholic lines. This difference between the members of small and large litters is also shown by the normal animals but the differences between members of large and small litters is ever so much greater in the alcoholic lines.

Inbreeding tends to emphasize the alcoholic effects. This is probably due to the related animals responding to the treatment in closely similar ways on accoimt of the similarity of their constitutions. Inbreeding as such, may be harmful. But inbreeding added to the alcohol eflfects produces a much more serious condition in the offspring than either inbreeding or alcoholism alone could do.

The data from alcoholized male lines indicates that the female offspring from alcoholic males are less viable and more frequently deformed than the male offspring. And heterogeneous matings of such male and female offspring further emphasizes the same inferiority on the part of the female offspring from treated males. This is a very significant fact.

The fact that the offspring of one sex differs in quality from those of the opposite sex and that the female offspring of an alcoholic male are inferior to his male offspring suggests at once a difference between the germ cells concerned in the production of the male and female young. Miss Stevens showed that the spermatocytes of the male guinea-pig contained a heteromorphic pair of chromosomes and half of the spermatozoa would be expected to receive one member, the x chromosome, of the heteromorphic pair and one-half of the spermatozoa the other member, the y chromosomes, of the heteromorphic pair. We now have two possibilities in explanation of the above facts. In the first place, it may be assumed that the alcohol acts similarly on all of the chromatin to injure it. Thus a mass action would cause the spermatozoa carrying

PROCEEDINGS 249

the larger member of the heteromorphic pair to deliver more injured chromatin and the other spennatozoa with a less total amomit of injured chromatin would deliver less when they fertilize eggs containing equal amounts of normal chromatin. The fertilized egg giving rise to the female, therefore, contams a greater proportional amount of alcoholic chromatin to normal chromatin than does the egg giving rise .to the male, and so the female product is actually more injured than the male.

A second possible explanation of these conditions may be that the x and y chromosomes themselves respond differently to the treatment, the X being the more sensitive of the two. But in either case the two classes of spermatozoa certainly seem to respond differently to the treatment and this shows a physiological difference in behavior to correspond with the well known morphological differences so often shown between the two groups of spermatids of many animal species.

The data from alcoholic female lines indicates that the male offspring from alcoholic females are inferior in qiuility to the female offspring. And heterogeneous m^atings of such male and female off spring further prove the inferiority on the part of the male offspring from treated females. This is also significant. How can it be put in accord with the above chromosomal explanations for the difference in quaUty between the female and male young of alcoholized fathers?

If we admit that all of the eggs arising from an alcoholized female guinea-pig are homomorphic and contain groups of chromosomes equal in mass, it follows that her male and female offspring receive the same amount of injured chromatin and should be affected by such chromatin to equal degrees. But this is only part of the case, the injured female chromatin is combined with normal chromatin from the normal father when the eggs are fertilized and here the difference arises. The female offspring receives from the normal father a larger amount of normal chromatin than do the male offspring. So that the female arises from an egg in which equal amounts of good and injured chromatin are present while the male offspring arises from an egg in which a larger amoimt of injured chromatin is united with a smaller amount of normal. Therefore, proportionally the male offspring has more injured chromatin in his entire bodily make-up than does the female and is comparatively in a more abnormal condition.

Another explanation of these differences between the male and female offspring of alcoholized females could be based on the possibility of the female being heterozygous for sex. This involves a very complex discussion but one for which there is some ground on the basis of the regulation of the sex ratio in these animals.

Finally then, the experiments show the hereditary transmission through several generations of conditions resulting from an artificially induced change in the germ cells of one generation. And they furnish data of importance bearing upon the pathological behavior of the carriers of heredity as well as the differences in behavior between the two types of germ cells produced by an animal carrying heteromorphic chromosomes.

250 AMERICAN ASSOCIATION OP ANATOMISTS

57. Development of the scala vestibuli and acala tympani and their communications in the human embryo. George L. Streetbr, Carnegie Institution, Embiyological Research, Baltimore.

The perilymphatic spaces are formed by the coalescence of mesodermal tissue spaces surrounding the membranous labyrinth and closely resemble in their formation the subarachnoid spaces. They cannot, however, be regarded as subarachnoid spaces that have invaded the region of the internal ear, because their development is in loco and independent of the latter. In the latest stage examined (130 mm. crown-rump length) the communication with the subarachnoid spaces is npt yet established.

In their formation the perilymphatic spaces follow a definite morphological plan. They spread from two foci; the larger one begins as a rounded sac lying opposite the foot-plate of the stapes and lateral to the sacculus, from which point it subsequently spreads upward over the utricle and also downward along the apical side of the cochlear duct to form the scala vestibuli; the other focus is near the fenestra rotundum from where it spreads along the basal side of the cochlear duct to form the scala tympani. These foci can be definitely outlined in fetuses 50 mm. long. In 85 mm. fetuses the two scalae extend spirally downward along the cochlear duct to a point three-fourths of its last turn from the tip of the duct and they do not commimicate with each other. In 130 mm. fetuses they extend to the tip of the cochlear duct and open into each other forming the helicotrema.

The semicircular canals acquire their perilymphatic spaces relatively late. In the 130 mm. fetus the lateral canal has a space throughout one-half its length. The remainder and that of the other canals are acquired after this.

58. The auproroptic canal, its morphology and arudomical relation to choked disc. Frederick Tilney, Department of Anatomy, Columbia University.

In a recent paper^ the writer called attention to the presence of a small canal extending out from the third ventricle above the optic nerve and chiasm. This I called the SuprorOptic Canal. It was first observed in studying the fore-brain of the domestic cat. • In this animal the canal projects laterad from the cephalic extremity of the ventricle across the optic chiasm and for a short distance into the optic nerve on either side. It is lined with ependyma and measures 0.75 mm. in length. It is situated upon the dorsum of the chiasm and in this position extends along the optic nerve. The canal is marked upon the surface by a ridge running along the dorsum of the optic chiasm and optic nerve. This is the Supra-Optic Ridge.

I have observed this canal in the following forms: Squalus acanthias, Mustelus laevis, Lepidosteus osseus, Rana pipiens, Menobranchue,

1 Morphology of the Diencephalic Floor: A contribution to the Study of Craniate Homology. Journal of Comparative Neurology, 1915, vol. 26, no. 3.

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Iguana tuberculata, Gallus gallus, Botaurus lentiginosus, DidelpKys quica, Bradypus tridactylus, Dipus aegyticxis, Dasjrprocata agouti, Mus deciunanus, Lepus sylvaticus, Sphingunis prehensilis, Mephitis mephitica, Odocoelus hemionus, Odocoelus virginianus, Orjrx beatrix, Ovis aries, Castor canadensis, Mirounga (Macrorhinus augustirostris), Nasua narica, Ursus hombilis, Canis latrans, Canis familiaris^ Genetta vulgaris, Felis domesticus, Felis pardus, Felis leo, Lemur macaco, Macacus cynomolgus, Nyctipithecus trivirgatus, Babuin cjniocephalus, Hylobates hoolock, Simla satyrus, and Homo.

The existence of a canal phyletically so constant as this one, must have some definite ontogenetic explanation. This was sought for in the embryology of .several forms and found to be thoroughly consistent in all of them, so that the conditions in the cat may serve as a paradigm for the vertebrate development of this accessory recess of the third ventricle.

At the early stage of eight somites in the cat embryo, the entire cephalic expansion of the neural tube is given over to the formation of the primitive optic vesicles which contain a large prosencephalic ventricle. A little later, these primitive vesicles show a constriction which causes the appearance of two small evaginations now recognizable as the definitive optic vesicles. Each of these evaginations contains a recess accessory to the prosencephalic ventricle.

The definitive optic vesicles then proceed to give rise to the eye-cup which retains its connection with the brain by means of an attenuated stalk. Through this stalk passes a narrow canal, the renmant of the former spacious connection between the fore-brain and the eye vesicle. Thus it is obvious that the embryonic basis of the supra-optic canal is to be foimd in this small tubular extension from the third ventricle. The fibers which form the optic nerve and optic chiasm grow into and through the optic stalk with the result that these fibers* take up a position ventral to the canal, thus causing the supra-optic recess of the ventricle to rest upon the dorsum of the optic nerve and chiasm.

In the majority of forms the proximal portion of the canal alone persists into adult life. Osborne, Herrick and Kingsbury, have shown, however, that the rudimentary condition of the eye in Necturus is accompanied by a similar condition of the optic nerve which retains in the adult the primitive lumen of the optic vesicle and is hollow for a considerable distance peripherad.

The history of this canal is clear, both from the embryological and the phyletic standpoints. It remains to inquire what clmical significance it may assume in cases where there is an accumulation and retention of fluid in the fore-brain ventricles. Obviously, such a canal placed in such relation to the optic chiasm and nerve might bring pressure to bear upon the fibres of these structiu'es and thus interfere with the normal hemal and non-hemal drainage in this part of the nervous system. Should the canal become dilated, there is butoneresult possible, namely, a compression of the optic fibres lying beneath it.

What happens to this canal in the event of dilatation is shown in a

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case of internal hydrocephalus in a child four years of age which presented a bilateral choked disc. There can be no question that the distention of the supra-optic canal caused profound changes as compared with the normal. The actual alterations are easily appreciated, as follows:

First, the obliteration of the supra-optic ridge by a flattening out of the roof of the canal; second, the thinning of the ependymal lining of the canal; and third, the evidence of compression of the optic fibers as they pass beneath the canal.

The appearance of choked disc in brain tumor is variously estimated as occurring in from 80 to 90 per cent of all cases. Papilledema, however, is much more constant than this in internal hydrocephalus. Opinion today is turning toward the belief that it is the complicating internal hydrocephalus, occasioned by the tumor, which produces the edema of the optic nerve. Such a view would explain the absence of choked disc in a certain percentage of brain tumor, for it is possible that some new growths may exist without compromising the ventricular chambers. This would be particularly true of the endotheUomata in which the surface pressure is often so diffuse as to cause no appreciable change in the size or relations of the ventricles. On the other hand, extremely small tumors if rightly placed, as for example, in the neighborhood of the aqueduct of Sylvius may occlude the passage between the third and fourth ventricles, thus causing retention of the fluid in the third and lateral ventricles.

The causation of choked disc from this viewpoint would require the establishment of an internal hydrocephalus. This does not in any way preclude the possibility of the production of papilledema due to increased pressure in the intervaginal sheath. As far as our knowledge permits today, it seems admissible to conclude that choked disc may be the result of the following mechanical disturbances in the anatomical relations.

1. Increased intercranial pressure operating particularly upon the fluid in the sub-arachnoid space and so causing distention of the intervaginal space aroimd the optic nerve (Manz-Schmidt-Rimpler theory); 2, internal hydrocephalus which produces a distention of the supraoptic canal, and in this way puts pressure upon the optic nerve and chiasm; 3, both of these mechanical disturbances may be operative at the same time.

As to the so-called toxic theory of Leber in the causation of choked disc, the facts at present seem to warrant its acceptance. But it should be held with some reservation since we are not yet in a position to state the exact modus operandi of such toxic substances as may cause a swelling of the disc It is possible for example, that such toxins should act directly upon the nerve to cause edema or again upon the ependyma of the ventricles causing a swelling of the ependymal epithelium, thus producing internal hydrocephalus which actually becomes the underlying cause of choked disc. This aspect in the pathogenesis of papilledema must receive still further investigation. On the other hand, with reference to the mechanical elements operative in the causation

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of choked disc, it now seems justifiable to supplement our previous conceptions by the rule which the supra-optic canal may play in the causation of papilledema as the direct result of internal hydrocephalus.

59, Observations on the mitochondrial content of the cells of the nuclei of the cranial nerves. ' M. DeG. Thurlow, Anatomical Laboratory, Johns Hopkins University. (Presented by E. V. Cowdry). The purpose of this investigation was to determine whether the amount of mitochondria could be used as a basis for classifying nerve cells into motor and sensory groups. The problem was suggested by the striking amount of mitochondria present in the cells of the mesencephalic nucleus of the V nerve, an amount sufficient to distinguish the cells in it from every other cell in the medulla and giving them an appearance similar to spinal ganglion cells.

White mice were used, and the observations were confined to the cells of the nuclei of the cranial nerves because these may more readily be classified into somatic sensory, visceral sensory, visceral motor and somatic motor divisions. The brains were fixed by a special method which permits of the study of both the Nissl substance and the mitochondria differentially stained in the same cell. The individual mitochondria were counted and the number per cubic millimetre of cytoplasm determined, this serving as the basis for the comparison.

The mesencephalic nucleus of the V, the cochlear and vestibular nuclei of the VIII, the nuclei of the VI and VII nerves were chosen for study because these nuclei seem to represent extremes in the amoimt of mitochondria. In the cells in individual nuclei the quantitative variations in mitochondria are but slight although the variation is great as between the cellsin different nuclei. The largest amount of mitochondria was found to be present in the mesencephalic nucleus of the V, the smallest amount in the nucelus of the VII. This would seem to indicate that sensory cells differ from motor cells by the amount of mitochondria within them, but this in reaUty is not the case because there are large amoimts of mitochondria in both sensory and motor cells (e.g., the cochlear nucleus of the VIII and the nucleus of the VI), while in striking contrast to this are other motor and sensory cells (e.g., the nucleus of the VII and the vestibular nucleus of the VIII) containing a greatly reduced amount. Furthermore, the motor cells of the VI nerve contain more than the sensory cells of the vestibular nucleus of the VIII, and the sensory cells of the cochlear nucleus of the VIII contain relatively more mitochondria than the motor cells of the VII nerve, so that we have examples of motor cells containing more than sensory cells, and of sensory cells containing more than motor cells.

It is evident that the mitochondrial content cannot be used as a basis of classification of sensory and motor cells, although the cells of certain nuclei of the cranial nerves can be recognized with certainty by the relative amounts of mitochondria within them. Variations in amount of mitochondria are not without significance in view of the nmnerous observations on changes in the amount of mitochondria, with activity, in other than nerve cells.

254 AMERICAN ASSOCIATION OF ANATOMISTS

60. The position and relations of the sex gland in early human embryos,

(Lantern.) John Warren, Harvard Medical School.

The object of this communication is to point out the changes in the position of the sex gland and the consequent variation in its relations to the abdominal organs in the earlier stages of development. The sex gland first appears as a mere superficial thickening covering the ventral and mesial surfaces of the Wolffian body, and does not exert any special impression on the neighboring organs apart from that due to the WolflBan body itself. In an embryo of 9.4 mm. this anlage has rather ill defined limit?, but extends approximately frpm the level of the sixth and seventh to a point between the tenth and eleventh thoracic nerves. The WolflBan body at this stage begins opposite the last pair of cervical nerves and ends near the last pair of thoracic nerves.

In an embryo of 10.2 mm. the sex gland has assumed definite proportions, and is raised above the surface of the WoflSan body so as to make distinct impressions on the organs in relation to it. The upper end of the WolflBan body lies between the second and third pair of thoracic nerves. The upper pole of the sex gland is opposite the fourth pair of thoracic nerves and immediately fallow the opening between the pleural and peritoneal cavities, while the lower pole terminates between the first and second pair of lumbar nerves. The cephaUc end of the gland is practically in relation to the lung on both sides Below this lung area anteriorly on the right gland comes an hepatic area forming an impression on the posterior surface of the right lobe of the liver and below the liver an intestinal area. Towards the lower end there is a mesial surface in relation to the mesentery. On the left side the gland is in relation anteriorly first to the greater curvature of the stomach, then to the great omentum and below the latter the anterior surface is free. The mesial surface is towards the mesentery.

In an embryo of 16 mm. these relations become more complicate and owing to the unusual size of the gland deeper mpressions are formed on the organs with which it is in contact. The upper end is at the level if the top of the ninth thoracic pair of nerves and ends below at the top of the second pair of lumbar nerves. The upper end of the mesonephric folds in which the WolflBan bodies develop, is prolonged above from the diaphragm to the top of the WolflBan body and lies just lateral to the opening between the pleural and peritoneal cavities. These folds which make deep impressions on the right and left lobes of the liver, form with the WolflBan bodies and sex glands a prominent ridge on the dorsal wall of the abdominal cavity, and this ridge is prolonged by means of the gubemaculum to the ventro-lateral wall of the abdomen. In this way the dorsal wall on either side of the mesentery of the intestine is divided into a lateral region external to the ridge and a mesial region internal, between it and the mesentery. Furthermore these ridges make longitudinal grooves in the various organs on either side of the general vertebral groove. The right sex gland is in relation anteriorly for its whole extent with the liver, being deeply imbedded in that organ. The Miillerian duct also causes a distinct impression on the liver external

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to that caused by the gland. The WolflSan body lies dorso-lateral and the adrenal and lower down, the cardinal vein dorso-mesial. While the greater part of the gland lies rather transversely, the lower part makes a half turn so that its long axis lies antero-posteriorly and takes a position between the Wolffian body and the intestine. On the left side the sex gland has the same posterior relations as the right, Vhile anteriorly it presents, in succession from above downward, first a ventromesial surface for the stomach and a ventro-lateral surface for the liver, which is later succeeded by a splenic surface, the spleen intervening between the gland behind and the liver and stomach in front. Below and internal to the spleen comes an omental surface and still lower a pancreatic. Below the pancreas the gland makes the half turn already described, and the gland then lies between the Wolffian body externally and the mesentery internally, while anteriorly the omentum covers it nearly to the lower end.

In an embryo of 25 mm. the upper end of the gland lies opposite a point between the eleventh and twelfth thoracic nerves while the lower end is opposite the second pair of lumbar nerves. As the upper pole has descended from the level of the ninth to that of the twelfth pair of thoracic nerves, the relations especially on the left are somewhat modified though the impressionson the various organs are more accentuated owing to the increased size of the gland. Both glands now have renal surfaces behind the relation to the primitive kidneys and lie much more free in the abdominal cavity, being attached to the dorsal wall by their mesenteries only. On the right side there appears a mesial surface in relation to the duodenum and lower a smaller area in contact with the head of the pancreas. The gland then turns mesially and is moulded against the intestine and its mesentery below the duodenum. On the left there is no longer any gastric surface, the gland being first in relation anteriorly to the hver and then to the spleen which separates it from the stomach. Below the spleen comes the pancreatic and omental surfaces, the latter organ receiving a deep impression from the gland. In other respects the relations are the same as in the preceding stage.

Beyond this stage the gland rapidly descends along the dorsal wall of the abdomen and in an embryo of 37 mm. extends from the third lumbar to the fifth lumbar and first sacral nerves. Only the upper ends of both glands lie in contact with the kidneys and rest below on the dorsal wall along the psoas muscle. The right gland is still entirely covered in front by the liver and more mesially by coils of intestine. The upper end of the left gland is just touched by the omentum but otherwise is imbedded in coils of the intestine. In the oldest stage studied, an embryo of 42 mm., the gland extends from the fourth lumbar to the fifth lumbar and first sacral nerves and is entirely below the kidneys. On the right side the gland is covered anteriorly by liver and mesially by intestine, while on the left the intestine practically surrounds it. The mesentery of the colon and part of the colon itself are folded over the top of the left gland and lie partly behind its upper end. Below the liver both glands are in relation mesially with the wall of the

256 AMERICAN ASSOCIATION OF ANATOMISTS

bladder and make impressions especially on that part of the lateral wall of the bladder in which lie the mnbiUcal arteries. Both glands occupy a shallow fossa in the angle between the dorsal and lateral walls of the abdomen. The upper end of the original mesonephric fold can be traced along the outer border of the kidney for a considerable distance and is continuous below with the mesentery of the gland which nms along the outer border of the psoas muscle and then obliquely across it towards the pelvic brim.

61. The establishment of the circulation of cerebrospinal fluid. Lewis

H. Weed, Johns Hopkins University.

Data regarding the establishment of the circulation of cerebrospinal fluid in the embryo may be obtained by replacing the existent fluid in the ventricles and in the central canal of the spinal cord by foreign substances. This replacement may be accomplished by a simple compensatory device which provides for the simultaneous introduction and withdrawal of fluid. As used in this work and found successful; such a mechanism may be constructed of two reservoirs suspended by cord over a pulley, so that as one is raised the other is lowered to an equal degree. These reservoirs are in turn connected by rubber tubing to two needles, held on brackets at the same level. One of these needles is inserted into the central canal of the spinal cord, the other into the mesencephahc or lateral ventricle. The replacement has been done on numerous living pig embryos of various stages; these embryos were then kept aUve for varying periods, up to two hours, by placing them in a 38** incubator.

It was felt that reUable results in any problem involving the possible passage of fluid through a membrane could only be studied by the introduction of a true solution, followed by the subsequent precipitation of the foreign salts in this solution. For this purpose, isotonic solutions of potassium ferrocyanide and iron-ammonium citrate in equal amoimts, were used, as these salts were found non-toxic, not attracted to specific cellular elements and in the strength used, non-coagulants of protein. The fixation of this material in a fluid containing dilute hydrochloric acid caused the formation of Prussian blue which was insoluble in the routine histological technique. For comparison, solutions of silver nitrate and suspensions of carbon granules (india ink) were employed.

With this method of investigation — the replacement of the existent medullary fluid by a true foreign solution without increase in pressure — evidence of a trustworthy character in regard to the course and distribution of the cerebro-spinal fluid may be obtained. The further spread of this replaced fluid must be caused by a normal intraventricular production of the normal fluid.

If in a living pig embryo of 8 mm. an injection (specimens of this size are somewhat too small for replacement) of the ferrocyanide solution be made into the central spinal canal, under mild pressure from a syringe, the distribution of the fluid (as evidenced by the precipitated granules of Prussian blue) , remains strictly intraventricular even

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though the embryo is kept ahve for some time after the injection. In embryos of 12 to 13 mm., replacements of the fluid may be made. At this stage, the results differ from those of the smaller embryos only in the collection of dense granules in an oval region in the superior central portion of the roof of the fourth ventricle. Histologically this aggregation of granules occurs against a rounded differentiated area in the ependymaJ liring of the ventricle.

The first evidence of an extra-ventricular spread of this replaced fluid occurs in pig embryos of over 14 mm. This extension of the solution from the ventricle occurs from the same differentiated area in the rhombic roof, against which the granules collected in the earlier stage. This spread is approximately coincident with the development of tufts in the choroid plexuses of the fourth ventricle. The area of ependymal differentiation, which has been termed the area membranacea superior, comes to lie in the superior half of this roof when the division caused by the laterally developing plexuses occurs.

In 18 mm. pig embryos subjected to similar replacements, the extraventricular spread is by no means extensive; two areas, however, of escape for the ventricular fluid are made out locally in the two divisions of the rhombencephalic roof. The superior of these points of fluid-passage is the same concerned in the primary outflow of the fluid while the inferior is a somewhat similar area of ependymal differentiation in the caudal half. The first microscopic evidence of this inferior area is found in pig embryos of 15 mm.

After this stage is passed, the further extra-ventricular extension of the fluid occurs rapidly. The peribulbar spaces are first filled with the fluid and from this region, a spread downward in the perispinal spaces occurs. Simultaneously with this caudal enlargement of the fluid spaces, the replaced fluid may be traced along the ventral surface of the mesencephalon. In pig embryos of 23 mm., these replacements show a total filling of the ventricular system with the blue and an almost complete surroimding of the cerebro-spinal axis with the granules. The superior portion of the mesencephelon and the cerebral hemispheres are the last parts of the nervous system to show evidence of a pericerebral investment. This final establishment of the adult cerebro-spinal relationship occurs in pig embryos of about 26 mm.

Similar stages in the filling of the spaces about the nervous system may be obtained by simple injections with this true solution into the central canal of the spinal cord under very mild syringe-pressure. The emplojmaent of the ordinarj^ moderate pressure of injection results in similar spreads of the fluid but always in earlier stages than are obtained by the replacement method. Strong syringe-pressure, below those causing obvious ruptures, result in the further decrease in the stage necessary for a given type of spread.

For comparison with these results, injections and replacements of india ink were made. No extraventricular spread of this granular suspension was obtained in any replacement experiment. With the use of syringe-pressures markedly above the normal, the ink could be

258 AMERICAN ASSOCIATION OF ANATOMISTS

forced outward from the roof of the fourth ventricle. The spread, however, was always much less extensive than after replacement with the ferrocyanide in an embryo of the same size.

The use of silver nitrate gave results valuable only for the sake of comparison. The power to coagulate protein apparently caused many artefacts and led to very limited spreads.

The two areas of ependymal differentiation in the two halves of the rhombic roof are both intact menibranes, apparently devised for the passage outward of the ventricular fluid . Both differentiate at a slightly earUer stage than that at which they function actively. In the smaller embryos the superior area membranacea fulfills by far the greater function but after the establishment of the lower point of fluid-passage it undergoes regressive changes. The area membranacea inferior continues to develop and function actively. It gradually forms, with increasing growth of the embryo, a caudal projection beneath the cerebellum. This inferior area in the roof gradually occupies with its differentiated ependyma the whole velum chorioidea inferior.

These results may be interpreted as affording evidence of the establishment of the circulation of cerebrospinal fluid in pig embryos of about 26 mm. The passage of this fluid from the ventricular system into the pericerebral and perispinal spaces occurred only from two localized areas in the two portions of the rhombic roof.

62. Blastolysis as a morphogenetic factor in the development of monsters.

(Lantern.) E. I. Werber, Osborn Zoological Laboratory, Yale

IJniversity.

On a previous occasion I have reported some experimental results of the action of products of pathologic metabolism on the developing egg of FunduVus heteroditus. It was then shown that a very great variety of monsters, similar to human and other mammalian monsters, can be produced with solutions of, particularly, two substances which are known to be present in the blood or urine of individuals suffering from disturbances of carbohydrate metabolism, namely, with butyric acid and acetone.

Although in each experiment a great diversity of morphological results has been obtained by the employment of the same method, it seems reasonable to conclude that at least some factors are common to the morphogenesis of all monsters resulting from a given experiment. A study of many deformed embryos from butyric acid and acetone solutions and analysis of existing morphological relations has, indeed, convinced me that in nearly all malformed embryos of my experiments evidence can be found of a common morphogenetic principle. For, in nearly all of them the action of some forces which tended to dissociate and destroy the germ's substance is apparent.

This action is, however, a 'complex component' in the sense of Roux, as it is probably conditioned by a number of factors present in a varying degree in individual eggs of the same experiment Analysis of these factors will for some time to come be one of the objects of my

PROCEEDINGS 259

investigations. A the present time it is the results of their collective action, to which latter I have appUed the term blastolysis, that I wish to call your attention.

Blastolysis either destroys part or all of the germ's substance, or it may spUt off and disperse parts of the latter. The numerous meroplasts resulting from my experiments and particularly such teratomata as the *soUtary eye' and the 'isolated eye' bear ample witness to this process. The presence on the yolk of a small fragment of nervous tissue with an eye at a great distance from a malformed embryo, or even without an embryo, would, in my opinion, seem to permit of no other interpretation.

While, as we see, some effects of blastolytic action are thus strikingly evident on examination of the eggs or embryos in toto, other effects can be recognized only by a study of sections of them. The latter I regard as very important, for it often reveals conditions which have a si^iificant bearing on the ontomechanics of the vertebrate organism. As we shall presently see, it also sheds Ught on the mode of formation of some terata, which has so far been the object of fruitless speculation.

The following are a few out of a great and constantly increasing number of facts which microscopic examination of malformed embryos may disclose. In the head re^on a lens may be found free, not associated with an eye, or a profusion of small lentoids may be met with, even though eyes may be entirely lacking. The complete absence of the spinal cord (amyelia) and of the notochord was noted in one embryo. That the latter condition is due to blastolytic elimination of the parts of the germ's substance which were eventually to form the spinal cord and notochord and not to an inhibition in the development of these parts would seem to be evidenced by the experience that both the spinal cord and the notochord are usually present in highly deformed embryos. Moreover, the notochord particularly, can not infrequently be found to be partially doubled — a condition indicating the action of a force which tends to dissociate parts of the early embryonic primordiimi (blastolysis).

Similarly, ectopia of some organs which may occasionally be found on microscopic examination of my experimental monsters would also seem to point decidedly to blastolytic action. Thus I have found in one embryo in secticms through the anterior part of the bead a tubelike structiu'e which made the impression of a fragment of the intestine, while in other embryos a rudimentary eye anl^e (optic vesicle) has been found on the ventral side of the posterior part of the brain, at, or beyond the level of the ear vesicles. Such findings indicate unmistEj^ably that dissociated parts of the embryonic primordium sometimes become dislocated and are apparently capable of independent further development and differentiation in their new surroundings.

A beautiful illustration of such blastolytic dispersion of minute parts of the embryonic primordium is presented also by some monstra anophthalmica asymmetrica, on sections of which I was able to find posterior to the single lateral eye and ventrally from the brain a very

260 AMERICAN ASSOCIATION OF ANATOMISTS

small fully differentiated fragment of an optic cup. In the latter the chorioid coat and aU layers of the retina including the rods and coneslayer are at the same stage of differentiation as these parts of the single eye. The bearing of these facts is evident. For they show us the nature of the formative cause imderlying the condition of single-eyedness. Thus in the case of lateral single-eyedness the potential optic anlage has been ekninated by blastolytic destruction, an event which occasionally may be betrayed by a dislocated optic cup fragment, the only remnant of the anlage of the lacking eye.

In the light of our theory of blastolysis, supported by these and many other facts, the ierata of the eye, such as cyclopia, synophthalmia and monophthalinia as3mametrica mitst, as Spemann has rightly pointed out, be regarded as due to a defect and not to an inhibition as has been claimed by others and notably by Dareste and by Stockard. This view is further strengthened by the condition of the brain of teratophthalmic embryos. In asymmetrically monophthalmic embryos the brain is on the side, which lacks the eye, usually distorted in its relation to the chief body axis and sometimes a dorsol-ateral part of it on the eyeless side is seen to be lacking, just as if it had been mechanically removed.

In S3aiophthalmic and some cyclopean embryos I have always found the forebrain to be exceedingly small and imilobed, while the rest of the brain may often be apparently normal. Here, evidently, the blastolytic defect involved the potential interocular area and parts of the potential ophthalmic anlagen.

The fused olfactory pits and the proboscis-shaped mouth of such embryos (the latter due to approximation and partial fusion of the potential maidllary and mandibular arches) furnish additional evidence for the correctness of the view according to which cyclopia or synophthalmia is due to a defect.

Some experiments with acetone in which solutions of low concentrations and long exposures have been employed, have yielded results which furnish further evidence for the blastolytic defect imderljdng the formation of ophthalmic terata. While some of the eggs so £reated have given rise to synophthamic or monophthalmic monsters, many of them have developed into embryos with two large, greatly protruding eyes. Examination of sections shows that these eyes are elongate and ovoid in shape, and it is difficult to escape the impression that during their development some forces must have been acting which tended to disrupt or disintegrate them (blastolysis). The blastolytic defect, although of a moderate degree in these cases, is strikingly exhibited by the small and wanting forebrain. Owing to this defect the eyes appear in posterior sections to be approximated. No fusion of the eyes has, however, occurred, for the part of the potential interocular area eliminated by blastolysis has not been large enough to cause a fusion of the potential eye anlgen. It could not be imagined that this approximation of the eyes is due to an inhibition which prevented the brain from pushing out the eye vesicles forwards and lateralwards, for these eyes are

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in the usual lateral position in the head, from which they so grotesquely protrude. They certainly have been pushed out by the brain, and even more so than is normally the case. Their approximation is secondary and is solely due to blastolytic elimination of an intermediate part.

As we see, the morphogenesis of some deformities, like those of the eyes and of other terata &ads a simple and rational explanation, if the principle of blastolyds is consistently applied in their analysis. More data in support of the correctness of this analysis and a full treatment of the subject will be found in forthcoming papers.

63. Teratogenesis of a human aihorax, acephalic, acardiac triplet with numerous ageneses. Ten figures: Macroscopic,* Microscopic, Photomicroscopic and Skiographic. H. O. WnriE, Anatomical Laboratory of the College of Physicians and Surgeons, Medical Department of the University of Southern California. ' Absence or maldevelopment of head.

Possible factors contributing or entirely responsible for the causation of such phenomena in the very early period of development. Opinions entertained at present and possible errors of such opinions. Suggestions ventured, based on our present knowledge of embryology.

Functional hyperactivity, compensatory hypertrophy, or primary developmental hyperplasia due to disturbance of normal co-ordination or possibly an intrauterine * Developmental Neurosis,' as possible causes of agenesia of two or more like parts or organs. Possible embryologic causes of aplasia.

Imperfect development not the cause of abnormal circulatory conditions.

Acardiaci may be caused by primary development defects, or secondary degenerations, or faulty development of one-half of a twin blastoderm, or imperfect or arrested segmentation.

Probable causes of total absence of internal genitals and presence of external in the same foetus.

BIBLIOGRAPHY

Gbogono, W. a. 1894-95 Acephalous acardiac foetus. Transactions Obst.

Soc. London, vol. xxxvi, p. 65. Kerb, J. L. 1896 Acephalous infants. Lancet, London, vol. ii, p. 380. RuDiNGER 1874 Ueber die willkurlichen Verunstaltungen des menschlichen

Korpers. Berlin. Med. Anz., May 28. Keith, W. H. 1900 The anatomy and nature of two acardiac acephalic foetuses.

Ibid., vol. xlii, p. 99. Leboucq, H. 1907 Description anatomique d'un acardiaque humain para cephalien. Ann. Soc. de Med. de Gand, vol. Iv, p. 39. LowRY, M. 1892 Ueber einen Fall von Acardiacus anceps. Prag. Med. Woch enschrift, vol. xvii, p. 157.

THB ANATOMICAL BBCORD, VOL. 10, NO. 3

262 AMERICAN ASSOCIATION OF ANATOMISTS

Ballanttne, J. W. 1892-93. Description of a foetus paracephalus dipiu

acardiacus. Edin. Med. Jahresb., vol. xxxviii, p. 842. Ballanttne,. J. W. 1895-96 Teratogenesis : An inquiry into the causes of

monstrosities. Transactions Edinburgh Obstetrical Soc, vol. xzi,

pp. 12; 220; 258. Spee. 1898 Referat liber Acardiacus und dessen GenesS. Schwalbes Jahres bericht, p. 227. Claudius. 1882 Die Entwicklung der herzlosen Misgel>urten. Wiener Med.

Jahresbericht. Hill. 1896 Bulletin of the Johns Hopkins Hospital, nos, 181 and 185. WnsscH. 1907 Wiener Med. Wochenschrift, vol. Ixxvii, p. 674. Bbnda and Nacke 1907 Zeutralblat fUr G3m&k, p. 468. Bretschneider. 1893 Drillinge und Acardiacus. Geb. Gesellsch. Leipzig.

Feb. 16. Bretschneider. 1903 Zeutralblat fur Gynftkologie, p. 668. Hauck. 1899 Ein Acardius, Aerzt. Prax. WQrzburg, Bd. xii, p. 246. Sltman, W. D. 1890 An acephalous acardiac monster of 6 month's gestation

with rudimentary heart. Transactions Obs. Soc. London, vol. xxxi, p.

258.

The copy of these Abstracts came in too late to have them placed in their alphabetical order.

64. The longitudinal musde in the colon of the pig embryo, P. E. Lineback, Harvard Medical School.

For piirposes of description the colon of the pig may be divided into two portions: first, that part from the valve of the colon to the right or hepatic flexure, and second, the part from the hepatic flexure to the anus. Early in growth the first part develops a sharp *U '-shaped coil not imUke a cochlea, making three and a half revolutions. In the 165 mm. embryo it has assumed its permanent shape.

In a pig embryo of 32 mm. there is no longitudinal muscle in the large intestine, although in the small intestine the corresponding layer is well defined.

At the 39 mm. stage the longitudinal muscle has made its appearance in the rectal region, but is not seen anywhere in the upper portion of the colon. It begms just inside the anal opening as two small bundles of fibers, flattened dorso-ventraUy, located in the median line, one dorsal and the other ventral to the tube. These layers spread laterally as they are followed upward and come together at the sides of the tube about half a millimeter from the point of origin. Above this level the layer of longitudinal muscle fibers completely surrounds the intestine and extends upward about 2 mm.

In a 60 mm. pig the dense and sharply defined muscle in the lower end of the rectum has grown upward only a short distance further, but a complete circle of longitudinal fibers can now be traced downward from the ilio-caecal junction. This upper portion is loosely formed, and is evidently rapid in the downward growth, already extending several millimeters from the valve of the colon.

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In an 80 mm. pig the downward growth hbs passed through the spiral, across the transverse colon, and a third of the way down the descending colon where it will be met by the upward growth.

At the 140 mm. stage of the longitudinal muscle is a well defined layer of fibers, completely surrounding the tobe and extending throughout its entire length.

65, A cytological study of connective tissue cells of animals stained vitally with acid azo dyes, Katherine J. Scott, Department of Anatomy, University of California. (Presented by H. M. Evans.) Goldmann's (1) description of Tyrrol-Zelle' and his conclusions that the staining of these cells results from the capacity of specific cell organs to react to the dye have tended to strengthen the belief of many workers that a true vital staining of cytoplasmic elments is possible. Whether or not this is due to a chemical imion, as Ehrlich (2) holds, or is a physical phenomenon could not be decided from the evidence presented here. But careful cytological studies should enable us to determine whether special cytoplasmic elements are affected by these dyes. This can be settled in the case of the mitochondria. Tschaschin (3) in 1912 and later, reports a staining of the true mitochondrial elements in the fibroblasts and clasmatocytes by isamine blue and trypan blue. This observation is in contradistinction to the theory that the vital dye granules represent merely deposits of the dye, as stated by Evans and Schulemann (4).

Without going into the more theoretical aspects of the interrelations between all possible granular structures and mitochondria in these cells, we have attempted a cytological study of the vitally stained elements using the familiar methods for the identification of mitochondria.

Observations on the resting cells stained with janus green, as well as study of the reaction of these cells to varjring amounts of dye and pictures during the processes of ingestion and egestion of the dye show that the respective identity of dye granules and mitochondrium is always maintained. The dye granules imdergo definite changes in accordance with the functional state of the cell. Such changes in functional state may also affect the mito-chondria, as for example, in the more rounded forms met with in cell degeneration in overloading with the dye. But under no conditions are the mitochondria of connective tissue cells stained vitally with acid azo dyes.

(1) GoLDMANN, E. E. 1909, 1912 '*Due fiussere und innere Selarction des

gesunden und kranken Organismus im Lichte der *vitalen Furbung.'^ Ttibingen; Uene Untersuchungen u.s.w., "Tubingen."

(2) Ehrlich, P. 1913 'Chemio-therapy.' Address in Pathology, 7th Inter national Congress of Medicine, British Medical Journal, August.

(3) TocHASCHiN, S. 1912, 1913 Folia Haematologica, Bd. 14, s. 295; Bd. 16, s.

247; Bd. 17, ss. 17.

(4) Evans and Schulemann 1913, 1914 Jahresb. d. Sch. Ges. f. vat. Kul.;

Science, vol. 34, f. 443; Deut. med. Wochenschr., vol. 13.

264 AMERICAN ASSOCIATION OF ANATOMISTS

66. On the behavior of the ovary and especially of the atretic foUide towards

vital stains of the aao group. Herbert M. Evans, University of

California.

The nonnal Graffian follicle of the ovary constitutes, as it were, an individual physico-chemical system for the liquor folliculi contains a higher concentration of vital dye than the tissue juices elsewhere in the ovary. This fact is peculiarly fortunate in so far as it enables us to detect the behavior towards the stain on the part of any and all of th6 cells concerned in the growth and atresia of the follice. Thus beginning degeneration of the granulosa cells is often marked by an abrupt change ia their reaction towards the dye. Normally resistant to the entry of the dye, the degenerating cells may house large 'droplets' of the vital color so that the whole follicular epithehiun is IfSen with it. This reaction, however,, is to be separated sharply from the so-called 'macrophage' reaction, inasmuch as the granulosa cells which behave in this way show chromatolysis and other degenerative changes and are short-Uved.

True macrophages play an important r61e in follicular atresia and they are electively stauued by vital dyes of this class.^ The later stages of follicular atresia are consequently marked by brilliant deposits of the vital stain due to the intense dye granules in the macrophages concerned here. The macrophages must be considered as utilizing the products of the disintegrating ovum, towards which they are attracted and which they invade by an active penetration of the zona peUucida. These are the cells first seen in this act by Pfltiger and by Lindgren.

^ The Macrophages of Mammals, Amer. Jour. Physiology, Vol. 37, No. 2, May, 1915.

DEMONSTRATIONS

/. Cell changes in the hypophysis of the albino rat after gonadedomy. W. H. F. Addison, University of Pennsylvania.

2, The origin and structure of a fibrous tissue formed in wound healing. George A. Baitsell (introduced by R. G. Harrison), Yale University.

5. a, A brain of a hyper-ontomorph and a brain of a meso-ontomorph. bj X-ray photograph of the abdomen of a hyper-ontomorph and of a mesoontomorph. Robert Bennett Bean, Tulane University of Louisiana.

4. Topographical study of the IS mm. chick embryo, Edward A. Boyden, Harvard Medical School, Boston, Mass.

5. A simple type of ether thermostat for the regulation of temperature in an electric oven. H. Saxton Burr, Anatomical Laboratory of the Yale University School of Medicine.

6. Loose connective tissue as seat of lympho-granulopoesis. Wera Danchakoff (introduced by W. H. Lewis), Rockefeller Institute, New York City.

7. The effects of light on the retina of the turtle and of the lizard. Samuel R. Detwiler (introduced by R. G. Harrison), Osborn Zoological Laboratory, Yale University.

8. Demonstration of an unu^sual shape assumed by human erythrocytes. V. E. Emmel, Washington University Medical School.

9. An anomalous origin of the obturator artery below the inguinal ligament. V. E. Emmel and P. L. Schroeder, Washington University Medical School.

10. Artificial daylight for the microscope. Simon Henry Gage, Cornell University, Ithaca, N. Y. The demonstration is to be so arranged that each observer can see the same specimen with unmodified artificial Ught, with artificial dayUght and with true dayUght.

11. Obliterated vessels in the thymus, a possible source of thymic corpuscles. J. F. Gudernatsch, Cornell Medical College, New York City.

1£. On the reversal of laterality in the limbs of amblystoma embryos. Ross G. Harrison, Osborn Zoological Laboratory, Yale University.

265

266 AMERICAN ASSOCIATION OP ANATOMISTS

IS, Demonstration of dissectionSy cleared specimens showing the injected vascular system, and stereoscopic photographs made from dissections and injections, of albino rat embryos, Chester H. Heuser, The Wistar Institute, Philadelphia.

The dissections were made of embryos which had been stained in diluted alum cochineal. In a properly stained embryo it is possible to focus a strong light upon the specimen and make dissections of very small structures under the binocular microscope. For the most careful work it is also essential to have the specimen firmly held in position; the embryo can be removed from 80 per cent alcohol, quickly rinsed in absolute and attached to a small piece of ground glass with celloidin. Embryos which are too small to be cut free hand can be split quite accm-ately through the sagittal plane by the following procedure. The piece of glass holding the embryo is moimted in the sUding microtome. The knife is replaced by two glass strips which extend one on either side of the embryo. The specimen is then oriented and raised between the glass strips so that the plane for sectioning coincides with the upp>er surface of the strips. The embryo is then covered with a few drops of celloidin. After the latter has been hardened by 80 per cent alcohol, which is allowed to drop on for a few minutes, the sectioning is done with a safety razor blade which is manipulated with the two index fingers and pushed in a see-saw movement over the glass strips. Since the embryo is oot infiltrated with celloidin the cutting can not be done with one sweeping stroke of the knife. If it is desirable not to have all organs cut in the sagittal plane, a para-sagittal section can first be made ; the cut surface covered with a thin layer of celloidin; and subsequent dissection done in a dish of alcohol under the microscope, using a very fine scalpel.

The study of injected embryos can be greatly faciUtated by varying the method of illumination. Transmitted Ught is no doubt best for the majority of vessels. Strong Ught focused upon the specimen makes it possible to see surface vessels better, at the same time bringing out the surface markings to better advantage. A concentrated beam of light entering the specimen from the side also produces an interesting picture.

14' A demonstration of the technique of cultures of chick tissues will he made at any time (by appointment) during or after the meeting, E. R. HosKiNS, Zoology Department, Yale University (Room 221, Osbom Zoological Laboratories).

15, Teased preparations showing the different types of renal tubules of birds (Chicken). G. Carl Huber, University of Michigan.

16. Teased preparations of the seminiferous tubules of birds (Chicken). G. Carl Huber. In mammals the seminiferous tubules occur in the form of arches or systems of arches all the ends terminating in the rete testis by means of tubuU recti. No anastomoses or blind

PROCEEDINGS 267

ends are present. In the bird the seminiferous tubules branch and anastomose freely, to such extent that it has been foimd impossible to delimit definite tubules; the embryonic rete-cord net being maintained in the fimctional gland. The later disappearance of the retecord net in phylogeny may argue for a late disappearance of this net in mammalian ontogeny.

17. Teasedpreparations showing single, striated, voluntary miLsde fibres. G. Carl Huber. Many of the volimtary fibres are of fusiform shape with fine attenuated ends.

18. An early human embryo. N. W. Ingalls, School of Medicine, Western Reserve University.

19. Specimens showing effect of inanition upon the structure of the thyroid gland. C. M. Jackson, University of Minnesota, Minneapolis, Minn.

iO. Models of embryonic brains showing the relaiions of the neuromeres to the cerebral nerves. Franklin P. Johnson, Department of Anatomy, University of Missouri.

21. Microscope preparations showing iniercalaXed discs in Limvlus heart musde. H. E. Jordan, University of Virginia.

£2. The prolonged gestation period in nursing mice.

25. The germ cell cycle in the mouse. W. B. Kirkham (introduced by R. G. Harrison), Osborn Zoological Laboratory, Yale University.

24. A quick method for preparing sections of dried bone. Frederic P. Lord, Dartmouth Medical School.

26. Tissue culture preparations. C. C. Macklin, Department of Anatomy, Johns Hopkins Medical School. Preparations will illustrate binucleate cells and amitotic nuclear division leading to their formation; also mitosis occurring in amitotic and double nuclei.

26. Models showing the development of the atrial septum and the sinus septum in pig embryos. C. V. Morrill, Cornell Medical College, New York City.

27. Artificial parthenogenesis in Cumingia. Margaret Morris (introduced by R. G. Harrison), Osborn Zoological Laboratory, Yale University.

28. Mounted preparations iUustraiing the growth of the mammary gland in the albino rat. J. A. Myers (presented by C. M. Jackson), University of Minnesota.

268 AMERICAN ASSOCIATION OF ANATOMISTS

29. Some phases of cell mechanics. Theophilus S. Painter (introduced by R. G. Harrison), Osbom Zoological Laboratory, Yale University.

80. The musde of Breschet in birds — a possible forerunner of the tensor tympani in mammals. A. G. Pohlman, St. Louis University. Breschet (1836) describes a muscle or muscle rudiment in the form of a fibrous string running from the tip of the extracolumella over the drum and down the Eustachian tube. This ligament was re-discovered by Smith in 1904 and was described as an elastic Ugament at the last meeting. Smith held the Ugament to be inconstant because hefoimd it in Gallus but not in Columba. Breschet describes it as constant. The writer agrees with the latter writer. It is wanting in Columba. The dissected specimens show the relations as they obtain in the pigeon, chicken, turkey, duck and goose. In the latter two mentioned, particularly in the last named, a well developed bundle of involuntary muscle fibres is added which may be followed down to the common opening of the tubes in a pad which I choose to call the Eustachian cushion. The cushion is made up largely of elastic fibres The presence of an involimtary muscle bimdle in the ear of some birds and its close relation to the position of the Tensor tympani in mammals is at once suggestive of two things: 1) that the path of the Breschet muscle may be followed in the migration of the slip of the pterygoid into the middle ear region; and 2) that we may account in part for the position of the autonomic otic gangUon situated on the motor branch to the Tensor tympani muscle in a more satisfactory manner. The latter ganglion is far more compUcated in its structure than usual text book description would indicate, (see sections of baby's head through this region).

31. Simple method of exposing the relations in the middle ear region.

A. G. Pohlman, St Louis University.

The method is as simple as it is compUcated to describe and therefore lends itself best to demonstration. The usual maUet and chisel are used and advantage taken of the cleavage planes in the human temporal bone. The gross relations of the structures in the middle ear may be exposed by the average student in about half an hour and but ten or fifteen minutes are required in experienced hands. The methods has been used for a number of years and will be glad to show how it is done to those interested in the dissecting room.

52. Differeniiaiion of endothelium and rhythmical cell division from studies • on the living blastoderm of the chick. Florence R. Sarin, Department of Anatomy, Johns Hopkins Medical School, Baltimore.

53. Photographs and microscope slides demonstrating the nerve cell changes caused by pure muscular exertion. Tamao Saito (Tokio), Phipps Psychiatric Clinic, Johns Hopkins Hospital, Baltimore, Md. (Introduced by E. V. Cowdry.)

PROCEEDINGS 269

On the right hand row of the pictures the brain (pyramidal cells of the fifth layer from the second anterior one-fifth portion of the convolution), the cerebellum (hemisphaere), the spinal cord (cells of the lateral group of the anterior horn of cervical portion) and the spinal gangUon (cells of foiulh Imnbal gangUon) of an experimented rat and on the left hand row the corresponding tissues from a not working animal are shown. Each section is taken in 45 and 1000 times magnifications. (Technic: alcohol fixation and carbol-thionin stain.)

The animal was put in a large basin containing water of 38°C. temperature 5 to 7 J hours every day for 7 successive days and then was set on a suspended glass rod for the rest of the time. Of the 149^ hours of the experiment he swam 50 hours and was set on the glass rod 93 hours, the remaining 6 J hours being time for feeding.

Two pairs of the microscope slides show cells of the lateral group of the lumbal cord of the experimented rats and of the not working rat, each pair being stained with the carbol-thionin resp. Cajal's method.

S4a. Drawings from dissections and reconstructions illustrating the anatomy of the nasofrontal region.

SJfb. Photographs of new laboratory apparatus and equipment. J. Pabsons ScHAEFFER, Daniel Baugh Institute of Anatomy and Biology of the Jefferson Medical College.

S6, Accessory nerve-endings in striated musde of Rana. H. D. Senior, New York University Medical College.

56. Model of the human lungs, trachaea, dorsal wall of pericardium, etc. B. Spector (introduced by H. D. Senior), New York University Medical College.

57. Cells of the yolk-sac and blood cells in fish embryos. Charles IL Stockard, Cornell Medical College, New York City.

58. Sections and two models to illustrate the development of the perilymphatic spaces in the internal ear of the human embryo. George L. Streeter, Carnegie Institution, Research in Embryology, Baltimore.

59. Specimens showing variations in the mitochondrial content of the cells of the nuclei of the cranial nerves. M. DeG. Thurlow (introduced by E. V. Cowdry), Anatomical Laboratory, Johns Hopkins University.

40. The position and relations of the sex gland in early human embryos. John Warren, Harvard Medical School.

4t. Drawings of Fundulus monsters in toto and microscopic perparations showing evidence of blastolysis as a morphogenetic factor in the development of monsters. E. I. Werber, Osbom Zoological Laboratory, Yale University.

4£. Studies on cell lamivaiion in the cortex of the sheep. Charles Baglet, Jr. (introduced by E. V. Cowdry), Neurological Laboratory, Phipps Psychiatric CUnic, Johns Hopkins Hospital. AMERICAN ASSOCIATION OF ANATOMISTS

OFFICERS AND LIST OF MEMBERS

Officers

President Hbnrt H. Donaldson

Vice-President Clabbncb M. Jackson

Secretary-Treasurer Charles R. Stockard

ExectUive Committee

For term expiring 1916 Arthur W. Meyer, Charles F. W. McClure

For term expiring 1917 Warren H. Lewis, C. Judson Hbrrick

For term expiring 1918 Hermann von W. Schulte, John L. BRBSiBR

For term expiring 1919 Euot R. Clark, Reuben M. Strong

Committee on International Congress F. P. Mall and G. S. Huntington

Honorary Members

S. Ram6n y Caj al Madrid, Spain

John Cleland Glasgow, Scotland

Camillo Golgi Pavia, Italy

Oscar Hertwig Berlin, Germany

Alexander Macalister Cambridge, England

A. Nicolas Paris, France ,

L. Ranvibr Paris, France

GusTAV Retzius Stockholm, Sweden

Wilhelm Roux Halle, Germany

Carl Toldt : Vienna, Austria

Sir William Turner Edinburgh, Scotland

Wilhelm Waldeyer .Berlin, Germany

Members

Addison, William Henry Fitzgerald, B.A., M.B., Assistant Professor of Normal

Histology and Embryology, University of Pennsylvania, S982 Pine Street,

Philadelphia, Pa. Allen, Bennet Mills, Ph.D., Professor of Zoology, University of Kansas, 1S29

Ohio Street, Lawrence, Kans. ■ Allen, William F., A.M., Ph.D., Instructor of Histology and Embryology,

Institute of AncUomy, University of Minnesota, Minneapolis, Minn, Alus, Edward Phelps, Jr., LL.D., Palais de Camoles, Mentone, France.

271

272 AMERICAN ASSOCIATION OF ANATOMISTS

Arey, Leslie B., Ph.D., Instructor in Anatomy, Northwestern University Medical School, 24M1 Dearborn Street^ Chicago^ III, Atwbll, Wayne Jason, A.B., Instructor in Histology, 1SS5 Geddes Avenue^

Ann Arbor J Michigan. Baqley, Jr., Charles, M.D., Phipps Institute, Johns Hopkins Hospital, Baltimore, Md, Baitsell, George Alfred, Ph.D., Instructor in Biology, Yale University, New

Haven, Conn, Baker, Frank, A.M., M.D., Ph.D. (Vice-Pres. '88-'91, Pres. '96^W), Professor

of Anatomy, Medical Department, University of Georgetown, 1901 Biltmore

Street, Washington, D. C. Baldwin, Wesley Manning, A.B., M.D., Professor of Anatomy, Albany Medical

College, Albany, N. Y. Bardeen, Charles Russell, A.B., M.D. (Ex. Com. '06-'09), Professor of Anatomy and Dean of Medical School, University of Wisconsin, Science Hall,

Madison, Wis. Badertscher, Jacob A., Ph.M., Ph.D., Assistant Professor of Anatomy,

Indiana University School of Medicine, 417 N. Lincoln Street, Bloomington,

Ind. Bartelmetz, George W., Ph.D., Assistant Professor of Anatomy, University

of Chicago, Chicago, III, Bates, George Andrew, M.S., Professor of Histology and Embryology, Tufts

College Medical School, 906 Central St., Avbumdale, Mass, Baumgartnbr, Edwin A., Ph.D., Instructor in Anatomy, Washington University

Medical School, St. Louis, Mo. Baumgartner, William J., A.M., Assistant Professor of Histology and Zodlogy,

University of Kansas, Lawrence, Kans, Bayon, Henry, B.A., M.D., Associate Professor of Anatomy, Tulane University,

2B12 Napoleon Avenue, New Orleans, La, Bean, Robert Bennett, B.S., M.D., Professor of Gross Anatomy, Tulane

University of Louisiana, Station SO, New Orleans^ La, Begg, Alexander S., M.D., Instructor in Comparative Anatomy, Harvard

Medical School, Boston, Mass, Bens LEY, Robert Russell, A.B., M.B. (Second Vice-Pres. '06-'07, Ex. Com.

'08-*12), Professor of Anatomy, University of Chicago, Chicago, III. Bevan, Arthur Dean, M.D. (Ex. Com. '96-*98), Professor of Surgery, University

of Chicago, 2917 Michigan Avenue, Chicago, III, Bigelow, Robert P., Ph.D., Assistant Professor of Zoology and Parasitology,

Massachusetts Institute of Technology, Bostori, Mass, Black, Davidson, B.A., M.B., Assistant Professor of Anatomy, Western Reserve

University, Medical Department, 1S5S East 9th Street, Cleveland, Ohio, Blair, Vilray Papin, A.M., M.D., Clinical Professor of Surgery, Medical Department, Washington University, 400 Metropolitan Building, St, Louis, Mo. Blaisdell, Frank Ellsworth, M.D., Assistant Professor of Surgery, Medical

Department of Stanford University, 16S0 Lake Street, San Francisco, Calif. Blake, Joseph Augustus, A.B., M.D., 40 Ave, Henri Martin, Paris, France. Bonne y, Charles W., A.B., M.D., Demonstrator in Anatomy, Jefferson Medical

College, Philadelphia, Pa.

MEMBERS 273

BoTDBN, Edward Allen, A.B., A.M., Teaching fellow, Histology and Embryology, Harvard Medical School, BoBton, Mass. Bremeb, John Lewis, M.D. (Ex. Com. '15^), Associate Professor of Histology,

Harvard Medical School, Boston, Mass. Bboadnax, John W., Ph.G., M.D., Associate Professor of Anatomy, Medical

College of Virginia, Richmond, Va. Brookoyer, Charles, Ph.D., Professor of Anatomy, University of Arkansas,

Little Rock, Arkansas. Brooks, William Allen, A.M., M.D., 167 Beacon Street, Boston, Mass. ^ Brown, A. J., A.B., M.D., Instructor in Anatomy, Colimibia University, 166 East

64th Street, New York, N, Y. Browning, William, Ph.D., M.D., Professor of Nervous and Mental Diseases,

Long Island College Hospital, 54 Lefferts Place, Brooklyn, N. Y. Brtce, Thomas H., M.A., M.D., Regius Professor of Anatomy, University of

Glasgow, No. 2, The University, Glasgow, Scotland. BiTLLARD, H. Hats, AM., Ph.D., Johns Hopkins Medical School, Baltimore, Md. Bunting, Charles Henrt, B.S., M.D., Professor of Pathology, University of

Wiscormn, £0B0 Chadhoume Avenue, Madison, Wis. Burr, Harold Saxton, Ph.B., Ph.D., Instructor in Anatomy, School of Medicine, Yale University, New Haven, Conn. Burrows, Montrose T., A.B., M.D., Acting Resident Pathologist, Johns Hopkins

Hospital, Baltimore, Md. Campbell, William Francis, A.B., M.D., Professor of Anatomy and Histology,

Long Island College Hospital, S94 Clinton Avenue, Brooklyn, N. Y. Carpenter, Frederick Walton, Ph.D., Professor of Zodlogy, Trinity College,

Hartford, Conn. Casamajor, Louis, B.A., M.D., Assistant Professor of Neurology, Colimibia

University, 4S7 West 59th Street, New York City, Chambers, Robert, Jr., A.M., Ph.D., Assistant in Anatomy, Cornell University

Medical College, New Yxyrk City. Chbevbr, David, A.B., M.D., Assistant Professor of Surgical Anatomy, Harvard

Medical School, BO Hereford Street, Boston, Mass. Chidester, Floyd E., A.M., Ph.D., Associate Professor of Zoology, Rutgers

College, New Brunswick, N.J, Child, Charles Manning, Ph.D., Associate Professor of Zo5logy, University of

Chicago, Chicago, III, Chillingworth, Felix P., M.D., Assistant Professor of Physiology and Pharmacology, Tulane University, New Orleans, La. Clapp, Cornelia Maria, Ph.D., Professor of Zoology, Mount Holyoke College,

South Hadley, Mass. Clark, Elbert, B-.S., Instructor in Anatomy, University of Chicago, Chicago,

ni.

Clark, Eleanor Linton, A.M., Research Worker, Department of Anatomy,

University of Missouri, Columbia ^ Mo. Clark, Eliot R., A.B., M.D., Professor of Anatomy, University of Missouri,

406 S. 9th Street, Columbia, Mo. Cob, Wesley R., Ph.D., Professor of Biology, Sheffield Scientific School, Yale

University, New Haven, Conn.

274 AMERICAN ASSOCIATION OF ANATOMISTS

CoGHiLL, George E., Ph.D., Associate Professor of Anatomy, University of Kansas Medical School, SS8 Illinois Street y Lawrence , Kane,

CoHN, Alfred E., M.D., Associate in Medicine, Rockefeller Institute for Medical . Research, SI 6 Central Park West, New York, N. Y.

Cohoe, Benson A., A.B., M.B., Associate Professor of Therapeutics, University of Pittsburgh, 705 North Highland Aventie, PiUsburgh, Pa.

CoNANT, William Mbrritt, M.D., Professor of Clinical Surgery, Tufts Medical School, 486 Commonwealth Avenue, Boston, Mass.

GoNGDON, Edgar Davidson, Ph.D., Assistant Professor of Anatomy, Leland Stanford University, School of Medicine, SSO Coleridge Avenue, Palo Alto, Calif.

Gonklin, Edwin Grant, A.M., Ph.D., Sc.D., Professor of Biology, Princeton University, 1S9 Broadmead Avenue, Princeton, N.J.

Gorner, George W., A.B., M.D., Assistant Professor of Anatomy, Anatomical Laboratory, Berkeley, Calif.

Gorning, H. K., M.D., Professor of Anatomy, BUndesstr. 17, Basel, Switzerland.

GoRSON, EixGENE RoLLiN, B.S., M.D., Surgeou, Lecturer on Anatomy, Savannah Hospital Training School for Nurses, 10 Jones Street, West, Savannah, Gn.

GowDRY, Edmund V., Ph.D., Associate in Anatomy, Anatomical Laboratory, Johns Hopkins Medical School, Baltimore, Md.

Graig, Joseph David, A.M., M.D., IB Ten Broeck Street, Albany, N. Y.

Grile, George W., A.M., M.D., Professor of Surgery, Western Reserve University, 22B Osborn Bldg., Cleveland, 0.

Gullen, Thomas S., M.D., BO E. Eager Street, Baltimore, Md.

Gunningham, Robert S., B.S., A.M., Johns Hopkins Medical School, 716 N. Broadway, Baltimore, Md.

GuRTis, George M., A.B., A.M., Acting Professor of Anatomy, Medical Department of the Vanderbilt University, 1047 South Srd Avenue, Nashville, Tenn.

Dahlgren, Ulric, A.B., M.S., Professor of Biology, Princeton University, B04 Guyot Hall, Princeton, N.J.

Danchakofp, Wbra, M.D., Rockefeller Institute, New York City.

Danforth, Gharles Haskell, A.M., Ph.D., Associate in Anatomy, Medical Department, Washir^ton University Medical School, St. Louis, Mo.

Darrach, William, A.M., M.D., Assistant Attending Surgeon, Presbyterian Hospital, Instructor in Glinical Surgery, Golumbia University, 47 West 60th Street, New York, N. Y.

Davis, David M., B.S., Johr^ Hopkins Medical School, Baltimore, Md.

Davjs, Hbnrt K., A.B., A.M., Instructor in Anatomy, Cornell University Medical College, Ithaca, N. Y. •

Dban, Bashford, Ph.D., Professor of Vertebrate Zodlogy, Golumbia University, Gurator of Fishes and Reptiles, American Museum Natural History, River^ dale-on-Hudson, New York.

Dbtwiler, Samuel Randall, Ph.D., Assistant in Biology, Yale University, New Haven, Conn.

Dexter, Franklin, M.D., £47 Marlborough Street, Boston, Mass.

Dixon, A. Francis, M.B., Sc.D., University Professor of Anatomy, Trinity GoUege, 7S Grosvenor Road, Dublin, Ireland.

MEBiBERS 275

DoDSON, John Milton, A.M., M.D., Dean and Professor of Medicine, Rush Medical College, University of Chicago, 6806 Blackston Avenue , Chicago , III.

DoLLEY, D. H., M.D., Professor of Pathology, University of Missouri^ Columhiaf Mo.

Donaldson, Henry Herbert, Ph.D., D.Sc, (Ex. Com. '09 -'13), Professor of Neurology, The Wistar Institute of Anatomy and Biology ^ Woodland Avenue and S6th Street,. Philadelphia, Pa.

Downey, Hal, M.A., Ph.D., Associate Professor of Histology, Department of Animal Biology, University of Minnesota, Minneapolis, Minn.

DuESBERG, Jules, M.D., Research Associate, Carnegie Institution of Washington, Johns Hopkins Medical School, Baltimore, Md.

Dunn, Elizabeth Hopkins, AM., M.D., Marine Biological Laboratory, Woods Hole, Mass.

Eccles, Robert G., M.D., Phar.D., 681 Tenth Street, Brooklyn, N. Y,

Edwards, Charles Lincoln, Ph.D., Director of Nature Study, Los Angeles City Schools, lOS^ West 99th Place, Los Angeles, Calif.

Eggbrth, Arnold Henry, Assistant in Histology and Embryology, University of Michigan, 1S24 Volland Avenue, Ann Arbor, Michigan.

Elliot, Gilbert M., A.M., M.D., Assistant Professor and Demonstrator of Anatomy, Medical School of Maine, 162 Maine Street, Brunswick, Me.

EiocEL, Victor E., M.S., Ph.D., Assistant Professor of Anatomy, University of Illinois College of Medicine, Congress and Honore Streets, Chicago, III.

EssicK, Charles Rhein, B.A., M.D., 1807 North Caroline Street, Baltimore, Md.

Evans, Herbert McLean, B.S., M.D., Professor of Anatomy, University of California, Berkeley, Calif.

EvATT, Evelyn John, B.S., M.B., Professor of Anatomy, Royal College of Surgeons, Dublin, Ireland.

Eycleshymer, Albert Chauncey, Ph.D., M.D., Professor of Anatomy, Medical Department, University of Illinois, Honore and Congress Streets, Chicago, III.

Ferris, Harry Burr, A.B., M.D., Hujtit Professor of Anatomy and Head of the Department of Anatomy, Medical Department, Yale University, 5P5 St. Ronan Street, New Haven, Conn.

Fbttbrolp, George, A.B., M.D., Sc.D., Assistant Professor of Anatomy, University of Pennsylvania, SSO South 16th Street, Philadelphia, Pa.

FiscHELis, Philip, M.D., Associate Professor of Hilatology and Embryology, Medico-Chirurgical College, 828 North 6th Street, Philadelphia, Pa.

Flint, Joseph Marshall, B.S., A.M., M.D. (Second Vice-Pres. '00-'04), Professor of Surgery, Yale University, 820 Temple Street, New Haven, Conn.

Frost, Gilman Dubois, A.M., M.D., Professor of Clinical Medicine, Dartmouth Medical School, Hanover, N. H.

Gage, Simon Henry, B.S. (Ex. Com. '06-' 11), Emeritus Professor of Histology and Embryology, Cornell University, 4 South Avenue, Ithaca, N. Y.

Gallaudbt, Bern Budd, AM., M.D., Assistant Professor of Anatomy, Columbia University, Consulting Surgeon Bellevue Hospital, 110 East 16th Street, New York, N. Y.

Geddes, a. Campbell, M.D., M.B., Ch.B., F.R.S.E., Professor of Anatomy, McGill University, Montreal, Canada.

276 AMERICAN ASSOCIATION OF ANATOMISTS

Gibson, James A., M.D., Professor of Anatomy, Medical Department, University

of Buffalo, 24 High Street, Buffalo, N. Y, GiLMAN, Philip Kinqsworth, B.A., M.D., Professor of Surgery, University of

Philippines, Suite 417-427 Kneedler Bldg., Manila, P. /. Globus, J. H., B.A., Assistant in Anatomy, Cornell University Medical College,

New York City, GoETSCH, Emil, Ph.D., M.D., Associate in Surgery, Johns Hopkins Hospital,

Baltimore, Md, Greene, Charles W., A.M., Ph.D., Professor of Physiology and Pharmacology,

University of Missouri, 814 Virginia Avenue, Columbia, Mo, Greenman, Milton J., Ph.B., M.D., Sc.D., Director of The Wistar Institute of

Anatomy and Biology, $6th Street and Woodland Avenue, Philadelphia, Pa, Gudernatsch, J. F., Ph.D., Assistant Professor of Anatomy, Cornell University

Medical College, New York Ciiy, Guild, Stacy R., A.M., Instructor in Histology and Embryology, University of

Michigan, 1511 Washtenaw Avenue, Ann Arbor, Mich. GuTBR, Michael F., Ph.D., Professor of Zoology, University of Wisconsin, 1S8

Prospect Avenue, Madison, Wis. Halsted, William Stewart, M.D., Professor of Surgery, Johns Hopkins University, 1201 Eutaw Place, Baltimore, Md, Ham ANN, Carl A., M.D. (Ex. Com. '02-04), Professor of Applied Anatomy and

Clinical Surgery, Western Reserve University, 416 Osbom Building, Cleveland,

Ohio. Hardesty, Irving, A.B., Ph.D. (Ex. Com. '10 and '12-'15), Professor of Anatomy and head of Department of Anatomy, Tulane University of Louisiana,

Station 20, New Orleans, La. Hare, Earl R., A.B., M.D., Instructor in Surgery, University of Minnesota,

62S Syndicate Building, Minneapolis, Minn. Harrison, Ross Granville, Ph.D., M.D. (Pres. '12-' 13), Bronson Professor of

Comparative Anatomy, Yale University, New Haven, Conn. Harvey, Basil Coleman Hyatt, A.B., M.B., Associate Professor of Anatomy,

University of Chicago, Department of Anatomy, University of Chicago, Chicago,

III, Harvey, Richard Warren, M.S., M.D., Assistant Professor of Anatomy,

Anatomy Department, University of California, Berkeley, Calif. Hatai, Shinkishi, Ph.D., Associate in Neurology, Wistar Institute of Anatomy

and Biology, Philadelphia, Pa. Hathaway, Joseph H., A.M., M.D., Professor of Anatomy, Anatomical Department, Detroit Medical College, Detroit, Mich. Hazen, Charles Morse, A.M., M.D., Professor of Physiology, Medical College of

Virginia, Richmond, Bon Air, Va. Heaqey, Francis Wenger, A.B., M.D., Instructor in Anatomy, Columbia

University, 437 West 69th Street, New York City. Heisler, John C, M.D., Professor of Anatomy, Medico-Chirurgical College,

S829 Walnut Street, Philadelphia, Pa. Heldt, Thomas Johanes, A.B., A.M., 200 East Lanvale Street, Baltimore, Md. Herrick, Charles Judson, Ph.D. (Ex. Com. '13-) Professor df Neurology,

University of Chicago, Laboratory of Anatomy, University of Chicago,

Chicago, III.

MEMBERS 277

Hertzler, Arthur E., M.D., F.A.C.S., Associate in Surgery, University of Kansas, 1004 Rialto Building, Kansas City, Mo.

Herzoo, Maximiuan, M.D., Professor of Pathology and Bacteriology, La Gola University, 1S68 Fulton Street, Chicago, III.

Heuser, Chester H., A.M., Ph.D., Fellow in Anatomy, Wistar Institute of Anatomy, S6th Street and Woodland Avenue, Philadelphia, Pa.

Hewson, Addinell, A.M., M.D., Professor of Anatomy, Philadelphia Polyclinic for Graduates in Medicine, f iJW> Spruce Street, Philadelphia, Pa.

Hill, Howard, M.D., 1010 Rialto Building, Kansas City, Mo.

Hill, James Peter, D.Sc, F.R.S., Todrell Professor of Zodlogy and Comparative Anatomy, University of London, University College, Gower Street, London, W.C., England.

Hilton, William A., Ph.D., Professor of Zoology, Pomona College, Claremont, Calif.

HoEVB, Hubertus H. J., M.D., Hoeve Hospital, Meherrin, Virginia.

Hooker, Davenport, M.A., Ph.D., Assistant Professor of Anatomy, Yale Medical School, New Haven, Conn.

Hopewell-Smith, Arthur, L.R.C.P., M.R.C.S., L.D.S., Professor of Dental Histology, Histo-Pathology, Comparative Odontology, University of Penn-sylvania Dental College, Philadelphia, Pa. •

Hopkins, Grant Sherman, Sc.D., D.V.M., Professor of Veterinary Anatomy, Cornell University, Ithaca, N. Y.

HosKiNS, Elmer R., A.B., A.M., Assistant in Biology, Yale University, Oshome Zodlogical Laboratory, New Haven, Conn.

Hrdli5ka, Ales, M.D., Curator of the Division of Physical Anthropology, United States National Museum, Washington, D. C.

HuBBB, G.Carl, M.D. (Second Vice-Pres. 'OO-'Ol, Secretary-Treasurer '02-' 13, Pres. '14-'15) Professor of Anatomy and Director of the Anatomical Laboratories, University of Michigan, ISSO Hill Street, Ann Arbor, Mich.

Huntington, George S., A.M., M.D., D.Sc, LL.D. (Ex. Com. '95-'97, '04r-'07, Pres. '99-'03), Professor of Anatomy, Columbia University, 4S7 West 59th Street, New York, N. Y.

Ingalls, N. William, M.D., Associate Professor of Anatomy, Medical College, Western Reserve University, Cleveland, Ohio.

Jackson, Clarence M., M.S., M.D. (Ex. Com. '10-' 14), Professor and Head of the Department of Anatomy, University of Minnesota, Institute of Anat' amy, Minneapolis, Minn.

Jenkins, George B., M.D., Professor of Anatomy, Department of Anatomy, University of Louisville, Louisville, Ky.

Johnson, Charles Eugene, A.M., Ph.D., Instructor in Comparative Anatomy of Vertebrates, Department of Animal Biology, University of Minnesota, Minneapolis, Minn.

Johnson, Franklin P., A.M., Ph.D., Associate Professor of Anatomy, University of Missouri, 408 South Ninth Street, Columbia, Mo.

Johnston, John B., Ph.D., Professor of Comparative Neurology, University of Minnesota, Minneapolis, Minn.

Jordan, Harvey Ernest, Ph.D., Professor of Histology and Embryology, Uni' versity of Virginia ^ University, Va.

THB ANATOMICAL BSCOKD. VOL. 10, NO. 3

278 AMERICAN ASSOCIATION OF ANATOMISTS

Kampmeier, Otto Fbbderick, A.B., Ph.D., .Vssistant Professor of Embryology and Comparative Embryology, Univerr'^y of Pittsburghy School of Medicine j Pittsburgh, Pa.

Kappers, Cornelius Ubbo Aribns, Director of the Central Ir^tittUe for Brain Research of Holland, Mauritskade 61, Amsterdam, Holland.

Kbillbr, William, L.R.C.P. and F.R.C.S.Ed. (Second Vice-Pres. '98-'99), Professor of Anatomy, Medical Department University of Texas, Staie Medical College, Galveston, Texas,

Kbith, Arthur, M.D., LL.D., F.R.C.S., F.R.S., Hunterian Professor of Anatomy, College of Surgeons, London, England.

Kbllt, Howard Atwood, A.B., M.D., LL.D., Professor of Gynecology, Johns Hopkins University, I4IS Eutaw Place, Baltimore, Md.

Ejdrnan, John D., Jr., A.B., M.D., Assistant in Anatomy, Columbia University, 4S7 West 59th Street, New York City.

Kbrr, Abram T., B.S., M.D. (Ex. Com. '10-'14), Professor of Anatomy, Cornell University Medical College, Ithaca, N. Y.

Kbt, J. a., B.S., Instructor in Anatomy, Creighton Medical College, Omaha, Neb.

EiNOERY, Hugh McMillan, A.M., Instructor in Histology and Embryology, Cornell University, Ithaca, N. Y.

Kingsbury Benjamin F., Ph.D., M.D., Professor of Histology and Embryology, Cornell University, SOB University Avenue, Ithaca, N. Y.

KiNOSLEY, John Sterling, Sc.D., Professor of Zodlogy, University of Illinois^ Urbana, III.

Kino, Helen Dean, A.B., Ph.D., Assistant Professor of Embryology, Wistar Institute of Anglomy, S6th Street and Woodland Avenue, Philadelphia, Pa.

Kirkham, William Barri, Ph.D., Instructor in Biology, Sheffield Scientific School, Yale University, New Haven, Conn.

Knower, Henry McE., A.B., Ph.D. (Ex. Com. '11-'16), Professor of Anatomy, Medical Department, University of Cincinnati, Station V, Cincinnati, Ohio.

KoFoiD, Charles Atwood, Ph.D., Professor of Zodlogy, University of California, Assistant Director San Diego Marine Biological Station, $616 Etna Street y Berkeley, Calif.

Kunkel, Beverly Waugh, Ph.B., Ph.D., Professor of Zoology, Lafayette College, Easton, Pa.

Kuntz, Albert, Ph.D., Assistant Professor of Histology and Biology, Department of Anatomy, University of St. Louis, SOU Castleman Avenue, St. Louis, Mo.

KuTCHiN, Harriet Lehmann, A.M., *^The Maplewood,** Green Lake, Wisconsin.

Kyes, Preston, A.M., M.D., Assistant Professor of Experimental Pathology, Department of Pathology, University of Chicago, Chicago, III.

Lamb, Daniel Smith, A.M., M.D., LL.D. (Secretary-Treasurer '90-*01, VicePres. '02-^03) Pathologist Army Medical Museum, Professor of Anatomy, Howard University, Medical Department, BII4 18th Street, N. W., Washington, D. C.

Lambert, Adrian V. S., A.B., M.D., Associate Professor of Surgery, Columbia University, 168 East 71st Street, New York, N. Y.

Landacre, Francis Leroy, A.B., Ph.D., Professor of Anatomy, Ohio State University, 8026 Inka Avenue, Columbus, Ohio.

MEMBERS 279

Lane, Michael Andrew, B.S., iJ?f S. California Avenue, Chicago, III,

Laurens, Hbnrt, Ph.D., Instructor in Biology, Yale University, New Haven, Conn.

Lee, Thomas G., B.S., M.D. (]Ex. Com. '08-'10, Vice Pres. '12-43), Professor of Comparative Anatomy, University of Minnesota, Institute of Anatomy, University of Minnesota, Minneapolis, Minn,

Lbidy, Joseph, Jr., A.M., M.D., 1S19 Locust Street, Philadelphia, Pa,

Lewis, Dean D., M.D., Assistant Professor of Surgery, Rush Medical College, People^ s Oas Building, Chicago, III.

Lewis, Frederic T., A.M., M.D. (Ex. Com. '09-'13, Vice-Pres. '14-), Assistant Professor of Embryology, Harvard Medical School, Boston, Mass.

Lewis, Warren Harmon, B.S., M.D. (Ex. Com. '09-' 11, '14-), Professor of Physiological Anatomy, Johns Hopkins University, Medical School, Baltimore, Md,

LiLLiE, Frank Rathay, Ph.D., Professor of Embryology, Chairman of Departnaent of Zoology, University of Chicago; Director Marine Biological Laboratory, Woods Hole, Mass., University of Chicago, Chicago, III.

LiNEBACK, Paul Eugene, A.B., M.D., Teaching Fellow in Histology and Embryology, Harvard Medical School, Boston, Mass,

LocT, William A., Ph.D., SoiD., Professor of Zodlogy and Director of the Zo5logiaal Laboratory, Northwestern University, 1^45 Orrington Avenue, Evanston, III.

Loeb , Hanau Wolf, A.M., M.D., Professor and Director of the Department of the Diseases of the Ear, Nose and Throat, St. Louis University, 5S7 North Qrand Avenue, St, Louis, Mo,

LoRD", Frederic P., A.B., M.D., Professor of Anatomy and Histology, Dartmouth Medical School, Hanover, N, H.

Lowrby, Lawson Gentry, A.M., Fellow in Neuropathology, Harvard Medical School; Pathologist, Danvers State Hospital, Hawthorne, Mass,

Macklin, C. C., M.B., Assistant in Anatomy, Department of Anatomy, Johns Hopkins Medical School, Baltimore, Md.

McCarthy, John George, M.D., Formerly Assistant Professor of Anatomy, McGill University, U^ St. Mark Street, Montreal, Canada.

McClitng, Clarence E., A.M., Ph.D., Professor of Zodlogy, University of Pennsylvania, Philadelphia, Pa.

McClure, Charles Freeman Williams, A.M., Sc.D. (Vice-Pres. 'lO-'ll. Ex. Com. '12-' 16), Professor of Comparative Anatomy, Princeton University, Princeton, N.J,

McCoRMACK, William Eli, M.D., Instructor in Embryology and Histology, University of Louisville, Medical Department, 101 W, Chestnut Street, Louisville, Ky.

McCoTTER, RoLLO E., M.D., Professor of Anatomy, Medical Department, University of Michigan, 809 E. University Avenue, Ann Arbor, Mich.

McFarland, Frank Mace, Ph.D., Professor of Histology, Leland Stanford Junior University, B Cabrillo Avenue, Stanford, Calif.

McGill, Caroline, A.M., Ph.D., M.D., Pathologist, Murray Hospital, ButU, Mont,

McKiBBBN, Paul S., Ph.D., Professor of Anatomy, Department of Anatomy, Western University, London, Ontario, Canada.

280 AMERICAN ASSOCIATION OF ANATOMISTS

McMuRRiCH, James Playpair, A.M., Ph.D., LL.D. (Ex. Com. *06-*07, Pres.

'08-'09), Professor of Anatomy, University of Toronto, 76 Forest Hill Road,

Toronto, Canada. McWhortbb, John E., M.D., Worker under George Crocker Research Fund,

College of Physicians and Surgeons, Colimibia University, 206 West 107th

Street, New York, N. Y. Mall, Franklin P., A.M., M.D., LL.D., D.Sc. (Ex. Com. '00-'05, Pres. »06-'07),

Professor of Anatomy, Johns Hopkins Medical School, Baltimore, Md. Mangum, Charles S., A.B., M.D., Professor of Anatomy, University of North

Carolina, Chapel Hill, N. C. M ALONE, Edward Fall, A.B., M.D., Assistant Professor of Anatomy, University

of Cincinnati, College of Medicine, Station V, Cincinnati, Ohio, Mark, Edward Laurens, Ph.D., LL.D., Hersey Professor of Anatomy and Director of the Zoological Laboratory, Harvard University, 109 Irving Street,

Cambridge, Mass. Matas, Rudolph, M.D., Professor of Surgery, Tulane University, 21^66 St. Charles

Avenue, New Orleans, La. Maximow, Alexander, M.D., Professor of Histology and Embryology at the

Imperial Military Academy of Medicine, Petrograd, Russia, Botkinskaja 2,

Petrograd, Russia. Mellus, Edward Lindon, M.D., 1$ Fuller Street, Brookline, Mass. Mercer, William F., Ph.M., Ph.D., Professor of Biology, Ohio University, Box

384, Athens, Ohio. Metheny, D. Gregg, M.D., L.R.C.R., L.R.C.S., Edin.— L.F.P.S., Glasg., Associate in Anatomy, Jefferson Medical College, 11th and Clinton Streets, Philadelphia, Pa. Meyer, Adolf, M.D., LL.D., Professor of Psychiatry and Director of the Henry

Phipps Psychiatric Clinic, Johns Hopkins Hospital, Baltimore, Md, Meyer, Arthur W., S.B., M.D. (Ex. Com. '12-' 16), Professor of Anatomy,

Leland Stanford Junior University, Stanford University, Calif. Miller, Adam M., A.M., Professor of Anatomy, Long Island College Hospital,

Henry and Amity Streets, Brooklyn, N. Y. Miller, M. M., Ph.D., Instructor in Anatomy, Vanderbilt University Medical

School, Nashville, Tenn, Miller, William Snow, M.D. (Vice-Pres. '08-' 09), Associate Professor of Anatomy, University of Wisconsin, 2001 Jefferson Street, Madison, Wis. MiXTER, Samuel Jason, B.S., M.D., Visiting Surgeon Massachusetts General

Hospital, 180 Marlboro Street, Boston, Mass. MooDiE, Roy L., A.B., Ph.D., Instructor in Anatomy, University of Illinois

Medical College, Congress and Honor e Streets, Chicago, III. Moody, Robert Orten, B.S., M.D., Associate Professor of Anatomy, University

of California, 2826 Garber Street, Berkeley, Calif. Morrill, Charles V., A.M., Ph.D., Instructor in Anatomy, Cornell UniverMy

Medical School, 1st Avenue and 28th Street, New York, N, Y, Muller, Henry R., A.B., M.D., Assistant in Anatomy, Johns Hopkins Medical

School, Baltimore, Md. MuNSON, John P., Ph.D., Head of the Department of Biology, Washington State

Normal School, 706 North Anderson Street, Ellensburg, Washington,

MEMBERS 281

MuRPHEY, Howard S., D.V.M., Professor of Anatomy and Histology, 6t9 Welch

Aventie, Station A , AmeSf la, Mtebs, Bubton D., am., M.D., Professor of Anatomy and Secretary of the Indiana University School of Medicine, Indiana University ^ Bloomington, Ind.

Mters, Jay A., M.S., Ph.D., Instructor in Anatomy, University of Minnesota y 624.' University Avenue, S, E,, Minneapolis, Minn.

Myebs, Mae Lichtbnwalner, M.D., Associate Professor of Anatomy and Director of the Laboratories of Histology and Embryology, Woman^s Medical College of Pennsylvania, North College Avenue and Blst Street, Philadelphia, Pa,

Nachtrieb, Henry Francis, B.S., Professor of Animal Biology and Head of the Department, University of Minnesota, 905 East 6th Street, S,E,, Minneapolis, Minn,

Nbal, EEerbebt Vincent, Ph.D., Professor of Zodlogy, Tufts College, Tufts College, Mass,

Noble, Harriet Isabel, 26B Pvinam Avenue, Brooklyn, N, Y,

NoRRis, H. W., A.B., Professor of Zodlogy, Grinriell College, Grinnell, Iowa,

Painter, Theophilus S., Ph*.D., Instructor in Biology, Sheffield Scientific School,. Yale University, New Haven, Conn,

Papanicolaou, George, Ph.D., M.D., Assistant in Anatomy, Cornell University Medical College, New York City,

Papbz, James Wenceslas, B.A., M.D., Professor of Anatomy, Histology and Embryology, Atlanta Medical College, 94 Butler Street, Atlanta, Ga,

Parker, George Howard, D.Sc», Professor of Zodlogy, Harvard University, 16 Berkeley Street, Cambridge, Mass,

Paton, Stewart, A.B., M.D., Lecturer in Biology, Princeton University, Princeton, N, J,

Patten, William, Ph.D., Professor of Zoology, Dartmouth College, Hanover,N. H,

Paterson, a. Melville, M.D., F.R.C.S., Professor of Anatomy, University of Liverpool, Liverpool, England,

Patterson, John Thomas, Ph.D., Professor and Chairman of the School of Zodlogy, University of Texas, University Station, Austin, Texas,

PiERsoL, George A., M.D., Sc.D. (Vice-Pres. '93-'94, '98-^99, '06-W, Pres. '10'11), Professor of Anatomy, University of Pennsylvania, 47^ Chester Avenue, Philadelphia, Pa,

PiERSOL, William Hunter, A.B., M.B., Associate Professor of Histology and Embryology, Biological Department, University of Toronto, Toronto, Canada.

PoHLMAN, Augustus G., M.D., Professor of Anatomy, Medical Department, University of St, Louis, 1402 South Grand Avenue, St, Louis, Mo,

Potter, Peter, M.S., M.D., Oculist and Aurist, Murray Hospital, Butte, Montana, 4it-4tS Hennessy Building, Butte, Montana.

Poyntbr, Charles W. M., B.S., M.D., Professor of Anatomy, College of Medicine, University of Nebraska, Omaha, Neb,

Prentiss, H. J., M.D., M.E., Professor of Anatomy, University of Iowa, Iowa City, Iowa,

Pryor, Joseph William, M.D., Professor of Anatomy and Physiology, State College of Kentucky, 261 North Broadway, Lexington, Ky,

282 AMERICAN ASSOCIATION OF ANATOMISTS

Radasch, Henry E., M.S., M.D., Assistant Professor of Histology and Embryology, Jefferson Medical College, Daniel Baugh Institute of Anatomy ^ liih and Clinton Streets, Philadelphia, Pa. Ranson, Stephen W., M.D., Ph.D., Professor of Anatomy, Northwestern University Medical School, 24^1 Dearborn Street, Chicago, III.

Reagan, Franklin P., A.B., Princeton University^ Princeton, N, J. *

Reed, Hugh Daniel, Ph.D., Assistant Professor of Zoology, Cornell University, 108 Brandon Place, Ithaca, N. Y.

Reese, Albert Moore, A.B., Ph.D., Professor of Zoology, West Virginia University, Morgantown, W.Va.

Retzer, Robert, M.D., Professor of Anatomy and Dean, Creighton College of Medicine, Omaha, Neb.

Revell, Daniel Graisberry, A.B., M.B., Professor of Anatomy, University of Alberta, Strathcona Post Office, Edmonton South, Alberta, Canada.

Rhinehart, D. a., M.D., Professor of Anatomy, University of Arkansas, Old State House, Little Rock. Ark.

Rice, Edward Loranitb, Ph.D., Professor of Zoology, Ohio Wesleyan University, Delaware, Ohio.

Robinson, Arthur, M.D., F.R.C.S. (Edinburgh), Professor of Anatomy, University of Edinburgh, The University, Edinburgh, Scotland.

Ruth, Edward S., M.D., Assistant Professor of Anatomy, University of the Philippines, College of Medicine and Surgery, Manila, P. /.

Sabin, Florence R., B.S., M.D. (Second Vice-Pres. '0S-'09), Associate Professor of Anatomy, Johns Hopkins University, Medical Department, Baltimore, Md.

Santee, Harris E., A.M., Ph.D., M.D., Professor of Anatomy, Jenner Medical College, and Professor of Neural Anatomy, Chicago College of Medicine and Surgery, 2806 Warren Avenue, Chicago, III.

ScAMMON, Richard E., Ph.D., Professor of Anatomy, Institute of Anatomy, University of Minnesota, Minneapolis, Minn.

Schaefer, Marie Charlotte, M.D., Associate Professor of Biology, Histology and Embryology, Medical Department, University of Texas, 701 North Pine Street, San Antonio, Texas.

ScHABPPER, Jacob Parsons, A.M., M.D., Ph.D., Professor of Anatomy, Jefferson Medical 'College, 11th and Clinton Streets, Philadelphia, Pa.

ScHOCHET, Sidney Sigsfried, M.D., Instructor in Anatomy, Dartmouth Medical School, Hanover, N . H.

ScHOEMAKER, Daniel M., B.S., M.D., Professor of Anatomy, Medical Department, St. Louis University, H02 South Grand Avenue, St. Louis, Mo.

ScHULTE, Hermann von W., A.B., M.D. (Ex. Com. '15) Associate Professor of Anatomy, Columbia University, 437 West 69th Street, New York, N. Y.

ScHMiTTER, Ferdinand, A.B., M.D., Captain Medical Corps, U. S. Army, Columbus Barracks, Columbus, Ohio.

Scott, John W., A.M., Ph.D., Professor of Zoology, University of Wyoming, Laramie, Wyo.

Scott, Katherine Julia, A.B., M.D., Assistant in Anatomy, University of California, Berkeley, Calif.

Seelig, Major G., A.B., M.D., Professor of Surgery, St. Louis University, Humboldt Building, 6S7 North Grand Avenue, St. Louis, Mo.

MEMBEBS 283

Selling, Lawrence, A.B., M.D., Selling Building^ Portland, Ore,

Seniob, Harold D., M.B., D.Sc, F.R.C.S., Professor of Anatomy, New York University, University and Bellevue Hospital Medical College, SS8 East 26ih Street, New York, N, Y.

Sheldon, Ralph Edward, A.M., M.S., Ph.D., Professor of Anatomy, University of Pittsburgh Medical School, Grant Boulevard, Pittsburgh, Pa.

Shields, Randolph Tucker, A.B., M.D., Dean, University of Nanking Medical School, Nanking, China.

Shxjfeldt, R. W., M.D., Major Medical Corps, U. S. A. (Retired), SS66 Eighteenth Street, N, W,, Washington, D. C.

Silvester, Charles Frederick, Curator of the Zodlogical Museum and Assistant in Anatomy, Princeton University, 10 Nassau Hall, Princeton, N. J.

Simpson, Sutherland, M.D., D.Sc, F.R.S.E. (Edin.), Professor of Physiology, Cornell University Medical College, Ithaca, N. Y.

SissoN, Septimus, B.S., V.S., Professor of Comparative Anatomy, Ohio State University, B74 Hth Avenue, Columbus, Ohio.

Slxtder, Greenfield, M.D., Clinical Professor of Laryngology, Washington University Medical School, S64^ Washington Avenue, St. Louis, Mo.

Smith, Charles Dennison, A.M., M.D., Superintendent Maine General Hospital Professor of Physiology, Medical School of Maine, Maine General Hospital, Portland, Me.

Smith, George Milton, A.B., M.D., Associate in Pathology, Washington University Medical School, St. Louis, Mo.

Smith, Grafton Elliot, M.A., M.D., F.R.S., Professor of Anatomy, The University, Manchester, England.

Smith, J. Holmes, M.D., Professor of Anatomy, University of Maryland, Green and Lombard Streets, Baltimore, Md.

Smith, M. DeForest, A.B., M.D., Assistant in Neurology, Columbia University, JiS7 West 69th Street, New York City.

Smith, Philip Edward, M.S., Ph.D., Department of Anatomy, University of California, Berkeley, Calif.

Snow, Perry G., A.B., M.D., Dean and Professor of Anatomy, School of Medicine, University of Utah, lOSl South l^th East Street, Salt Lake City, Utah.

Spitzka, Edward Anthony, M.D., 63 East 91sf Street, New York, N. Y.

Stbensland, Halbert Severin, B.S., M.D., Professor of Pathology and Director of the Pathological Laboratory, College of Medicine, Syracuse University, 309 Orange Street, Syracuse, N. Y.

Stewart, Chester A., A.M., Assistant in Anatomy, University of Minnesota, Minneapolis, Minn.

Stiles, Henry Wilson, M.D., Professor of Anatomy, College of Medicine, Syracuse University, 309 Orange Street, Syracuse, N. Y.

Stockard, Charles Rupert, M.S., Ph.D. (Secretary-Treasurer '14), Professor of Anatomy, Cornell University Medical College, New York, N. Y.

Stotsenburg, James M., M.D., Instructor in Anatomy, Wistar Institute of Anatomy and Biology, Philadelphia, Pa,

Strebter, George L., A.M., M.D., Research Associate in Embryology, Carnegie Institution, Johns Hopkins Medical School, Baltimore, Md.

Stromsten, Frank Albert, D.Sc, Assistant Professor of Animal Biology, University of Iowa, 943 Iowa Avenue, Iowa City, Iowa.

284 AMERICAN ASSOCIATION OF ANATOMISTS

Strong, Olivbb S., A.M., Ph.D., Instructor in Anatomy, Columbia University, 4S7 West 59th Street, New York, AT. Y,

Strong, Reuben Myron, A.M., Ph.D., Professor of Anatomy, University of Mississippi, Urdversity, Miss,

SuNDWALL, John, Ph.D., M.D., Professor of Anatomy, University of Kansas, Lawrence, Kans.

StJTTON, Alan Callender, A.B., Student, Johns Hopkins Medical School, Baltimore, Md.

Symington, Johnson, M.D., F.R.S., Professor of Anatomy, Queens University, Belfast, Ireland,

Swift, Charles H., M.D., Ph.D., Associate in Anatomy, Department of Anatomy, University of Chicago, 663S Maryland Avenue, Chicago, III,

Taintor, F. J., M.D., Assistant Professor of Anatomy, St, Louis University, St. Louis, Mo.

Taylor, Edward W., A.M., M.D., Assistant Profefeflor of Neurology, Harvard Medical School, 467 Marlboro Street, Boston, Mass,

Terry, Robert James, A.B., M.D. (Ex. Com. '08-42), Professor of Anatomy, Washington University Medical School, St, Louis', Mo,

Thompson, Arthur, M.A., M.B., LL.D., F.R.C.S., Professor of Anatomy, University of Oxford, Department of Human Anatomy, Oxford, England,

Thorkblson, Jacob, M.D., Dillon, Mont.

Thro, William C, A,M., M.D., Assistant Professor of Clinical Pathology, Cornell University Medical School, 28th Street and 1st Avenue, New York, N. Y.

TnttRiNGBR, Joseph M., M.D., Assistant in Histology and Embryology, Harvard Medical School, Boston, Mass.

Thyng, Frederick Wilbur, Ph.D., Assistant Professor of Anatomy in the University and Bellevue Hospital Medical College, SS8 East B6th Street, New York, N. Y,

TiLNEY, Frederick, A.B., M.D., Professor of Neurology, Columbia University, 161 Henry Street, Brooklyn, N. Y.

ToBiE, Walter E., M.D., Professor of Anatomy, Medical School of Maine, S Deering Street, Portland, Me.

Todd, Thomas Wingate, M.D., Ch.B. (Mane), F.R.C.S. (Eng.), Professor of Anatomy, Medical Department Western Reserve University, Cleveland, Ohio.

Tracy, Henry C, A.M., Ph.D., Professor of Anatomy, Marquette Medical School, Fourth and Reservoir Street, Milwaukee, Wis.

TUPPBR, Paul Yoer, M.D., Clinical Professor of Surgery, Washington University Medical School, Wall Building, St. Louis^ Mo.

Waite, Frederick Clayton, A.M., Ph.D., Professor of Histology and Embryology, Western Reserve University School of Medicine, 1S6S East 9th Street, Cleveland, Ohio.

Walker, George, M.D., Instructor in Surgery, Johns Hopkins University, corner Charles and Center Streets, Baltimore, Md.

Wallin, Ivan E., B.S., M.A., Assistant Professor of Anatomy, Marquette University School of Medicine, Milwaukee, Wis.

Warren, John, A.B., M.D., Associate Professor of Anatomy, Harvard Medical School, 240 Longwofd Avenue, Boston, Mass.

Waterston, David, M.A., M.D., F.R.C.S.Ed., Butte Professor of Anatomy, University of St. Andrews, St. Andrews, Fife, Scotland.

MEMBERS 285

Watkins, Richard Watkin, B.S., Assistant in Anatomy, University of Chicago ^

Chicago, III, Watt, Jambs Ceawfobd, B A., M.B., Lecturer in Anatomy, University of Toronto,

20 Hawthorne Avenue, Toronto, Canada. Weed, Lewis Hill, A.M., M.D., Associate in Anatomy, Johns Hopkins Medical

School, Baltimore, Md. Wbidenbeich, Franz, M.D., a.o. Professor and Prosector of Anatomy, 19 Vogesen

Street, Strasshurg, i. Els, Germany, Wbrbbr, Ernest I., Ph.D., Sessel Research Fellow, Oshom Zoological Labor atory, Yale University, New Haven, Conn. West, Charles Ignatius, M.D., Associate Professor of Anatomy, Medical

Department of Howard Xftiiversity, 9i4 M Street N, W,, Washington, D. C, West, P. A., B.A., Johns Hopkins Medical School, Baltimore, Md, West, Randolph, A.M., Student, College of Physicians and Surgeons, Columbia

University, 4^7 West 69th Street, New York, N. Y. Wheeler, Theodora, A.B., Medical Student, Johns Hopkins Medical School,

Baltimore, Md. White, Harry Oscar, M.D., Professor of Anatomy, Histology and Embryology,

Medical Department, University of Southern California, 616 E, Washington Street, Los Angeles, Calif, Whitehead, Richard Henry, A.B., M.D., LL.D., Professor of Anatomy and

Dean of Medical Department, University of Virginia, University P. 0., Va. Wilder, Harris Hawthorne, Ph.D., Professor of Zodlogy, Smith College,

Northampton, Mass. Williams, Stephen Rigos, A.M., Ph.D., Professor of Zodlogy and Geology,

Miami University, SCO East Church Street, Oxford, Ohio. Willard, William A., A.M., Ph.D., Professor of Histology and Embryology,

University of Nebraska, College of Medicine, 4£d Street and Dewey Avenue,

Omaha, Neb. Wilson, J. Gorden, M.A., M.B., CM. (Edin.), Professor of Otology, Northwestern University Medical School, 24S7 Dearborn Street, Chicago, III. Wilson, James Thomas, M.B., F.R.S., Challis Professor of Anatomy, University,

SydneT^, Australia. Wilson, Louis Blanchard, M.D., Director of Laboratories, Mayo Clinic, 8S0

West College Street, Rochester, Minn. WisLOCKi, George Bern ays, A.B., Student, Johns Hopkins Medical School,

Baltimore, Md. WiTHERSPOON, Thomas Casey, M.D., S07 Granite Street, Butte, Mont. Worcester, John Locke, M.D., Instructor in Anatomy, University of Michigan, 1214 Willard Street, Ann Arbor, Mich. SEA WATER AS A MEDIUM FOR TISSUE CULTURES

M. R. LEWIS

Department of Embryologyy Carnegie Institution

POUR FIGURES

As early as 1878 L. Fredericq demonstrated that the blood and haemolymph of invertebrate marine forms is isotonic with the sea water and it has long been known that Locke's solution and Ringer's solution contain the same proportion of certain salts as does the sea water, only in different concentrations (Loeb, '06). Macallum ('08) has shown that not only does the body fluid of the lower forms of marine life correspond in composition with that of sea water, but also that the plasma of higher animals is not changed in composition from sea water, but is simply more dilute.

Loeb ('06) in his work on the antagonistic action of certain salts, demonstrated the necessity for the presence of the various salts in the composition of sea water, also the importance of the . proportion of the various salts one to another in order to obtain normal behavior of certain marine animals.

Although the composition of the sea water varies slightly for different localities, on the whole the salt content remains surprisingly constant, and while an analysis of the sea water for practically any given locality has been determined, it is in general that given by Henze ('10) (taken from Roth) :

Ocean surface


To 1000 parts


NaCI


26.862


KCl


0.582


MgCU


3.239


MgSO« 


2.196


CaSO*


1.350


Rest


0.070



34.299


AHATOHICAL RECOBD, VOL. 10, NO. 4


FEBBUART, 1918 CI. 18.999


287

288


.8


M.


R. LEWIS


Middle ocean


To WOO parts NaCl


30.292 KCl


0.779 MgCU


3.240 MgS04


2.638


CI.


19.<


CaSO


1.605 Rest


0.080

38.634

Herbst, C, '03-04, makes artificial sea water as follows:

To 100 cc. of distilled water NaCl 3.00 g. KCl 0.08 g.

MgS04 0.66 g. CaCU 0.13 g.

To 100 cc. of above he adds 1 cc. of a 4.948 per cent solution of NaHCO,. Loeb, J., '13, uses 100 molecules NaCl, 2.2 molecules KCl, 7.8 molecules MgCU, 3.8 molecules MgSO^ and to this he adds 1-2 molecules CaCU. This solution is diluted until specific gravity corresponds with that of the sea water normal for the animal and for regeneration problems Loeb adds to 100 cc. of the above solution 1 cc. of a 3/8 m. NaHCOs solution.

While the ratios of certain salts are quite constant, there are other variations which influence the osmotic pressure of the sea water in different localities, as for instance Loeb, J., '13, states that the free alkalinity is higher in the sea water at Woods Hole than at Pacific Grove.

The osmotic pressure of a solution such as the sea water or the plasma can be determined according to Carrey ('15) from the depression of the freezing point of the solution by means of the formula : osmotic pressure = 22.4 a A/1.85.

Carrey ('15) states that the determinations of the sea water at Woods Hole for six different years gave an average A = 1.81*^C., and that this is isotonic with sodium chloride 0.52 m; magnesium chloride 0.29 m; cane sugar 0.73 m and Van't Hoff's

SEA WATER AS A MEDIUM FOR TISSUE CULTURES 289

TABLE 1— GARREY


8BA WATBB FBOM

Xaples

Arcachon

Pacific Grove, Cal

Pacific Grove, Cal

Woods Hole

Beaufort, N. C

Helgoland

In the Kattegat

Open Baltic Sea

Kiel Harbor

Newport River near Beaufort


A-*c


-.229

-1.89

-1.925

-1.90 -1.81 -2.04 -1.90 -1.66 -1.30 -1.093

-1.707


OBSBRVSR


Bottazzi

Rodier

Greene

Garrey

Garrey

Garrey

Dakin

Garrey

Dakin

Dakin

Garrey


RETERBNCX


Arch. ital. de biol. 1897 v. 28, p. 61.

Trav. dee Lab. d'arcachon, 1899, 103.

Bull. U. S. Bureau Fisheries, 1904, V. 24, p. 429.

Biol. Bull., 1905, V. 8, p. 257.

Biol. Bull., 1905, V. 8, p. 257.

1911.

Bio.-Chem. Jour., 1908, 269.


Biol. Bulletin, vol. 28, no. 2, 1915.


solution made up from half molecular solutions according to the formula given by Loeb 43 (see above).

Garrey also states that by his findings the sea water of Pacific Grove is about 5 per cent more concentrated than that of Woods Hole, while Beaufort, N. C, is about 12 per cent more concentrated than Woods Hole.

The osmotic pressure of the plasma of many animals can be determined from the A (depression of freezing point of their plasma) given below.

In addition to the above Garrey '15 has determined the freezing point for various dilutions of sea water and also the concentration of pure salt which has a corresponding freezing point.

Thus it can be seen that if the A for the plasma of any given animal is known, it is an easy matter with the aid of the above table to find the dilution of sea water which is isotonic with the plasma in question and thus one can obtain a solution, which contains the salts in the same proportion as the plasma and at the same time isotonic with the plasma.

This general formula does not hold for the selachians, for the blood of these animals contains a large amount of urea and

TABLE 2


Invertebrate Marine Animal.

Alcyonium palmatum

Asteropecten aurantiacus.

Holothuria tubuloea

Sipunculus nudus

Maja squinado

Homarus vulgaris

OctopuB macropus

Limulus polyphemus

Limulus polyp bemus

Limulus polyphemus


A - "C.


Vertebrate Marine Animal.

Elasmobranchee

Torpedo marmorata

Mustelus vulgaris

Trygon violacea

Charax puntazxo

Cerna gigas

Crenilabrus pavo

Box salpa

Chelonia mydas

Colpochelya kempi

Caretta caretta

Thalassochelys caretta

Chelonia caonana

Cetacians

Pinnipedians

Invertebrate freeh water anima.

Anodonta cygnca

Astacus fluviatilis

Dytiscus marginalia

Hirudo modicinalis

Libelien larva

Libellen Imagin

Vertebrate fresh water animals

Anguilla vulgaris

Barbus fluviatilis

Leuciscus dobula

Cyprinus carpio

Tinea vulgaris. Elsox Ijcius

Salmo trutta

Polyodon spathula

Scaphirhynchus platyrhynchus

Lepidosteus osseous (L.) ("bar' ) —

Amia calva (L.) (landlocked)

Catostomus teres

Perca fluviatilis

Fresh water ganoids all have blood identi cal in concentration with fresh watei telcosts.

Rana eaculenta

Salamandra niaculata

Erays europaoa

Pseudemys elegans


Man

Cow... Horse. . .

Pig

Rabbit.

Dog

Cat

Sheep. .


-2.196

-2.312

-2.315

-2.31

-2.36

-2.29

-^2.24

-1.82

-2.03

-1.71


-2.182

-2.26

-2.36

-2.44

-1.04

-1.035

-0.74 -0.76

-0.82 -0.88

-0.675

-0.687-0.7

-0.69 -0.625

-0.61

-0.60

-0.65 -0.7

-0.65 -0.7

-0.20

-0.80

-0.57

-0.40 -0.43

-0.6

-0.65


-0.58 -0.475 -0.45

-0.512

-0.512

-0.62

-0.486 -0.503 -0.487 -0.608

-0.51 -0.498


-0.465

-0.479

-0.474

-0.48

-0.526

-0.585

-0.564

-0.615

-0.592

-0.571

-0.638

-0 619


0.69 0.558


0.50 0.507 •0.52

0.52 0.51


Woods Hole Beaufort

Newport river near Beaufort

Beaufort


Beaufort Beaufort Beaufort


Mississippi river Mississippi river .Vi ississippi river Mississippi river Mississippi river Mississippi river


Mississippi valley


OBSBRVBR


BottasEi

Bottassi

Bottazzi

Bottazxi

Bottaszi

Bottaxzi

Bottaszi

Garrey

Carrey

Garrey

Carrey HOber HSber Hober H6ber Hober Hober Hober Garrey Garrey Garrey Bottazzi


Hober Hdber H6ber Hober Hdber Hober

Hober Hober Hober Hober H6ber H6ber Garrey Garrey Garrey Garrey Garrey Garrey Carre V


Bottazzi

Bottazzi

Bottaszi

Garrey

Hamburger


290

SEA WATER AS A MEDIUM FOR TISSUE CULTURES


291


TABLE 2— Continued


IQ08 AND EICBBY08


A


OBSERVER


Frog Blood of frog


-0.46

-0.46

-0.045

-0.42

-0.215

-0.215

-0.230

-0 405


Backman and Runnstrom


Ovarian egg


Backman and Runnstrom


Fertilized unsegmented egg


Backman and Runnstrom


Earlv flrastrula


Backman and Runnstrom


Late gastrula


Backman and Runnstrom


Early medullary plate


Backman and Runnstrom


5 day embryo


Backman and Runnstrom


20-25 dav embrvo


Backman and Runnstrom


Chick Blood of chick


-0.63 -0.63 -0.45 -0.508


Bialaszewicz


Ovarian egg


Bialaszewicz


EfTor volk


Bialaszewicz


Embrvo incubated 6 davs


Bialaszewicz


8 days


-0.517 -0.523 -0.557 -0.560 -0.566 -0.601


Bialaszewicz


10 days

12 days


Bialaszewicz Bialaszewicz


14 days


Bialaszewicz


16 days


Bialaszewicz


18 days


Bialaszewicz

The records of Bottazzi are quoted from Hober ('06).

probably the culture medium most favorable for these animals would be one made up according to Fiihner, H. ('08).

To 1000 parts distilled H2O.

Sodiiun bicarbonate . 2 gr.

Calcium chloride dry . 2 gr.

Potassium chloride . 1 gr.

Sodium chloride 20 .

Urea 25.0

or else one made up from 66f sea water + 33^ distilled H2O (which has the same A as a 2 per cent NaCl solution) with sodiiun bicarbonate 0.02 g. and urea 2.5 g.

Also A. R. Koontz, who is now working on the fresh water muscles finds that manganese is probably necessary for certain fresh water muscles.

By actual experiment, as can be seen below, a solution which is slightly hypotonic to the blood plasma forms a more successful tissue culture medium than one which is exactly isotonic.

292


M. R. LEWIS


TABLE 3— GARREY


dilution: woods hole

8BA WATER \ + C.Cll.


' DISTILLED WATBB C.CM.


A-'c.


DENSITIES OF SEA

WATER DILUTIONS AT

21.5»C. (RBF. HtO

AT 21.5»C.)


NaCl WITH SAME QMS. IN 100 OC. or SOLUTION


ce.


re.


Undiluted -1.81


1.02426


3.04


85


15


-1.54



2.6


75


25


-1.35



2.275


66}


33i


-1.20



2.00


60


40


-1.09



1.81


50


50


-0.915


1.0123


1.58


45


55


-0.82



1.4


40


60


-0.73


1.0096


1.21


35


65


-0.64



1.07


33i


66}


-0.61


1.008


1.02


32


68


-0.595



1.00


30


70


-0.547


1.0073


0.91


25


75


-0.460


1.0062


0.76


20


80


-0.37


1.0046


0.60


10


90


-0.187


1.0023


0.30


Loomis '94 (quoted from Hober).


Mol per liter


Percentage of NaCl


A


0.01


0.058


-0 036


0.02


0.117


-0.071


0.03


0.176


-0.106


0.04


0.234


-0.141


0.05


0.293


-0.176


0.06


0.351


-0.211


0.07


0.410


-0.245


0.08


0.468


-0.280


0.09


0.527


-0.314


0.10


0.585


-0.348


0.20


1.17


-0.687


This is not actually true, for when all operations are carried on in a moist chamber, the isotonic solution is satisfactory, but since tissue cultures are usually made in the dry air of the laboratory where extensive evaporation takes place, the hypotonic solution gives more successful results. In fact a solution which is quite hypotonic may give good growth for the cells appear to absorb large quantities of water without injury, but a hypertonic solution retards the growth of the cells.

SEA WATER AS A MEDIUM FOR TISSUE CULTURES 293

For animals the i^ of whose plasma has not been determined, the sodium chloride content of the plasma may be used by means of table S-Garrey to find the dilution of sea water which is isotonic with that percentage of sodium chloride. The sodium chloride content of the plasma of many animals may be found by means of the table of Fiirth ('02), who gives an analysis of the plasma of many lower animals.

As Loeb, J., ('06) has suggested, the above solutions are in reality more protective than nutritive in their action and it is necessary to add some substance for the nourishment of the tissue, provided the culture is to be kept under observation for several days. Dextrose 0.1 per cent, 0.25 per cent and 0.5 per cent and a bouillon containing peptone^ serves for this nutritive substance.

One other point must be kept in mind in regard to a medium for tissue cultures and that is the fact that acid, even a slight trace of acid, interferes with growth and in order to keep the culture medium alkaline it is necessary to add about 0.02 per cent sodium bicarbonate.

The temperature at which the tissue cultures should be kept is that indicated by the body temperature of the animal in question, i.e., warm blooded animals at 39"^ C. and cold blooded at more or less the temperature of the normal environment of the animal. Although the temperature of the ocean is quite constant, necessarily that of the aquarium and also of tissue culture varies decidedly according to the room temperature and, while no tests were made, certain of the tissue cultures of cold blooded animals grew well at a room temperature which must certainly have been much higher than that normal for the environment of the animal.

^ 500 grams (one pound) finely chopped muscle is placed in a liter of distilled water and kept on ice for twenty-four hours. It is then cooked, strained and filtered and sufficient distilled water added to bring the fluid up to a liter again. 10 grams of peptone, pure (Witte's) 5 grams of NaCl are added and the medium heated to dissolve the peptone. It is then carefully neutralized, filtered and sterilized. This bouillon can be kept for days. In place of bouillon, 0.1 per cent or 0.2 per cent peptone alone may be used with satisfactory results.

294 M. R. LEWIS


SUMMARY


From the above it can be seen that a medium for tissue cultures can be formed as follows: 90 cc. of the dilution of sea water (or of Lockers solution), which is isotonic with the plasma of the animal from which the cultures are to be made, + 10 cc. of bouillon made from the muscle of the animal in question. It has been found that about 10 cc. of the bouillon, in a few cases 15 cc. or even 20 cc. were necessary, make the solution sufficiently hypotonic to offset any evaporation which may take place and at the same time to furnish the necessary nitrogenous food for the tissue. To the above 90 cc. of diluted sea water + 10 cc. of bouillon is added 0.02 grams of NaHCOs to neutralize the acid formed by the culture and 0.25 grams of dextrose to supply the energy for growth of the tissue. The cultures must be aseptic and should be kept in a warm chamber at 39° C. when made from the tissue of a warm blooded animal, or at the temperature normal for the animal when the cultures are made from tissue of a cold blooded animal.

EXPERIMENTS

Chick. In the case of the chick embryo (5 to 9 days) the sea water was made up from dry sea salt and possibly thus contaminated with various foreign substances. However that may be, the medium thus obtained gave successful growth, although on the whole the growth was neither as large nor did it contain as many mitotic figures as the growth obtained from a piece of chick limb bud when explanted into Locke's solution. Pieces of chick embryo heart explanted into the sea water medium did not survive as well as those in Locke's solution. The media most favorable for growth were 1) 30 cc. sea water + 60 cc. distilled water + 0.02 per cent NaHCOa + 0.25 per cent dextrose + 10 cc. chicken bouillon, and 2) 90 cc. distilled water + 10 cc. chicken bouillon + 0.9 per cent sea salt + 0.02 per cent NaHCOs + 0.25 per cent dextrose.

The growth from explanted pieces of the embryo limb bud is composed of abundant connective tissue and some muscle fibres

SEA WATER AS A MEDIUM FOR TISSUE CULTURES 295

(fig. 1), and in some cases a patch of epithelial membrane grows out in the midst of the connective tissue cells. The growth in sea water continued active for from 4 to 6 days and many growths were kept in a healthy condition for as long as 14 to 20 days by washing the culture every other day with a fresh drop of the same culture medium.

Contraction of the muscle fibres was not observed as was observed when the fibers grew out from a piece of chick limb bud and explanted into Locke's solution (Lewis, M. R., '15).

Fundulus, The media were made up from fresh sea water and the dilution which gave the largest growth was 40 cc. sea water to 60 cc. of distilled water. This is much more dilute than that given by Garrey (45 cc. sea water to 55 cc. distilled water) for the fundulus, but the above gave more successful results than did that of Garrey.

A fundulus egg, which contained an embryo just about to hatch, was placed in several successive dishes of sterile sea water by means of a sterile pipette and then from the sterile sea water into a dish of sterile medium 80 cc. of diluted sea water (40 cc. sea water + 60 cc. distilled water) + 20 cc. fundulus bouillon + 0.02 per cent NaHCOs + 0.25 per cent dextrose. In this dish the egg membrane was torn away as rapidly as possible and the embryo at once transferred to another dish of sterile medium, in which the embryo was cut into about six small pieces and each piece again transferred to a dish of sterile medium from which the hanging drop cultures were made in the usual manner (Lewis and Lewis, '15).

The cut surface of many of the explanted pieces simply became covered over by a growth of epithelial cells, but the pieces from the body of the embryo, especially those from the region where the yolk sac was attached, gave rise to the typical tissue culture growth along the cover slip. This growth spread out from the body wall as a thick, firm membrane. The epithelial cells, easily distinguished by their characteristic markings, formed the lower layer of this membrane, and over this grew numerous other cells, some of which were connective tissue cells and some appeared to be blood cells. The growth remained active for

296 M. R. LEWIS

SEA WATER AS A MEDIUM FOR TISSUE CULTURES 297

from 3 to 6 days. The muscle fibers continued to twitch spasmodically, but did not grow out as they did in the case of the chick. Few mitotic figures were seen. No effort was made to prolong the life of the cultures by washing it with fresh medium.

Hermit crab. The medium most favorable for growth was as follows: 90 cc. sea water + 10 cc. crab bouillon which contained 1 per cent NaCl + 0.02 per cent NaHCOa + 0.25 per cent dextrose. Very few of the tissues of the hermit crab grew as tissue cultures. The best results were those from the first claw which was undergoing regeneration, although the membrane which forms the breaking joint and also the lining of the shell gave rise to good growth when explanted.

Figure 4 shows the type of growth which arises from an explanted piece of the membrane of the breaking joint or from the lining of the shell. These cells are much smaller than those of either the fundulus or of the chick growths. They never form a membrane. Pieces of the regenerating first claw explanted into the above medium gave rise to extensive growth composed of a small cell either connective tissue or epithelium and a few muscle fibers. Unfortunately no good fixed preparations of these growths were obtained for photographs.

Limulus. The explanted pieces of shell lining gave about the same results as did those of the crab and the medium used was the same as that for the crab. The heart and skeletal muscle

Fig. 1 Photograph of a 4-day growth from the limb bud of a 6-day chick embryo explanted in sea salt 0.9 per cent -h NaHCOa 0.02 per cent -h dextrose 0.25 per cent -f 90 cc. distilled HjO -f 10 cc. chicken bouillon. Connective tissue and radiating muscle fibres. Osmic acid vapor fixation and iron haematoxylin stain. X 38.

Fig. 2 Photograph of a 3-day growth from a piece of fundulus embryo explanted into 80 cc. of diluted sea water (40 cc. sea water -f 60 cc. distilled H2O) H- 20 cc. fundulus bouillon -f 0.02 per cent NaHCOs -f 0.25 per cent dextrose. The membrane is not contracted. Osmic acid fixation and iron haematoxylin stain. X 38.

Fig. 3 Same as figure 2 only the edge of this membrane is contracted.

Fig. 4 Photograph of the growth from a piece of hermit crab breaking joint membrane explanted into 90 cc. sea water -h 10 cc. crab bouillon which contained 1 per cent NaCl -h 0.02 per cent NaHOCj + 0.25 per cent dextrose. Osmic acid fixation and iron haematoxylin stain. X 50.

298 M. K. LEWIS

gave rise to only a few wandering cells and the pericardium gave rise to many active wandering cells, the exact nature of which was not determined.

Sea anemone. The medium used was as follows: 95 cc. sea water + 5 cc. distilled water + 0.02 per cent NaHCOs + 0.25 per cent dextrose + 0.1 per cent peptone. Pieces of the septa explanted into this medium gave rise to an abundant growth, which was composed of a substance probably mesogloea and numerous scattered endoderm cells. The cilia along the edge of the septa remained active for many days in the cultures.

Grasshopper. As there was no analysis of the grasshopper's plasma foimd, the most favorable dilution of the sea water for growth of the grasshopper tissue was determined by a series of trials of various dilutions. 30 cc. sea water + 50 cc. distilled water + 20 cc. grasshopper bouillon + 0.02 per cent NaHCOs + 0.25 per cent dextrose was the medium which gave the best results.

The only tissue studied carefully was that of the testis, and it was found that not only the cell within the follicles, but also the isolated sperm cells, remained in a healthy condition and continued to divide in this medium. The behavior of the cytoplasmic structures of the sperm cells were studied carefully and will be given in detail in a separate publication.

The results from the cultures made do not justify any conclusions as to the comparative value of any one medium for tissue cultures, but they do show that the dilution of sea water affords a simple and exact way by means of which can be obtained a medium, which is not only isotonic with the plasma of any given animal, but which also contains the necessary salts in the same proportion as does the plasma of the animal.

Woods Hole. Maaearhuselts September, 1915

SEA WATER AS A MEDIUM FOR TISSUE CULTURES 299

LITERATURE CITED

Abderhalden, E. 1906 Lehrbuch der Physiologischen Chemie.

Fredericq, L. 1878 Recherches sur la Physiologie der poulpe commun (Octopus vulgaris). Arch, de Zool. experim. T. 7, p. 535.

FOhner, H. 1908 UbereineSpeisungsflussigkeitfiirselachierherzen. Zeitschr. f. allg. Physiol. Bd., 8, p. 485.

FthiTH, Otto 1903 Chemiche Physiologie der niederen Tiere.

Garret, W. E. 1905 Twitching of skeletal muscle produced by salt solution with special reference to twitching of mammalian muscles. Amer. Jour, of Physiol., vol. 13, 3.

1905 Biol. Bull., vol. 8, p. 257.

1915 Some cyoscopic and osmotic data. Biol. Bull., vol. 27, no. 2, p. 77.

Hamburger. 1902 Osmotischer Druck und lonenlehre. Bd. 1, p. 459.

Henze, M. 1910 Untersuchungen an Seetieren. Handbuch d. Biochem. Arbeitsmethoden, vol. 3, p. 1109.

Herbst, C. 1903-04 Uber die zur Entwicklung der seeigellarven notwendigon anorganischen Stoffe, ihre Rolle und ihre Vertrebarkeit. Arch, fiir Entwickmechan., Bd. 17, p. 306.

HdBER, R. 1906 Physikalische Chemie der Zelle and der Gewebe.

Lewis, M. R. and Lewis, W. H. 1915 Mitochondria und other cytoplasmic structures in tissue culture. Amer. Jour. Anat., vol. 17.. no. 3, p. 339.

Lewis, M. R. 1915 Rhythmical contraction of the skeletal muscle observed in tissue culture. Amer. Jour. Physiol., vol. 37, no. 1, p. 153.

Loeb, J. 1906 The dynamics of living matter. 1913 Artificial parthenogenesis. EXPERIMENTAL MESOTHELIUM

WILLIAM COGSWELL CLARKE

Department of Surgery j College of Physicians and Surgeons j Columbia University,

New York

ELEVEN FIGURES

In a normal animal that survives, repair and sometimes regeneration follows the destruction of tissue. The phenomenon of regeneration, that results in the restoration of structure and function has long been studied and discussed.

Following a physical injury which destroys the free surface cells of the peritoneum or pleura, or the lining cells of blood vessels, two possibilities exist as to how regeneration of the damaged zone proceeds. 1) Cells may grow from the periphery of the given denuded area, taking origin from adjacent, previously existing and intact flat surface cells; or 2) the exposed connective tissue cells of the floor of the injured area may proliferate, be changed in form and become flattened. In the second possibility, the pressure of the opposed surface of the smooth normal peritoneum covering either intestine or other organ is the physical agent, acting continually, that is premised as tending to flatten the surface cells of the damaged area. The pressure and friction of the rushing stream of blood is premised as acting in a similar manner upon the exposed proliferating cells following the destruction of the lining endothelium of a vessel. If there exists a parietal coagulum, reparative cells may invade it and become flattened by the blood stream when the surface of the coagulum is reached. If no coagulum is attached to the denuded area the proliferating cells are immediately exposed to the pressure and friction of the stream of rushing blood. Active friction and pressure between surfaces physically dissimilar, will always result in a mutual change in form until a physical equilibrium is established between them.

301

302 WILLIAM COGSWELL CLARKE

The experiments were undertaken in reference to the second possibiUty; namely, that the exposed deep connective tissue cells making up the floor of the injured area proliferate, change in form and become flattened; thus resulting in the so-called regeneration of a lining membrane in the repair of the surface cells. In other words, an attempt was made to ascertain what change in form takes place in the investing living connective tissue cells in contact for a time with the surface of various, relatively non-irritating, foreign bodies.

EXPERIMENTS

Celloidin is readily moulded into any shape, and after drying may be sterilized by boiling. At the same time celloidin, after the alcohol and ether have evaporated, sets free no chemicals in the dry state when placed in the tissues. Since 1910 more than sixty experiments have been done in which celloidin has been placed in the tissues of several varieties of animals and allowed to remain even as long as one year before removal for study. Its use has always resulted in the formation of a minimum amount of adventitious tissue about the foreign body. After the cellular gathering had disappeared, that is found in and about tissue debris immediately after the operative procedure, neither leucocytes nor so-called 'giant cells' were found in proximity to the celloidin. Celloidin was therefore accepted as relatively ideal non-irritating foreign body for use in operative procedures and experimentations in living tissues.

Experiment No, 1. Celloidin foreign body. Duration 28 daysDog. Animal No. 33. Operation October 3, 1913. Through a small wound of the skin of the abdominal wall, the subcutaneous tissues were divulsed by thrusting into the aerolar tissue a closed artery forceps, opening the instrument, and withdrawing it while forcibly held open. This injury produced a long, relatively dry tract. Following the introduction of a thin, sterile, smooth sheet of celloidin 2" xY '^ Vy the tissues immediately collapsed permitting an easy closure of the skin wound. The wound healed by primary union and the thin strip of celloidin could be felt beneath the skin.

The foreign body together with the adventitious tissue, was removed October 31, 1913, and the specimen (Surg. Path. No. 2642) was fixed in formalin. Paraffin embedding. Sections were cut at

EXPERIMENTAL MESOTHELIUM 303

right angles to the free surface. The surface cells, as the section shows, were elongated, closely disposed and parallel to the free surface. Tangential sections were also cut to demonstrate the free surface cells or those in contact with the foreign body, the celloidin strip. The cells were close together and were proven to be flat since the two planes in which the sections were cut were at right angles to each other. Compare figures 2 and 3 with figure 1.


Fig. 1 Photomicrograph, mag. 900. Experiment 28 days. Cross section, showing surface cells in contact with a solid, smooth, flat, foreign body (celloidin). Specimen from subcutaneous tissue of dog No. 2642. See A. A.

Since a histological criterion of mesothelium is a mosaic of black silvered lines, which results when the fresh peritoneum and certain other free surfaces are treated with silver salts, this test was made in the next experiment.

Experiment No, 2. Celloidin foreign body. Duration 21 days. Dog. No. 104. Operation December 5, 1913; a repetition of Experiment No. 1, up to the point of excision of foreign body and adventitious tissue which was done on December 26, 1913. The tissue (Surg. Path. No. 2744) enclosing the celloidin was split open, particular care being taken not to physically injure the specimen. The tissue in contact with the smooth surface of the celloidin was smooth and glistening. A square of the lining surface was placed for one hour in a protargol solution of about 20 per cent. The specimen was then placed in 40 per cent alcohol and exposed to the sunlight. As soon as the

THE ANATOMICAL RECORD, VOL. 10, NO. 4

304 WILLIAM COGSWELL CLARKE

tissue had become a deep brown, frozen sections were cut tangential to the free surface that had been in contact with the celloidin plate. The mosaic of silvered Unes in this free surface is shown in figure 4.


Fig. 2 Mag. 450. Experiment 28 days. Tangential section showing surface cells on the flat in contact with a solid, smooth, foreign body (celloidin). Specimen from subcutaneous tissue of dog No. 2642.

EXPERLMENTAL MESOTHELIUM 305

This silver mosaic demonstrated conclusively that the surface is covered by a complete layer of cells, and that the intercellular substance, as far as the silver reaction is concerned, is similar chemically to the


Fig. 3 Mag. 1000. Experiment 28 days. Tangential section showing surface cells on the flat in contact with a solid, smooth, foreign body (celloidin). Specimen from subcutaneous tissue of dog No. 2642.

306 WILLIAM COGSWELL CLARKE


Fig. 4 Mag. 500. Microphotograph experiment. Silver mosaic in the free surface of tissue in contact with the smooth surface of celloidin. Specimen from the subcutaneous tissue of dog No. 2744. See A.

.* ' intercellular substance of the peritoneum, pleura and endothelium of blood vessels.

Paraffin has been used for years to fill cavities in the tissues, and this material forms a solid foreign body, comparativ/gly non-irritating.

Experiment No, 3. Paraffin foreign body. Duration 19 days. Rabbit. Operation December 18, 1911. Melted paraffin was in-jected through a fine needle into the substance of the cornea. This injection mass spread out in a triangle with the apex at the sclerocorneal junction, the point where the needle had been introduced, and extended outwards over the pupil. The paraffin presented a feathery

EXPERIMENTAL ME80THELIUM 307

appearance in the transparent cornea. The paraffin had been split up into small isolated columns and rod-like shapes when shot into the tissues. These shapes had been preserved when the paraffin solidified. For four days new blood vessels running in from the sclero-corneal junction were seen on the outer margin of the paraffin mass. All these vessels diminished continuously and had disappeared when the cornea was removed, January 6, 1912 (Specimen Surg. Path. No. 1838).


Fig. 5 Photomicrograph, mag. 600. Section of cornea, at pupil. The connective tissue cells (syncytium) surround particles of paraffin. No. 1838. See A.

Microscopic examination: Serial sections through the cornea over the pupil show spaces in the tissue, elongated, oval or round. All these spaces have a distinct outline. They correspond to the situation of the needle-like masses of paraffin in the cornea. Either cells or syncytial masses form definite walls to these spaces (fig. 5). Though the connective tissue cells of the cornea are parallel to its surface, all the cells and syncytial masses bounding the paraffin, conform to the shape of the foreign bodies. In certain of the syncytial masses the nuclei are so numerous and so disposed as to form a structure often noted as a giant cell, of the type known as 'foreign body giant cell.'

This experiment demonstrates that cells become flattened, form syncytium, and adapt themselves to solid foreign bodies; also that giant cells form in tissue purely fibroblastic. Even

308 WILLIAM COGSWELL CLARKE

though blood vessels appeared in the cornea on the margin of the sclero-corneal junction, microscopically but few capillaries were found in the sections. The left hand opening A, in fig. 5, is undoubtedly a flat syncytium around a paraffin mass.

In addition to the experiments in which solid foreign bodies were used, fluids were also tried. The mucous secretion from the gall bladder, free from bile, seemed fitted for such an experiment. The following experiment, No. 4, was tried, therefore, in order to observe the cells that would line a canal, carrying a bland fluid, constructed through the mesodermal tissues of the abdominal wall.

Experiment No. 4. Foreign body fluid. Duration 28 days. Mucous fistula. Dog. No. 162. Operation December 23, 1912. After the cystic duct had been obliterated between two ligatures, a fistula was established from the gall bladder into and through the abdominal wall to a point in the animal's left flank. This biliary fistula was produced by suturing a rubber tube, -^ inch in diameter, into the gall bladder and permitting it to discharge at the skin opening. At the end of five days the tube was removed. There was a copious discharge of sticky mucous which at times was clear, at other times cloudy.

January 20, 1913, second operation. Two inches of tract were excised (Surg. Path. No. 2289) and fixed in Mann's fluid. Celloidin sections were cut across the tract.

Microscopic examination showed that there was a semi-circular opening in the tissue. The stroma around the canal was exceedingly cellular and contained many small blood vessels. In this stroma, polymorphonuclear leucocytes were numerous, and were grouped about the fistulous tract. Within the tract there were also aggregations of leucocytes. The walls of the fistula were distinct and sharply outlined. The cells of what was certainly the walls of the tract in the living animal were large, oval or elongated, and flattened, with large nuclei.

These cells were so arranged that the long axis was parallel to the wall of the tract. The connective tissue cells beneath the surface were more nearly round and were arranged without reference to the wall of the fistula. The surface was almost exclusively cellular, though at isolated points the wall consisted of a Aground' or intercellular substance. In places, as shown in figures 6 and 7, the lining was a single layer of cells with elongated nuclei. The tissues of the fistular wall was here actually syncjrtial in character since no intercellular margins were made out. Fifty serial sections, eight micra in thickness, demonstrated that closely disposed cells lined a larger part of the canal. At points the lining wall was either debris and leucocytes or coagulated exudate, so massed that the actual structure of the wall could not be made out.

EXPERIMENTAL MESOTHELIUM 309


C

o


c o


o

. <

CO

00 . CM 05


g 6


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bb S 03 CO

^ 00

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Fig. 7 Mag. 600. Experiment 28 days. Cross section of walls of mucous fistula showing surface cells. Specimen from subcutaneous tissue of dog. No. 2289. See A. A.

310

EXPERIMENTAL MESOTHELIUM 311

In order to obtain a cavity containing serum and not fibrin, 'dead spaces' were studied that frequently obtain where there has been slight inequality in the coaptation of the tissues when operative wounds are closed. Blood clots generally obliterate the greater part of such wound clefts, but the subsequent contraction of the clot and the consequent accumulation of exudate fluid often gives rise to these serum filled spaces. The following operative himnan case, and experiment No. 4 on a dog, demonstrate such conditions in the tissues, and show the type of lining cells of such cavities.

Case 1, Fluid in contact with tissue seven days. *Dead space,' Incised wound of thigh. Human adult. Operation by Dr. J. A. Blake, November 21, 1906. Exploratory incision through soft tissues of thigh to the periosteimi of the femur. No clinical signs of inflammation developed. Amputation at the hip November 28, 1906, for subperiosteal sarcoma. Specimen (Surg. Path. No. 233) fixed in formalin. Cross section of skin and subcutaneous tissues with exploratory wound revealed a fluid filled space, superficial to deep fascia, about 0.3 cm. in diameter. This space was the result of infolding or puckering of the walls of the wound at the time of the hurried closure. It contained fluid exudate with but little fibrin formation.

Microscopic examination: The wall of the space at points is made up of fibrin and masses of leucocjrtes. Elsewhere the tissue is thrown into hillocks. The majority of the structural cells are elongated fibroblasts which in numerous places parallel the walls of the dead space. Many of the cells on the surface are elongated fibroblasts, so disposed as to make a definite cellular wall or lining of the space. The stroma beneath the surface consists of large, round, connective tissue cells, with relatively large nuclei at right angles to the plane of the free smface of the space. The actual surface is a layer of syncytium, apparently fibroblastic in character. The nuclei are elongated, stain deeply, are close together in this surface structure, and show no signs of mitotic division. The general appearance of the lining cells is similar to that of the lining cells described in No. 1932 and in No. 2144, shown in figures 8 and 9.

Experiment No. 5. Foreign body: fluid. Duration 7 days. Dog. No. 33. Operation laparotomy, October 2, 1912. Experimental intestinal resection. Uneventful recovery. Autopsy October 9, several hours after death. Gross examination. The abdominal wound was not infected and the healing was by primary union. A section (Surg. Path. No. 2144) across the site of the operative wound demonstrated the irregular but well defined zone leading from the skin through the abdominal wall to the peritoneum, the tract of the healing wound. In the tissues just superficial to the deep fascia there was a cavity or

312 WILLIAM COGSWELL CLARKE

Mead space' measuring 0.8 by 0.5 cm. and extending along tract of wound. This cavity contained fluid. Specimens were cut from the wall. Parafl&n embedding. Microscopic examination. Observation with low power revealed a moderate number of coarse anastomosing strands of fibrin that but partially filled the cavity. Where the fibrin chanced to be attached to the wall of the dead space, the strands extended into the tissues between the cells; as in all healing wounds containing a blood clot. The fibroblasts were directed into the cavity wherever this fibrin served as a support. Where the wall was free from fibrin, sections demonstrated that topographically the surface presented oval or rounded elevations. High power observations of cross sections of the actual surface showed that the cells were large


\.


lig. 8. Fig. 9.

Figs. 8 and 9 Duration of experiment 7 days.

Fig. 8 Cross section of wall of 'dead space' showing at A A a complete surface layer of cells.

Fig. 9 Tangential section showing same surface cells, proving that the surface cells are closely apposed and are flattened. See A A.

and elongated, with relatively large, lightly staining nuclei. These cells are tangentially disposed on the free surface of the cavity, and therefore did not reach or point outwards, as did the surface cells at the point where fibrin strands were attached (fig. 8). Sections were cut tangential to the free surface through the wall of the cavity, where it was free from fibrin, to show the surface cells in an opposite plane to that of a cross section. The cells were found closely arranged and varied in shape from oval, elongated, to round; the nuclei were large, elongated and faintly staining, similar to the nuclei of the deeper fibroblasts. Thus it was positively shown that the surface cells were flattened and so disposed as to make a definite cellular covering to the wall (fig. 9). On comparison of a cross section of free surface cells with a tangential section, the cells are seen to be narrower in the plane

EXPERIMENTAL MESOTHELIUM 313

parallel to the free surface than in the plane at right angles to the free surface.

The two following experiments are with living tissue grown in the 'observation incubator/ Here it was possible to know absolutely from what tissue the cells were derived that finally enclosed a drop of fluid. In the first experiment, the photograph is of the stained tissue, but in the last experiment the tissue is photographed while alive and unstained.

Experiment No. 6, Cultivation of embryonal tissue in vitro. Drs. McWhorter and Whipple. Tissue from chick of 72 hours^ incubation. Specimen (Surg. Path. No. 2374), a small fragment of ectoderm and mesoderm was planted in coagulated plasma. Both the epithelium and the connective tissue proliferated, forming the type of structure shown in figures 10 and 11. As the connective tissue grew and advanced, it was observed encircling a vacuole in the plasma. The serum formed this vacuole as the fibrin around contracted. Since there was no fibrin within the vacuole, the proliferating connective tissue cells met with no support, and reacted to the vacuole as does tissue elsewhere in contact with a non-irritating solid or a fluid foreign body. This specimen was stained en masse and photographed.

Experiment No, 7. Cultivation of tumor tissue in vitro. Drs. McWhorter and Whipple. A small fragment of tissue (Surg. Path. No. 1942) from a sarcoma of a St. Bernard dog was planted in coagulated dog plasma. The connective tissue stroma of the tumor proliferated radially. At one point a vacuole in the mass of coagulated plasma was encountered. This vacuole apparently contained serum which had exuded from the coagulum. Figure 11 of the unstained living tissues demonstrates that the cell structure surrounds the vacuole as a syncytium. The large cell in the centre of the vacuole was moving on the surface of the cover slip, and not on the same plane as the wall of the vacuole. The focal distance of the lens at the magnification of 150 times makes it possible to show both the wall of syncj^tium enclosing the vacuole and the superimposed cell.

In this change in form of the cells that occurs in the tissues when in contact with a 'foreign body' it is probable that the cells are responding to a physical or chemical reaction in progress in the tissues. In substantiation of this belief the statement of Drew^ may be quoted, who working with pecten maximus experimentally implanted ovary in muscle tissue. He says

Drew, G. Harold. Experimental Metaplasia. Jour, of Exp. Zool. No. 10, 1911, p. 349.

3J4 WILLIAM COGSWELL CLARKE

Briefly summarised my results show that after implantation of a portion of the ripe ovary into the adductor muscle a layer of fibroblasts is formed around it and coincidently the ovarian tissue is invaded by phagocjrtes and degenerates. After the lapse of about six days no trace or organized ovarian tissue remains, but there is left a cyst surrounded by fibroblasts, and containing blood corpuscles and a


Fig. 10 Photomicrograph, mag. 150. Plant from chick embryo, in vitro culture. Stained mesenchymal cells surround a vacuole in the coagulated plasma. No. 2374.

quantity of small granules having the orange color of the yolk substance. After the lapse of about 20 days more, the innermost fibroblasts gradually change their shape, and form a layer resembling columnar epithelial cells which later become ciliated. Eventually the whole cyst becomes lined with well defined ciliated epithelium, which persists at least for 120 days, which is the longest period I have yet succeeded in keeping the animals alive in the experimental tanks. I

EXPERIMENTAL MESOTHELIUM 315

consider that there is some evidence to show that this change of the fibroblasts into ciliated epithelium is a reaction to the presence of some definite chemical substance within the cyst.

He further believes the fibroblasts of the implanted mass die and that the cyst lining is formed from the fibroblasts of the site of injection. He undertakes experiments to prove that the lining


Fig. 11 Photomicrograph, mag. 150. In vitro culture. Unstaiued growing tissue. The connective tissue cells surround a vacuole that occurred in the culture media. (Coagulated plasma) No. 1942.

cells of the cyst have not originated from the piliated cells of the transplanted oviduct, but from the cells of the host, at the site of injection.

Drew and.DeMorgan^ performed some experiments in pecten maximus to determine the origin and method of formation of the

Drew, G. Harold and De Morgan, W. The origin and formation of fibrous tissue produced as a reaction to injury in Pecten Maximus. Quart. Jour. Micro. Sci., vol. 55, part 3.

316 WILLIAM COGSWELL CLARKE

fibrous tissue formed as a capsule or wall about foreign bodies. They believe these structures arise from the fibroblasts in the walls of blood spaces and in the intermuscular connective tissue. The cells divide, migrate and arrange themselves in a concentric manner about the foreign body. These fibroblasts thicken and later present a somewhat stratified appearance. These observers believe the reaction to injury in the animal forms used is exactly similar to that which occurs in vertebrates.

In conclusion it may be stated the subcutaneous connective tissues react to the presence of a smooth surfaced, non-irritating foreign body, i.e., celloidine, in such a manner that there results a distinct pavement layer of flattened cells, the outlines of which are demonstrable by the impregnation of their intercellular substance with a silver salt. The tissues of the cornea, free from blood vessels, in contact with paraffin form similar flattened cells. Cells that are flattened and arranged to form a definite wall, lined the mucous fistula and the 'dead spaces' containing serum. It should be observed that all these results occurred in the connective tissue stroma of the structures injured by the experiment. Drew and DeMorgan obtained a reaction in the connective tissue stroma when foreign bodies had been introduced. This encapsulation of foreign bodies by connective tissue is a well known reaction of the tissues. The experiments with living tissues in the 'observation incubator' showed a similar cellular lining in the vacuoles in the media of coagulated plasma.

The above experiments show after repair is complete that the free surface cells of accidental spaces in the tissues are flattened and form a pavement. In other words after an injury, the connective tissue cells that are exposed become changed in the above manner. Therefore the second hypothesis premised in this article is tenable, namely, that the flat cells of serous surfaces and those lining blood vessels may regenerate from deep connective tissue cells and do not necessarily arise from adjacent intact mesothelial or endothelial cells.

yd

Ik' 1


A STUDY OF THE MORPHOLOGY OF THE INFERIOR

OLIVE

GEORGE B. JENKINS

Department of Anatomy, University of Louisville

SIX FIQURES

-r^"fc the suggestion of Dr. Mall I am making a preliminary repxiz^xl; on the morphology of the inferior olivary nucleus and its acc^^^sory nuclei; the portion of the work embodied in this repox-t was completed in the laboratory of Anatomy at Johns Hopkirx^ University in 1912 and 1913, the subjects used for this study w ex^^ selected from Dr. Mall's collection of human embryos.

T'iiis work was undertaken in the attempt to establish as defi^"*^^l3^ as possible, the external form and configuration of these ^^^^^^ar masses and their relations to each other, as a preliminary to ^ further study of the human brain-stem.

^"^ study of these masses, as found in the new-born, has already

J^^^"^ included by Miss Sabin, in her comprehensive work on the

^^^lla and Midbrain^ and a careful presentation of the adult

^T>^ y^y Weed,2 ^Qt^ workers having conducted their researches

^"^is laboratory. So it was determined to use, in this instance,

. - ^'^'i^lier stage of development as a comparison to ascertain, what,

^^^^ differences, would be manifested in the various ages. ^ ""^-^g upon the advice of Dr. Essick, who was familiar with the

g *^|^ cjoUection, Embryo No. 491 was chosen for this study, this

/(-^^^i^^^rien was a transsection of the brain-stem of a 280 mm.

g^ ~V^^ fetus, that had been cut, serially 40 micra thick and

^^^d with haematoxylin, and which offered a clear cellular

bjr f^^^^ atlas of the medulla and midbrain, by Florence R. Sabin, M.D., published

~ A^ Friedenwald Co., Baltimore, Md. bf^5 reconstruction of the nuclear masses in the lower portion of the human

li^^^^^^^tem, by Lewis H. Weed, M.D. Publication No. 191 of the Carnegie ^^^tion of Washington.

317

318 GEORGE B. JENKINS

differentiation, and such a stage of development as not to differ too widely from the subjects used by the other workers mentioned. This specimen was studied and modelled as described below; but since it only presented the results as seen upon crosssection, as did the work of both investigators mentioned, I planned to make a series of reconstructions, one in each plane, with the double purpose of gaining a more comprehensive knowledge of these nuclei, and at the same time checking up the accuracy of my work and the more certainly approximating the true form and appearance of the nuclei. Accordingly two other feti were selected, viz., No. 619, which was 200 mm. crown rump length; and No. 625 which was 220 nun. crown rump length. Both were chosen because they presented the nearest measurements to the original selection obtainable, the brain stem was removed, hardened, embedded in paraffin and sectioned serially 40 micra thick, in each instance. No. 619 was cut in the sagittal plane. No. 625 in the coronal plane, and both were stained with haematoxyhn and counterstained with erythrosin. These were then carried through the same steps as was No. 491, every other section was selected and marked, projected, traced, cut and piled in the same way as the original model, thus reconstructions of the nuclei were secured representing all three planes and a sufficient similarity in results was obtained to warrant the assumption that the original model was an accurate representation of the nuclei.

In the study and reconstruction of Embryo No. 491 every other section was drawn at a magnification of twenty-five diameters throughout the limits of the nuclei. In making each drawing the left half of each section was traced, extending the peripheral lines well beyond the median raphe the median line was carefully marked; a piece of stiff cardboard was cut 2 cm. in width, with parallel straight edges, one edge was fitted along the median Une, as marked on the tracing, along the other edge a line was drawn, thus making a straight-edge to pile by, each drawing was controlled by a comparative study with a higher magnification. Following this the drawings were traced upon wax plates, 2 mm. in thickness. Every fifth tracing was made upon

MORPHOLOGY OP THE INFERIOR OLIVE 319

a plate in which lamp-black had been incorporated with the wa-x mixture, each plate was then carefully cut around the periphery and numbered in serial order; (this method of reconstruction was first recommended by Bom, and has been described by Bardeen' and others as followed in this laboratory.

Having completed the tracings, a wooden form was built with one perpendicular, flat surface accurately squared with its base; upon this upright surface two parallel, perpendicular lines were drawn 10 cm. apart.

After all the plates had been cut out, they were piled in numerical order, orienting by the external form and by squaring the straight edges, which in the completed pile formed a flat surface. This side was then squared with the corresponding surface of the wooden upright and marked with two parallel cuts to correspond to the lines which had been drawn upon the wooden form. Following this, the nuclei were cut out in the first plate, leaving 'bridges' connecting the masses to the surrounding wax, and a plate fixed upon a glass slab. The wooden form was then placed against the straight edge so that the lines upon the box fitted against the cuts upon the wax plate, the base of the box indelibly outlined upon the glass slab and the box removed. After each succeeding plate had been cut and piled, orienting by the outline configuration, the form was placed in its marked position to complete the orientation, thus overcoming any tendency to 'puir the model one way or another. The form was then removed and the plate fixed to the pile with a hot knife, pins being used after a sufficient number of plates had been piled to afford the necessary bulk ; as the plates were piled cuts were made, front and back, so that a portion of the shell could be removed and the olive modelled to better advantage. This procedure was controlled by drawing a line across each side where the cut was to be made then in replacing the piece it could be fitted accurately by recompleting the broken lines. These methods were followed carefully throughout the

Bom's method of reconstruction by means of wax plates as used in the Anatomical Laboratory of the Johns Hopkins University, by Charles Russell Bardeen. Johns Hopkins Hospital Bulletin, vol. 12, 1901. THB ANATOMICAL BCCOBD, VOL. 10, NO. 4

320 GEORGE B. JENKINS

reconstruction. Frequent comparisons were also made, both with the tracings and with the section from which they w^re made. After completing the reconstruction, the model of the main nucleus was removed and studied in toto, the accessory nuclei had been anchored to the shell in order to separate them from the main nucleus when it was removed. It was found that the cephalic extremity of the ventromesial plate of the accessory nucleus was continuous with the upper part of the ventral lip pf the main nucleus. This connection had to be cut through before the olive could be removed from the shell. This same point of fusion was noted in all three reconstructions.

The completed model of the main nucleus (figs. 1, 2 and 3) is a flattened oblong mass, presenting ventromesial and dorsolateral surfaces, dorsomesial and ventrolateral borders, and upper or cerebral, and lower or spinal extremities; its greatest vertical measurement is 210 mm., its greatest width is 204 mm., and its greatest thickness 64 mm.

The ventromesial surface is convex in outline, both vertically and transversely, and narrows considerably in its lower threefourths, due to the encroachment of the hilus upon the ventral lip of the dorsomesial border. In the upper portion the two lips of the hilus are parallel for approximately a fourth of the way down, below this point the ventral lip recedes gradually, thus narrowing this surface below.

The dorsolateral surface is flattened in outline, though very irregular, as the convolutions and sulci are more marked upon this surface than elsewhere. This surface is more extensive than the ventromesial, and is quadrilateral in shape.

The ventrolateral border is broad, rounded and marked by the alternate ridges and depressions of the obliquely directed convolutions and sulci; this border, extending lateral to the pyramid forms the prominence on the ventrolateral aspect of the medulla, the olivary eminence. The rootlets of the hypoglossal nerve, pass first, between the hilus of the main nucleus and the accessory bodies, then curve lateralward passing between the pyramid and the ventromesial surface of the olive to appear superficially along the groove that separates the olivary eminence

MOHPHOLOGV OF THE INFERIOR OLIVE 321

from the pyramid, marking the position of the ventral margin of the ventrolateral border. This border is continuous with the surfaces along its rounded margins.


Fig. 1 Shows the ventroincsial surface. Note the narrowing due to the deficient ventral lip of the hiliis. //, marks the superior and 7', the inferior extremities. :!/, the dorsomesial and />, the ventrolateral borders. All the figures are from the olive of specimen Xo. 491 and they are enlarged 12^ diam.

The dorsomesial border, thinner than the ventrolateral, is the site of the hilus, which is a long, narrow, slit like opening between the leaflets, occupying almost the entire extent of this border. The hilus looks dorsomedialward and is embraced by the curv 322 GEORGE B. JENKINS

ing mass of the accessory nuclei. It is bounded by a prominent, rounded dorsal lip, which is practically a straight-edge, and is marked by numerous transverse elevations and depressions. The depressions are continuous with the tortuous sulci

H.


Fig. 2 Shows the ventrolateral border, //, marks the superior and T, the inferior extremities; V, the ventromcsial and D, the dorsolateral surfaces.

of the interior, and the elevations correspond more closely with the convolutions of the exterior, than with those upon the interior. This arrangement leaves a continuous, fairly smooth margin along the more dorsal portion of this lip. The ventral

MORPHOLOGY OF THE INFERIOR OLIVE 323

lip is prominent and parallel to the dorsal lip for some 50 mm. below the cephalic extremity, from this point the lip becomes thin and gradually recedes until it reaches a plane 42 mm. lateral to the plane of the dorsal lip. This widest measurement is found


L.


T. Fig. 3 Shows the dorsolateral surface. The lettering is the same as figure 1.

40 mm. above the lower extremity of the olive. From this point to the lower end of the hilus, the lip curves slightly medialward. The ventral lip, in its upper, prominent portion is similar in appearance to the dorsal lip, from this point downwards

324 GEORGE B. JENKINS

it is thin and fairly smooth upon both its internal and external surfaces along a narrow rim, exhibiting but little tendency to conform to the wrinkling so noticeable in the rest of the surface. At the point named on this border, 50 nun. below its cephalic extremity, it was continuous with the upper end of the ventromesial plate of the accessory bodies. Between the olive and the ventromesial plate, the intermediate accessory plate is interposed, this plate is narrow above at the point of fusion and is separated from the ventral lip of the main nucleus by a narrow interval. The width of this interval remains comparatively unchanged throughout so that the receding ventral lip is compensated for by the widening intermediate accessory plate. A study of a younger specimen, modelled at a later date than the subject of this report, shows the ventral lip of the hilus to be drawn medialward and united with the remainder of the ventromesial leaflet along a much thinned line, this line of tenuity corresponding to the future interval between the receding ventral lip and the lateral edge of the intermediate accessory mass. The fibre bundles of the hypoglossal nerve, which are increasingly prominent along the level of the lower three-fourths of the oUve, pass through this interval in their lateralward curve and probably cut away this accessory plate from the ventral lip of the hilus. The hilus extends from a point 24 mm. below the highest part of the upper extremity, to a point 22 mm. above the lowest part of the lower extremity of the main nucleus.

Both extremities are blunt and rounded, the inferior being less extensive than the superior, owing to the deficiency of the ventral lip below, where so much of the main nucleus has been sacrificed to form the accessory bodies. Both extremities present deep clefts and prominent elevations which are continuous with the gyri and sulci upon the surfaces, those of the lower extremity being more pronounced though less numerous than those of the upper.

The greater portion of the caudal extremity of the main nucleus is covered by the accessory bodies, which extend considerably lower in the braiii-stem than is the case with the main nucleus.

MORPHOLOGY OF THE IXF^ERIOK OLIVE 325

The general form of the olive, is, as Miss Sabin has so aptly described it, "that of a hollow shell with a wrinkled wall; the thinness of the shell being such that each external depression causes a corresponding elevation upon the interior, where the intervening depressions produce in turn the external convolutions; the tortuous outline on cross section is very marked as shown in figure 4 (which is from 491-16-3-1).

In describing the outline configuration of this nucleus, whilst there is a noticeable general similarity, certain features which are fairly constant, there is no hard and fast rule which will apply in every instance, since each nucleus presents certain modifi


Fig. 4 Emb. 491, slide 16, row 3, section No. 1 and shows tlie complicated pattern on cross-section.

cations of the general plan which are peculiar to itself. All of the inferior olivary nuclei, after an advanced stage of development is reached, present a complex pattern, due to the tortuous windings of its elevations and depressions, which have been more or less appropriately compared to those of the cerebral cortex, and like the cerebral pattern, it differs not only in different subjects, but also upon the two sides of the same subject, consequently the minute and detailed descriptions based upon the study of reconstructions are only to be considered as applying with any considerable degree of accuracy to the one model which has been the subject of the study. The following description is that of the model of Embryo No. 491, which was approximately

326 GEORGE B. JENKINS

an eight months fetus, and consequently, reasonably close, as to size and degree of development, to that of the new-born, and possibly not differing very widely from the adult type.

One marked difference between the cerebral pattern and that of the olive is, that due to the thinness of the wall of the nucleus, every marking upon the exterior has its complement upon the interior. The reverse being equally true, this fact presents an additional difficulty in the attempt to subdivide the surface of the olive into definitely outlined areas.

The markings upon the ventromesial surface and the ventrolateral border, are, for the most part, arranged as folds with intervening sulci, both of which are directed obhquely upward and lateralward from the ventral lip of the hilus. Upon reaching the dorsal margin of the ventrolateral border, they take a general transverse course across the dorsolateral surface toward the dorsal lip of the hilus, upon this surface though the sulci are deeper, more irregular and present munerous branches, and the folds or convolutions are more tortuous, being much broken by sulci, both by the main ones and their branches and by smaller unconnected ones, thus rendering the pattern more complex and more difl&cult to follow. In order to trace the pattern to better advantage, 1 had the entire exterior of the olive drawn as a continuous flat surface, just as one would open a book and lay it face down in order to gain a complete view of its backs (fig. 5). Reading from left to right with the caudal extremity of the nucleus toward you, there will be seen to be eight fairly continuous convolutions, separated by seven main sulci. The first sulcus is limited to the dorsolateral surface, beginning well forward upon the lower extremity. At the lateral margin of the surface it passes obliquely upward and medialward to the middle of this surface, then turns directly upward to end beneath an annectent gyrus which connects the first and second convolutions at a point a little above the junction of the middle and lower thirds of the olive. A small branch is given off from the medial side of this sulcus which curves upward in the first convolution. The first sulcus separates the first convolution from the second, save at the point mentioned. The second sulcus begins on the ven MORPHOLOGY OF THE INFERIOR OLIVE 327

tromesial surface, well back from the hilus, and passes obliquely upward and lateralward, then curving around the ventrolateral border passes upward and medialward, to end in a transverse fissure on the dorsolateral surface. This latter fissure limits superiorly, the annectent gyrus which connects the first and second convolutions, the second sulcus separates the second con Sup.


X

}.L.


Inf.

Fig. 5 The plane drawing of the entire exterior of the olive, the numerals 1 to 8 inclusive mark the convolutions; the letters a to ^ inclusive mark the sulci, Xf y and z mark the first, second and third transverse fissures upon the dorsolateral surface. Sup, marks the upper and InJ. the lower extremities; V.L. the ventral and D.L. the dorsal lips of the hilus. The continuous vertical lines indicate the margins of the ventrolateral border.

volution from the third. The third sulcus begins upon the ventromesial surface just short of the ventral lip passing obliquely upward and lateralward curves around the ventrolateral border with a marked upward trend, to end in the transverse fissure on the dorsolateral surface. A collateral is given off from the third sulcus which ascends and is lost in the third convolution. The third sulcus separates the third convolution

328 GEORGE B. JENKINS

from the fourth. The transverse fissure on the dorsolateral surface is very deep and extends well across this surface a little below its middle. It receives the second and third sulci and separates the third, and the connected first and second convolutions from the fourth. The fourth sulcus has the same course as the third until it reaches the middle of the ventrolateral border, where it divides into an upper and a lower limb, the upper limb is better defined and has a more marked upward trend. At the dorsal margin of this border it turns sharply downw^-rd and medialward to join the distal extremity of the lower limb in a second transverse fissure; the lower limb of the fourth sulcus, less distinct than the upper, pursues the same general course, but is almost effaced where it turns over the dorsal margin of the ventrolateral border, becoming more distinct on the dorsolateral surface, where it joins the upper limb in the second transverse fissure. The fourth sulcus audits transverse portion, separate the fourth convolution from the fifth. The second transverse fissure, really only the continuation of the united distal extremities of the two limbs of the fourth sulcus, is more broken than the first transverse fissure, and extends entirely across the dorsolateral surface at the junction of its upper and middle thirds. The fifth sulcus begins like the preceding one and presents a more marked upward trend. At the ventral margin of the ventrolateral border it gives off a lower limb which ascends and is lost in the fifth convolution, the main sulcus continuing obliquely upward and dorsalward to near the upper extremity of the olive, where it divides into a 'U' shaped sulcus, the short limbs of which embrace the curve of an annectent gyrus which connects portions of the fifth and sixth convolutions, the main portion of the sulcus separating these two convolutions. Upon the dorsolateral surface there is a secondary sulcus which has a downward and medialward course from the annectent gyrus mentioned, across the center of which a faint line apparently connects this sulcus with the fifth, this accessorj^ sulcus completes the separation of the fifth and sixth convolutions. The sixth sulcus begins almost at the margin of the ventral lip and is confined to the ventromesial surface. It ascends with only a slight

MORPHOLOGY OF THE INFERIOR OLIVE 329

lateralward inclination. At the junction of the upper and middle thirds of the olive this sulcus gives off a branch which ascends obliquely in the sixth convolution, curving on to the ventrolateral border to end near the upper extremity of the olive in a faint connection with the ventral limb of the *U' of the fifth sulcus. The main limb of the sixth sulcus ascends to near the upper portion of the ventromesial surface, where it unites with a transverse fissure; the main limb of the sixth sulcus and the transverse fissure separate the sixth convolution from the seventh. The transverse fissure marks the margin delimiting the upper extremity from the ventromesial surface, it is deep and irregular and continuous laterally with the main limb of the sixth sulcus. The seventh sulcus is deep and irregular, and pursues an almost vertical course from near the middle point of the ventral lip, to an annectent gyrus which connects the seventh and eighth convolutions. This gyrus separates the sulcus from the transverse fissure, and gives off from its medial side, a lower deep, and an upper shallow collateral sulcus which mark the eighth convolution. The seventh sulcus partially separates the seventh convolution from the eighth. Beginning well forward on the upper portion of the ventrolateral border is a deep cut which crosses the upper extremity and runs downward and medialward upon the dorsolateral surface, becoming irregular, and sending off numerous side branches. This sulcus at its beginning, splits the upper end of the sixth convolution, and ends beneath an overhanging prominence on the medial portion of the dorsomesial border. This prominence is continuous, over the upper end of the hilus, with the eighth convolution, and has been considered as a portion of that fold; the sulcus thus subdivides the sixth convolution and separates a portion of that convolution from the eighth.

The first convolution is irregular, marked by numerous short, deep depressions, rather semilunar in shape and includes almost the whole of the lower extremity and approximately the lower third of the dorsal lip of the hilus, and is separated from the second convolution by the first sulcus save at its two extremities, where it is connected by annectent gyri with the second convolu 330 GEORGE B. JENKINS

tion. The second convolution begins upon the lower extremity and ventrolateral border, where it is connected to the first convolution, it then turns upward upon the dorsolateral surface to end below the first transverse fissure by connecting with the superior extremity of the first convolution. This second fold is less irregular than the first, and is subdivided on the dorsolateral surface by a collateral of the second sulcus, and is bounded by the first and the main limb of the second sulcus upon its two sides, and by the first transverse fissure above. The third convolution is smaller than its predecessors. It is but faintly represented upon the ventromesial surface prominent on the ventrolateral border, and ends in a blunt, rounded extremity upon the dorsolateral surface. It is subdivided into three rolls by a fairly well-defined secondary sulcus below, and by the lower limb of the third sulcus above; separated from the second convolution by the deep second sulcus — ^which ends in the transverse fissure above — and from the fourth convolution by the main limb of the third sulcus. This limb turns around the upper extremity of the convolution to end in the transverse fissure thus sharply outlining this fold. The fourth convolution begins well forward upon the ventromesial surface, ascends obliquely, curving over the ventrolateral border to extend at first slightly downward, then transversely upon the dorsolateral surface. It is bounded by the third and fourth sulci and the first and second transverse fissures. At its beginning it is fairly smooth, being partially subdivided above by the lower limb of the fourth sulcus, but upon the dorsolateral surface it is broken and subdivided by secondary depressions into four irregular, connected folds. The fifth convolution presents a marked similarity to the preceding one, beginning as a fairly smooth prominence upon the ventromesial surface, it has a more marked upward trend than the fourth, and is broken above by a collateral from the fifth sulcus. It then becomes irregular in outline and curving over the dorsal margin of the ventrolateral border turns sharply downward, then transversely across the dorsolateral surface, where it becomes still more irregular, being broken into smaller folds by the offshoots from the second transverse fissure below, and from a secondary

MORPHOLOGY OF THE INFERIOR OLIVE 331

fissure above, this convolution is connected to the sixth near the upper extremity of the ventrolateral border by an annectent gyrus. The fifth convolution is bounded below by the fourth sulcus and the second transverse fissure, above, by the fifth sulcus and the secondary sulcus. The sixth convolution beginning upon the ventromesial surface passes almost directly upward to the upper extremity of the olive where it is split into two portions, the more ventral of which extends medially along the upper extremity to join the eighth convolution. The more dorsal fold turns sharply downward upon the dorsolateral surface, at the middle of which it turns upon itself to end in a rounded extremity beneath the overhanging eight by convolution. The sixth convolution is bounded by the fifth sulcus and the secondary sulcus previously described, above, by the sixth sulcus and the transverse fissure upon the upper extremity of the ventromesial surface. The seventh convolution is a narrow fold, limited entirely to the ventromesial surface. Beginning at the middle of the olive well forward upon the ventral Up, it ascends with a medial curve to the upper extremity of the surface where it is joined to the eighth convolution by an annectent gyrus just below the transverse fissure. This convolution is comparatively smooth, and is bounded laterally by the sixth and mesially by the seventh sulci and is limited above by the transverse fissure and the sixth sulcus at their junction, where it is connected to the eighth fold. The eighth convolution is of triangular shape and forms the mesial half of the ventromesial surface of the olive above. It is bounded laterally by the seventh sulcus, medially by the ventral lip of the hilus. At the upper extremity of the olive, of which it forms the highest part, it curves over on to the dorsolateral surface above, forming slightly more than the upper fifth of the dorsal lip of the hilus. The main mass of the convolution is very irregular where the tortuous collaterals of the seventh sulcus mark its surface. It is connected to both the sixth and seventh convolution by annectent gyri, the former appearing upon the upper extremity, the latter upon the ventromesial surface.

To gain an adequate conception of the wrinkling of the surface of the olive, it is necessary to study the individual sections

332 GEORGE B. JENKINS

themselves (fig. 4). These differ in complexity not only with the varying changes in the nuclear outline, but with the degree of shortening of the ventral lip. This shortening, most apparent in the lower levels, occurs upon the ventromesial surface, which is


Pf


VR


LP ---DR


- -IK X:-


Fig. G Drawing of the accessory nuclei. / is the inferior and *S the superior extremities. V.P. the ventromesial plate, T.P., the intermediate plate, and D.P. the dorsal plate. -V marks the position of the lower extremity of the olive. P.F. the point of fusion of the ventromesial plate with the ventral lip of the hilus of the main nucleus.

applied against the dorsum of the pyramid, being separated from that tract by the accessory nuclei.

The accessory bodies (fig. 0) are irregular, flattened plates, and are not wrinkled as is the case with the surface of the main nucleus. Collectively they consist of three plates, more or less

MORPHOLOGY OF THE INFERIOR OLIVE 333

fused in such manner as to embrace and form an incomplete covering for the hilus of the main nucleus. The combined mass of the accessory nuclei has the same vertical measurement as the main nucleus, viz., 210 mm. long, beginning above 34 mm. below the upper extremity of the olive and extending 34 mm. below the lower extremity, this lower extension of the accessory bodies is an irregular, fused mass presenting two isolated particles. One, the larger, is 41 mm. long, rounded in shape and situated upon the dorsolateral aspect of the olive, in line with and 27 mm. below the narrowed lower extremity of the dorsal plate. The other is a small, isolated nodule placed lateral to the mass. The main portion of the mass below the olive lays to the ventromesial side and is continuous above with both the ventromesial and intermediate plates.

The three plates which make up the mass of the accessory nuclei may be named from their relation to the dorsomesial border of the olive, ventromesial, intermediate and dorsal. The ventromesial plate is a long, narrow mass, flattened from without inward and is separated from the olive by the intermediate plate. The ventromesial plate is continuous below with the bulky lower mass of the intermediate plate passing upward. It crosses mesial to the intermediate plate to become continuous along its dorsal margin with the upper part of the dorsal plate, and on its ventral margin becomes continuous with the ventral lip of the hilus of the main nucleus as mentioned. The intermediate plate, broad and thick, fonns the bulky mass which extends below the main nucleus, extending upward ventromesial to the olive. This plate occupies the site of the deficient ventral lip of the hilus, being separated from the main nucleus by the hypoglossal fibres, as is also the ventromesial plate. Extending upward the intermediate plate is again fused on its mesial surface with the ventromesial plate above, and is joined by a narrow bridge, to the mesial margin of the dorsal plate. The intermediate plate forms the greater bulk of the accessory bodies. The dorsal plate begins farther cerebralward than the remainder of the accessory nuclei, and from this point extends downward as a broad, thin lamina

334 GEORGE B. JENKINS

which curves up over the dorsalateral surface of the ohve. It is contmuous at the upper part of its mesial margin with the ventromesial plate, and opposite the middle of the main olive is connected with the intermediate plate by an oblique bridge. This plate narrows at its lower extremity, the apex being in line with the isolated mass below.

Both the main accessory mass, as described, and the small associated bodies, present the same structural elements as are found making up the main nucleus.

HERNIA OF THE SMALL INTESTINE INTO A PERSISTENT GREAT OMENTAL CAVITY

JOHN LOCKE WORCESTER Anatomical Lahoratoryy University of Michigan

In the regular course of dissection in the Anatomical laboratory of the University of Michigan there was noted by the writer a subject in which after opening the abdominal cavity — with parts still in situ — it was observed that the greater part of the small intestine was lodged in a persistent cavity of the great omentum ; entrance to which had been gained by an opening in the left half of the transverse mesocolon in the region between the greater curvature of the stomach and the transverse colon. The rarity of this observation induced a search in the literature pertaining to similar conditions and it was found that relatively few cases presenting like relations and position of the intestines have been placed on record. A search of the literature was made difficult by view of the fact that the recorded cases are listed under various titles, such as internal hernias, hernia bursa omentalis, hernia Foramen of Winslow, hernia intermesocolica, Treitz hernia, etc. The author thinks that these cases should be called hernias of the bursa omentalis.

Since all cases of more marked deviations from the normal in the position of the different parts of the alimentary canal and the mesenteries has an interest both from the developmental and from the gross anatomical view points, the latter more particularly with reference to the diagnosis of pathological conditions and to surgery of the intestine, the case under consideration seemed worthy of record. The pertinent literature is given in very brief review and this before the case in question is presented.

By way of a very general summary, it may be stated that in only five of the recorded cases was a persistent cavity of the great

335

THE AXATOMICAl. RECORD, VOL. 10. NO. 4.

336 JOHN LOCKE WORCESTER

omentum noted; in four others, a partial separation of the adherent layers of the great omentum was mentioned; in twentythree of the recorded cases, the hernia entered the bursa omentalis by way of the epiploic foramen; in the same number of cases through openings in the transverse mesocolon; in three cases through openings in the walls of the great omentum; and in one case through a hiatus in the gastro-hepatic omentum. The small intestine was the viscus producing the hernia in forty-two cases, portions of the large intestine in three cases, and portions of both large and small intestines in five cases.

The cases recorded in the literature may be grouped into the following subdivisions of Hernia of the bursa omentalis:

a) Herniae entering the bursa omentalis through the foramen epiploic.

b) Herniae entering by way of an opening in the transverse mesocolon.

c) Herniae through openings in the walls of the great omentum.

d) Herniae entering the bursa omentalis by way of an opening in the gastro-hepatic omentum.

The first record of a case in which the hernia entered through the epiploic foramen was made by Blandin ('34). He observed at an autopsy a large segment of the small intestine in an opening in the transverse mesocolon, its point of exit from the bursa omentalis. These loops of intestine had entered the bursa omentalis through the epiploic foramen. Rokitansky (*42) reported having seen a large portion of the small intestine strangulated in the epiploic foramen. Treitz ('57) records a case in which two loops of jejunum were found in the lesser peritoneal sac, which was entered through the epiploic foramen. He stated that he had often seen parts of the large intestine, especially a hepatic flexure or abnormal loop of the transverse colon in tliis foramen. J. Wilson Moir ('68), as quoted by Cheine, observed most of the small intestine within the lesser sac having passed in through the epiploic foramen. Xovello ('81) reported a case where two meters of small intestine had passed through the epiploic foramen into the bursa omentalis. Mojoli ('84) observed a loop of the transverse colon in the epiploic opening. Elliott-Square ('86) found at an operation "eight inches of ileum, two feet from its termination," in the epiploic foramen. This foramen was large enough to admit two fingers. Treves ('88) reported an operation at which he found the caecum, ascending, part of the transverse colon, and between two and three feet of ileum, had passed through the epiploic foramen. He noticed that the caecum was of the undescended type, and had led the hernia into the lesser sac. A part of the hernia reappeared in the greater sac through an opening in the gastro-hepatic omentum. The appendix was found at the lesser

HERNIA OF THE SMALL INTESTINE 337

curvature of the stomach, near the oesophagus. Gangolphe (*90) found the greater part of the small intestine within the bursa omentalis which it had reached by way of the epiploic foramen. Neve ('92) observed at an operation that the ascending and part of the transverse colon had passed through a ring situated behind the stomach and to the right of the median line. It was large enough to admit two fingers, but he beheved it to be the epiploic foramen. Rehn ('92) reports having seen parts of thd small intestine in the epiploic foramen. Picardo ('93) saw a case where part of the small intestine was herniated through the epiploic foramen into the lesser sac. Stecchi ('94) had a case with small intestine coils within the small peritoneal sac which they had reached by way of the epiploic opening. Reynier ('97) writes of a case operated on by Dr. Wall and himself in which they found the greater part of the small intestine within the bursa omentalis. The epiploic foramen was the passage way for the hernia. Mori ('98) observed a case where part of the colon had passed through the epiploic foramen into the lesser sac. Rawitsch-Schtscherbo ('99) found in their case a diverticulum of the small intestine, nine centimeters long, in the foramen of Winslow. Groves and Martin ('00) had a case where parts of both small and large intestine were in a bursa omentalis hernia, entering by way of the foramen of Winslow. Adajaroff ('01) reported a strangulation of part of the small intestine in the epiploic foramen. Jeanbrau and Riche ('02) had a case where the duodeno-jejunal angle was the part in the epiploic foramen. Delkeskamp ('05) reports a case of a woman who after normal labor developed intestinal obstruction. At operation, it was found that all of the large intestine, except the sigmoid colon, and also coils of the small intestine had passed through the epiploic foramen into a ^^ peritoneal sac of the shape of a dilated stomach." He found a common ileo-caecal mesentery and a very long sigmoid mesocolon. Carwardine ('08) writes of a case in which he found two and a half feet of ileum forming a bursa omentalis hernia by passing through the epiploic foramen. Borsz^ky ('12) reported a case in which he found part of the small intestine passing through the epiploic foramen into the lesser sac and out through a slit in the gastro-hepatic omentum into the greater sac.

Of the recorded cases of hernia of the bursa omentalis in which entrance to the lesser sac was found through an opening in the transverse mesocolon, Loebl ('44) was the first reporter. He observed an opening the size of the fist about the middle of the transverse mesocolon through which the greater part of the small intestine had passed into the '* cavity of the great omentum." Diettrich ('47) reported a case in which there was an op?ning in the transverse mesocolon through which "all of the movable small intestine had passed into the bursa and out again through an opening in the omentum magniis (ligament gastro-colic) so that they hung in front of the transverse colon and free part of the great omentum." Deville ('51) observed a case in which an opening in the transverse mesocolon was found opposite to the body of the fourth lumbar vertebra. Through this had passed

338 JOHN LOCKE WORCESTER

coils of the small intestine into the lesser sac. The transverse colon descended as a sling to the level of the pelvis. Treitz ('57) reported two cases with openings in the transverse mesocolon through which the greater parts of the jejunums had reached the lesser sacs and lay l)etween the layers of the great omenta. Rembold ('65) saw^ in a case an opening in the transverse mesocolon through which two and a half feet of small intestine had passed into a space " between the liver and an hour-glass stomach" (bursa). Boettcher (76) in a case found an opening larger than the size of a fist in the transverse mesocolon. It had smooth borders, and was bounded by blood vessels. Most of the small intestine had passed through into the bursa, and lay between the layers of the free part of the grfe'at omentum — persistent great omental cavity. Furst ('94) reported a case with a nine centimeter opening in the transverse mesocolon through which one-third of the small intestine had entered the bursa. Hakonson ('94) saw in a case of hourglass stomach great defects in the transverse mesocolon and gastrohepatic omentum. Through the first of these openings, part of the colon and coils of the small intestine had entered the lesser peritoneal sac and returned to the greater sac through the second opening to hang down in front of the stomach. Laurer ('94) writes of a case possessing similar openings. The patient had an hour-glass stomach. Part of the small intestine passed through an opening in the transverse mesocolon and out of the bursa by means of the opening in the lesser omentum. Sundberg ('97) reports a case with a defect of the transverse mesocolon measuring five by ten centimeters. Through this the jejunum and most of the ileum entered the omental bursa. Here again the coils reappeared in the greater peritoneal sac through an opening in the lesser omentum. Akerman ('02) saw an opening the size of a fist in the transverse mesocolon of a patient at an operation. The greater part of the ileum, the caecum, and the lower end of the ascending colon had passed through this opening into the omental bursa and then part of the hernia returned to the greater sac by means of the epiploic foramen. Narath ('93) names the opening into the bursa in the case recorded l>y him the ^^recessus duodeno-jejunalis." Schumacher suggests designating it "the radix of the mesocolon transversum." Most of the coils of the small intestine passed through this opening into the bursa oment^lis, and then reappeared in an opening in the gastro-hepatic omentum, certain of the coils hanging in front of the stomach. Bastianelli ('04) describes a case of retroposition of the colon. The short transverse mesocolon had an opening through which a coil of the small intestine had passed. Schwalbe ('(H) found in a subject at autopsy a five centimeter opening in the transverse mesocolon through which most of the small intestine had passed into the omental bursa. Two coils occupied a separation of the layers of the great omentum at its upper part, the lower part of the membrane adhering to the abdominal wall. The transverse colon was of a long U-shape reaching to the pelvis. The limbs of the U ran parallel to the ascending and descending colon. The hepatic and splenic flexures were normal. Ihseche ('04) saw in a

HERNIA OF THE SMALL INTESTINE 339

case a four centimeter opening in the transverse mesocolon through which most of the small intestine reached the lesser peritoneal sac. Some of the coils returned to the greater sac through an opening in the gastro-hepatic omentimi and were in relation with the lesser curvature of the stomach. Chalmers ('05) reported a case as a "duodenal hernia through the recessus intermesocolica.'* He describes an opening in the transverse mesocolon bounded by blood vessels, and measuring about five centimeters in diameter. Two coils of small intestine passed through it into the lesser sac. Perman ('96) records a case having an opening four fingers wide in the transverse mesocolon to the right of the middle line of the body. Through this opening, all of the jejunum and most of the ileum passed into the bursa omentalis. A large opening in the lesser omentum permitted coils to reenter the greater peritoneal sac. Enderlin and Gasser (^06) report cases of intestine lying in the lesser sac. These were most likely far advanced types of "herniae mesocolic media." Mayo ('09) describes a case in which all of the movable small intestine, except the first three and the last twelve inches entered the bursa omentalis through the mesocolic omentum, and escaped through an opening in the gastro-hepatic omentum. In a second case he writes of an opening in the transverse mesocolon in front of the ligament of Treitz, measuring four inches. Five feet of jejummi passed through this opening, carrying with it a layer of the peritoneiun derived from transverse mesocolon and bulging out the gastro-hepatic omentum over the lesser curvature of the stomach. This last described hernia having other coverings than the walls of the bursa should probably not be classed as a true bursa omentalis hernia, but rather with those mentioned of Enderlin and Gasser. Prutz ('09) observed a case in which all of the movable small intestine except a terminal twenty inches of ilexim had passed through an opening in the transverse mesocolon into the bursa omentalis and then out through an opening in the lesser omentum to hang in front of the stomach. Schumacher ('09) reported finding in a patient an opening measuring five long by one and a half centimeters broad in the transverse mesocolon by way of which the jejunum entered the bursa omentalis and lay between the layers of the great omentum. Stoltzenberg ('10) saw in a case an opening measuring five inches wide in the transverse mesocolon through which the ileum had passed into the bursa. Many of the coils returned to the greater sac by an opening in the lesser omentum. The transverse colon was of a long U-shape, and reached the pelvis as in Schwalbe's case.

Under the variety of bursal herniae that enter by way of an opening in the walls of the great omentum, Baugrand ('60) reports a case with an opening in the great omentum through which the ileum had passed into its interior. Wandel ('03) had a patient with volvulus. At autopsy, he found a conamon mesentery and an opening in the front wall of the great omentum through which coils of the small intestine entered a cavity between the layers of the great omentum. Hilgreneiner ('03) presents the case of a similar hernia, except that the opening was in the lower part of the posterior wall of the great omentum.

340 JOHN LOCKE WORCESTER

The relative frequency in which openings in the gastrohepatic omentum give exit to herniae which have entered the bursa omentalis by means of openings in the transverse mesocolon makes one using such an opening to enter the bursa of special interest. Berg (^97) records such a case. He found a large opening in the gastro-hepatic omentum through which a meter of small intestine and a coil of the colon had entered the bursa.

A number of other cases were found in the literature consulted by the writer which were so indefinite in anatomic data that they are omitted from this brief review.

DESCRIPTION OF THE AUTHOR'S CASE

The subject was a well developed male; the record given to us gave his age as fifty-seven; the cause of death as rheumatism. Besides the condition about to be described, very interesting developmental defects were found in the vascular and urogenital systems. The heart received a right as well as a left superior vena cava, and the kidneys possessed double hili and ureters.

Upon opening the abdomen, part of the colon was observed lying anterior to the exposed part of the liver and stomach. On tracing this part of the colon beneath the left costal margin, it was determined that the gut was folded on itself at the oesophageal end of the stomach and then descended in front of the stomach and right portion of a very large great omentum. This descending part was thus parallel to the ascending colon. WTien this descending loop reached the level of the fourth lumbar vertebra it was folded under until it reached the right margin of the great omentum beneath which it disappeared.

Upon raising the great omentum to follow the colon it was discovered that all of the small intestine except the duodenum and the terminal ten centimeters of the ileum were within the great omentum, which thus formed a hernial sac. The intestine had entered this sac by an opening in the transverse mesocolon corresponding to the ventral surfaces of the third and fourth lumbar vertebrae. The dorsal border of the opening was found to be a thickened band, running parallel to which was an anastomosing branch between the middle and left colic arteries. On the anterior surface of the great omentum were relatively large arteries derived from the right and left gastro-epiploics. The colic

HERNIA OF THE SMALL INTESTINE 341

attachment of the great omentum was lunited to the left portion of the transverse colon, as seems to be the rule m cases with very long transverse colons.

Returning to the tracing of this last structure, it was found to cross the vertebral column behmd the omental sac and ascend parallel to the descending colon to a normal splenic flexure. Thus the transverse colon was of the U-shape described in the cases of Schwalbe, Stoltzenberg, and others. The caecum, ascending, and all of the transverse colon except the last described ascending limb were very much dilated and devoid of saculation. The colon measured one and a half meters, one-half of the length being in the transverse colon. The small intestine was normal except in relations. The stomach was small and concealed by the colon as previously described, except for a sinall area of the left side.

A long V- or U-shape transverse colon is not very rare. Huntington states that the relatively large fetal liver gives an oblique dkection to the developing transverse colon. With the latter's continued growth, it may form an arch with the summit in the pelvis. This long arch usually disappears with the relative decrease in size of the liver in the later stage of development. Where it persists in the adult it usually, as in the writer's case, forms a large part of the extra length of the large intestine found in these individuals. Transverse colons of this shape are normal in the baboon.

In the reported cases of bursal hemiae passing through openings m the transverse mesocolon in relatively few cases was a V- or U-shape transverse colon noted, so the author doubts any causal relationship.

From a study of recorded cases, it seems clear that more than one factor is responsible for a hernia of the bursa omentalis. Moynihan speaks of the frequency of four abnormalities noticed in these cases, viz., the presence of a common mesentery; the absence of fusion of the ascending mesocolon to the primary peritoneum of the posterior abdominal wall; unusually large epiploic foramen, and a very long mesentery. Unfortunately, so few of the reporters have given suflScient anatomical data

342 JOHN LOCKE WORCESTER

in their papers to suffice for statistics, that the writer finds himself unable to state that these conditions are frequent. None of the four abnormalities were present in the case under consideration.

The majority of the observers assign as the reason for the existence of the openings in the transverse mesocolon and omenta an atrophic process involving the area of the future opening and, this atrophy occurring so frequently in essentially the same places has led certain of these observers to postulate also that a slight embryonic defect in these membranes must mark the site for this atrophy.

Stoltzenberg suggests that such a defect in the transverse mesocolon would result from the rotation of the colon He reports having found in a four month fetus a small opening in the transverse mesocolon against which lay the two highest loops of the jejunum. Also that there was observed a relatively thin area in this location in the membranes of all fetuses and young infants he had been able to investigate at post mortems. Schwalbe also writes of having observed such depressions. The possibility has suggested itself to the author that such depressions might be explained by assuming that the arteries as they pass ventral in the membrane to reach the viscera cause slight ridges to appear in the mesenteries, the areas between appearing as depressions. The space between the anastomotic branches of the middle and left colic arteries might thus become such a slightly depressed area in the peritoneum. A number of observers have noted that these two arteries bound the opening found in the transverse mesocolon in these cases.

Stoltzenberg has pointed out that this area between these vessels may perhaps present a region in which the abdominal pressure would find a path of least resistance to what he terms the stomach-liver niche, located above the transverse colon. He fully appreciates that this pressure would be great in the relatively small abdominal capacity for the rapidly developing coils of the intestine as found in a fetus.

On the other hand, these bursal hemiae have so frequently returned to the greater peritoneal sac through an opening in

HERNIA OF THE SMALL INTESTINE 343

the gastro-hepatic omentum not bounded by vessels; in Berg's case, the hernia entered the lesser sac at this place, and in Blandin's case, in which the hernia, after having entered by way of the epilotic foramen, emerged by an opening in the transverse mesocolon; that the writer thinks this explanation of fetal pressure in an upward direction on vessel bounded depressions is inadequate. Postnatal pressure as a cause has perhaps been more frequent than prenatal. The clinical data in these cases show the frequency of ulcer, cancer, and hour-glass stomach in these subjects; conditions in which one would have violent vomiting and peristalsis to increase abdominal pressure.

The fact that in only a few cases have these hernias occupied the cavity of the great omentum which exists during the first and often the second year of life seems to the writer a strong point in favor of postnatal origin. Why may not gravity bring the intestinal coils of such a hernia down into this space instead of their remaining in the retrogastric portion of the bursa omentalis as is true in most of these cases? The presence of long and common mesenteries noted in a number of these cases would make this possible. Again, this form of hernia has not been observed in any subject in the first few years of life. The vast majority have been adults.

It seems plausible that in the cases where the hernias were found in a cavity of the great omentum these individuals possessed a cavity long after the normal time of fusion of the lamellae of the great omentum. When pressure atrophy effected an opening into the bursa omentalis of such subjects coils of the intestine would gravitate into the lowest part of that cavity.

344 JOHN LOCKE WORCESTER

LITERATURE CITED

Certain of the references given, it has been impossible to consult in the original.

AcKERMAN, J. H. 1902 Intraabdominaller Bruch durch eine oeffnung im mesocolon transversura. Nord. Med. Archiv. Chir. Heft 2, No. 9.

Adajarofp, Chr. I90I Operationoto letchenie na zapletenita tcherva. Medizinski Napredak. Nr. II and 12, p. 632.

Bastianelli, p. 1904 Strozzamento acuto di anse del tennue attraverso ad una fessura congenita del mesocolon traverso essendo il colon traverso in retropositione. II. Policlinico, sez. chir., vol. II, p. 56.

Baugrand 1860 Quoted from Duchaussoy, Etanglements internes. Mem. de Tacad. imp. de med. T. 24. p. 357.

Bero 1907 Zwei Falle von Achsendrehung des Magens, Verhandl. der Deutsch. Gesellach fur Chir., 33 Kongress, S. 213.

Blandin, Ph. Fred. 1834 Traite d'anatomie topographique ou Anatomic du Corps humain. 2 edit.

BoETTCHER, A. 1878 Hernia bursae omentalis mit im mesocolon tranversum befindlicher Bruchpforte. Archiv. fur Pathol. Anatomic (Virchow). Bd. 72, S. 642.

BoRsz^KY, Karl 1912 Pathogenese der Hernien der Bursa omentalis mit norraaler Bruchpforte. Beitrage zur klin. Chir., Bd. 77, S. 438.

Carwardine, T. 1908 Hernia through the foramen of Winslow. London Lancet. Part 2, p. 1213.

Chalmers, A. J. 1905 Duodenal hernia through rccessus intermesocolica. Jour, of Path, and Bact., vol. 10, p. 287.

Delkeskamp 1905 Zur Kasuistik der inneren Hernien, speciall der Hernia foraminis Winslowii. Beitrage zur Klin. Chir., Bd. 47, No. 3, S. 644.

Deville 1851 Bull, de la Societ^' anat. Paris, 26.

DiETTRiCH. 1847 Beitrage zur path. Anatomic. Viert^ljahrsschr. f. prakt. Heilkunde. Prag., No. 1, S. 125.

Elliott-Square, J. 1886 A case of strangulated internal hernia into the foramen of Winslow. Brit. Med. Jour., vol. 1, p. 1163.

Enderlen und Gasser 1906 Stereoskopbildcr zur Lehre der Hernien. Jena, S. 72.

FuRST 1894 Ett Fall af hernia intraabdominalis. Nord. Med. Arkiv. N. F., Bd. 4, H. 3.

Gangolphe, M. 1890 Hernia 6tranglee d travers 1' hiatus de Winslow. Lyon Medical, v. 64, p. 607.

Groves, H. J. F. and Martin, R. H. 1900 A case of hernia into the foramen of Winslow. Australian Med. Gaz., Sidney, No. 19, p. 413.

HERNIA OF THE SMALL INTESTINE 345

Hakonson. 1894 Fall af ventriculus bisaccatus med torsion af pyloris afdelningen samt egendomliga lage forandringar af narliggandc viscera. Nordiski Mcdicinski Arkiv. N. F., Bd. 4, H. 4, Nr. 21.

HiLGRENEiNER 1903 Zur Kasuistik der Hernia bursae omentalis. Prager Med. Wochenschr., v. 28, Nr. 43.

Huntington, Geo. S. 1903 Anatomy of the peritoneum and abdomen. Lea Bros., Philadelphia.

Ihseche, O. 1904 Innaugural dissertat on. Halle. Quoted by Stoltzenberg.

Jeanbrau, E. et Riche 1906 L'occlussion Intestinale. Rev. de Chir., v. 33, p. 618.

Laurer 1894 Ein Fall von Hernia mesocolonialis. In. Diss. Greifswald.

Loebl 1844 Zeitschr. d. k. k. Gesellschaft d. Aerzte zu Wein. 1. Jahrg. Bd. 1, S. 151-154.

Mayo, Wm. J. 1909 Mesocolic or rctrogastric hernia. Annals of Surgery, Philadelphia, no. 49, p. 487.

MoiR, J. Wilson 1868 Quoted by Cheine. Anatomical description of a case of intraperitoneal hernia. Jour, of Anat. and Physiol., vol. 2, p. 218.

MoJOLi, G. 1884 Storia di une occlusione lenta dell' intestine cagionate dal passagio e dallo strozzamento di un'ansa del crasso attraverso il foramen del Winslow. Rivista Clin, di Bologna, No. 4, p. 605-622.

Mori. 1898 Ernia pel foramen di Winslow. Gass. Med. Lombardo. Milano. V. 57, p. 257.

MoYNiHAN, B. G. A. 1906 Retroperitoneal hernia. London.

Xarath. 1903 Zur Pathologia und Chirurgie der Hernia Duodeno-jejunalis. Arch. f. klin. Chir., Bd. 71, H. 4.

Xevb, \. 1892 Hernia into the foramen of Winslow. London, Lancet, \o. 1, p. 1175.

XoRVELLO, A. 1881 Gas. med. Ital. prov. Venete. Padova. No. 38, p. 313-315.

Perman. 1906 Ett fall af intraabdominelt br&ck genom en oppning i mesocolon transversum och omentum minus. Hygiea, 2 F., Bd. 6, H. 5, p. 467.

PiCARDO, J. S. 1893 Un caso de hernia interna retroperitoneal por el hiatus de Winslow. Rev. de la Socied. Med. Argentina, Buenos-Ayres.

Prutz, W. 1910 Verletzungen und Krankheiten des Netzes und Mesenteriums. Deutsche Chirurgie. Lief. 46k, S. 104.

Rawitsch-Schtscherbo 1899 Ein Fall von Undurchgangigkeit des Darmes bedingt durch Einklemmung eines Diinndarmdivertikels in Foramen of Winslow. Wojenno Medicinski Shurnal. April, p. 627.

Rehn 1892 Ueber Eehandlung des acuten Darmverschluss. Archiv. de Langenbeck, Bd. 48, p. 310.

346 JOHN LOCKE WORCESTER

Rembold 1865 Ein fall von Achsendrehung des Duodenum. Oester. Zeitschr. fur prackt. Heilkunde. Nr. 6u. 7, S. 108-124.

Reynier, Paul 1897 Hernia do Tintestin grele dans Thiatus de Winslow. Soc. de Chir., p. 627.

RoKiTANSKY, Carl 1842 Handbuck der Pathol. Anatomy. Wein, S. 218.

ScHUMAKER, E. D. 1910 Dcf Hernien der Bursa omentalis mit abnormen Eintrittspforten. Beitrage zur klin. Chir., Bd. 66, p. 507.

Stect'hi, R. 1894 Ernia retroperitoneale atraverso il forame del Winslow. La Clin. Chir. Milano, v. 2, p. 653-664.

Stoltzenberg, F. 1910 Uber Hernia bursa omentalis mesocolica. Arch, fur path. Anat.— Virchow. Bd. 201, S. 470.

Sundberg 1897 Hernia bursae omenti med brachport i mesocolon transversum och sekundart genomenbrott af br&cket genom omentum minus. Upsala lakaform. Bd. 2, H. 9, S. 562.

8chwalbe, E. 1904 Intraabdominal Hernia der Bursa omentalis bei geschlossenem Foramen Winslovvii. Arch. f. pathol. Anat. — ^Virchow. Bd. 177, S. 561.

Treitz, E. W. 1857 Hernia retroperitonealis, ein Beitrage zur Gesicht inneren Hernien. Prague.

Treves; Fredrick 1888 Hernia into the foramen of Winslow. London, Lancet, October 13, p. 701.

Wandel 1903 Uber volvulus des Caecum und colon ascendens. Mitt, aus den Grcnzgeb der Med. u. Chir. Bd. 11, H. 2, S. 53.

THE DEVELOPMENT OF ERYTHROCYTES FROM HEMOGLOBIN-FREE CELLS AND THE DIFFERENTL\T10N OF HE.\RT MUSCLE FIBERS IN TISSUE CULTIVATED IN PLASMA

P G. SHIPLEY

Anntomical Laboratory^ John^^i Hopkins University

TWO FKJURES

The results of the small amount of hematological mvestigation which has been done by the use of tissue cultures shows the enormous possibilities of this method when applied to the investigation of problems for the solution of which the examination of smears and the section technique have proved inadequate.

Among a series of cultures made last winter were some for which the explanted tiss.ue was taken from the area opaca of chick embryos — before the formation of blood islands and the elaboration of hemoglobin. All of these cultures were planted in the blood plasma of an adult hen after Harrison's method, and in some of them, hemoglobin-bearing cells developed from amoeboid colorless elements apparently of mesoblastic origin.

In all of the cultures (one hundred and twenty) there was vigorous cell proliferation and migration which began within an hour after the culture sUdes were placed in the incubator and continued for from three to four days. This growth was so exuberant and cell migration was so rapid that in twenty-four hours the layer of new cells was perceptible without the aid of a lens, and in forty-eight hours a ring of new tissue three or four mm. in diameter was formed about the original explant. In some cases long cords of cells grew off from the edge of this ring for a distance of two or three mm., and the cells at the ends of the cords spread out on the cover glass to form a secondary growth or colony connected to the original transplant and its surrounding ring of new tissue by a thick strand of cells.

347

348 p. G. SHIPLEY

It was probably this over-luxuriant cell growth with the resulting proportionate increase in the bulk of the culture and the accumulation of the waste products of the metabolism of the new and rapidly growing cells, which caused the appearance of signs of degeneration and the cessation of growth in such a relatively short time. "^Cultures of ordinary embryonic mesoderm in a blood plasma medium will grow without signs of degeneration for seven or eight days without subculturing or plasma renewal but the initial signs of growth and migration rarely appear before the culture is twenty-four hours old. Throughout the life of the transplant, growth is much slower in cultures of mesoderm than in these cultures from the area opaca of very young embryos.

Microscopic examination revealed the presence of mesenchymal cells in the new growth, but by far the greatest bulk of culture grown tissue was composed of cells which were evidently descended from the endoderm of the yolk sac wall. These (fig. 1) were huge cells spread out in a rapidly broadening syncytium-like membrane in which there were no visible boundary lines between the cells. Easily distinguishable from the mesodermal cells by their size, their huge nuclei, and the lacy appearance of their cytoplasm, these endodermal elements may be seen to be full of fat and yolk granules. In fixed preparation?? stained with Giemsa^s stain, a fine open-meshed spongiobasi plastin network was apparent, the threads of which had a granular appearance. The huge nuclei, homogeneous in the fresh cell, finely granular in preparations fixed in osmic acid vapor, were marked by the presence of two or three darkly staineci chromatic masses or basophil nucleoli.

The endodermal cells in fresh preparations had, in common with all other cells grown in plasma, a clear homogeneous ectoplasm at their periphery surrounding a granular endoplasm in which the paraplastic structures and cytoplasmic granulations were contained.

Scattered about among these endodermal cells and in the clear area of plasma about the transplant, young erythrocytes developed in many cultures from undifferentiated, highly amoeboid,

DEVELOPMENT OF ERYTHROCYTES 349

very small mononuclear elements, with a strong basophilic, sometimes vacuolated cytoplasm, and a small round nucleus with a coarse chromatin network (fig. 2a). The process of erythrogenesis was initiated by a cessation of amoeboid movement.

1

V ft


tU


Fig. 1 Edge of a 48 hour culture from the area opaca of an 18 hour chick. Fixed in absolute methyl alcohol. Stained with Giemsa. Zeiss drawing prism. Kompens ocular No. 8 and 4 mm. apochromatic objective. Three large cells from the yolk sac wall and a large number of culture grown erythrocytes, e, nuclei of the endodermal cells; pblz, primitive blood cells; peb, primitive erythroblasts; rbcj abnormal forms of young erythrocytes; ne, normal chick red blood cell.

The cell withdrew its processes and became round, the cytoplasm, however, still retained its affinity for basic stains (fig. 2b). The round hemoglobin-free cells still had the power of multiplying by mitosis, but usually hemoglobin was elaborated in

350 p. G. SHIPLEY

their cytoplasm without further division, with the resulting formation of the hemoglobin-bearing erythrocytes.

In this way a small number of normal red cells were produced in cultures, but most of the erythrocytes differed in many respects from the red blood cell as one jfinds it in the vessels of the embryo when it has developed under normal conditions in the4 S 6


C. i


$ M « 

T 8 9 10 11 u

15

}>


Fig. 2 Young forms of blood cells from the above preparation. Zeiss drawing prism. Kompens ocular No. 8 and 2 mm. apochromatic oil immersion objective, a, primitive blood cells. No. 1 is dividing by mitosis and has a vacuolated cytoplasm. 6, Primitive erythroblasts. The nuclei still have a definite chromatin network and the cytoplasm is polychromatiphilic. No. 6 is dividing mitotically. c, abnormal red blood cells with pyknotic nuclei.

blood islands or vessels, connective tissue or the embryonic liver, bone marrow or spleen (fig. 2c). In the first place, the erythrocytes were very much smaller than the normal corpuscle of the chick embryo, the average diameter being about 4 m. Again, they had not the oval shape which characterizes the red blood cell of the chick. Most of them were spherical, or round discs. Poikilocytes were common and the cytoplasm of some

DEVELOPMENT OF ERYTHROCYTES 351

was perforated by vacuole-like apertures. A few had polychromatiphil cytoplasm but none showed any evidence of stippUng.

Not less pecuUar were the results of changes which took place in the nucleus of the primitive blood cell and the young erythrocyte. In none of these small red blood cells does one see the characteristic long, oval nucleus, with its rounded ends, but the nucleus is represented by one or more small homogeneous deeply stained fragments or rests (fig, 2c). The chromatin of the original functioning nucleus seemed to be concentrated into masses within the nuclear area, the breaking down of which released them to scatter about through the cell cytoplasm. A . cell may contain six or seven of these small chromatic masses, but usually two or three are found in one cell.

The morphology of the abnormal young forms is remarkably like that of young mammalian red blood cells with pyknotic nuclei and 'Jollykorper' or homogeneous chromatin rests, but the physiological significance of this aberration from the normal is uncertain. The dwarfing of the blood elements has its beginning in the primitive blood cells which are very much below the size of the corresponding element when developed under normal conditions, biit the agents which produce this dwarfing we can only guess at.

The appearance of undersized, atypical forms among red blood cells developed under cultural conditions is not surprising. Indeed it is not to be expected that the development and differentiation of the highly speciaUzed cells of warm blooded vertebrates should proceed unaltered when cells as low in the scale as the protozoa exhibit pronounced deviations from the normal when removed from their accustomed environment and forced to grow on media to which they are unused.

These erythrocytes have come into being in a medium clogged with the waste products of the metabolism of a large number of rapidly growing cells, and furthermore, were, in all probability, insuflSciently supplied with oxygen, since oxygen penetrates these clots of plasma with diflBculty. Both of these conditions may have contributed toward the production of atypical dwarf

THE ANATOinCAL RECORD, VOL. 10, NO. 4

352 p. G. SHIPLEY

forms, but it will take further and careful study to determine which factor was chiefly responsible.

In connection with the genesis of dwarfed, abnormal red corpuscles in culture, it is interesting to note certain peculiar, probably degenerative changes in the form of normal chick red blood cells which have remained in vitro for some days in plasma clots. They are conamonest in cultures from the embryonic spleen after three of four days incubation, and they consist of the drawing out of the poles of the afifected cell into one or more long processes. One or both poles of the erythrocyte may be affected. These long, flagella-like projections appear, when examined in a fresh condition, to be beaded for all or part of their length and to terminate in a small round knob. They are, actively motile, whipping back and forth at the end in a manner suggestive of the movement of cilia. They are similar to, but probably not identical with, the flagella which Kite has described as occurring under special conditions about the periphery of the manamalian erythrocyte.

In some of these cultures, parts of the embryonic body were included in the transplants, and in these an interesting phenomenon was observable. After an incubation period which varied from twenty-four to thirty hours, the heart muscle developed, assimied its function, and began to beat rhythmically. Regularity in beat was not maintained for over twenty-four hours, after which time the rate became slower and slower until it ceased. Even after cessation, however the pulsation could, for a time, be reinduced by mechanical or thermal stimulation. Especially were the heart muscles sensitive to cold. Removal from the incubator to the cold stage of the microscope was answered by renewed and vigorous beating long after the embryonic cardiac muscle had ceased to respond to other stimuli. Biurows and others have shown often that heart muscle cells will contract rhythmically when isolated from contact with others of their kind, but these cultures show that the embryonic cell which is destined to become heart muscle will differentiate and begin to function even though removed from its normal environment before differentiation has begun.

DEVELOPMENT OP ERYTHROCYTES 353

While the blood corpuscles developed in these cultures of the primitive area vasculosa were for the most part abnormal in size and shape, at least a study of this series of cultures indicates that development and differentiation of hemoglobin-bearing cells is possible in tissue transplants removed from the normal environment to a culture medium^ and shows that with an improved technique we may be able to approximate a norm closely enough to solve by this method many of the problems of erythroand leukocytogenesis. ^Also the development of erythrocytes from amoeboid colorless ancestors and the speciaUzation and assumption of rythmic contracticity by embryonic mesoderm cells under cultural conditions, together with Harrison's demonstration of the outgrowth of the nerve fiber, show that the life of cells in culture is not merely a series of ^survival phenomena^ on a downgrade of progressive differentiation. THE USE OF EARLY DEVELOPMENTAL STAGES IN THE MOUSE FOR CLASS WORK IN EMBRYOLOGY

C. H. DANFORTH Department of Anatomy, Washington University Medical School

The primary object of the course in embryology oflfered in medical schools is undoubtedly to give the student an idea of the principal features of human development. Human material, however, is available for embryological study to only a limited degree. Consequently the instructor must look to other mammals and frequently to the lower vertebrates, or even invertebrates, for illustrative material. This means that a course in 'human embryology' must of necessity be to some extent a course in comparative embryology, but one in which, owing to the usual briefness of the course, each lower form is sUghted as much as possible in order to leave time for considering higher forms. Such a procedure robs the course of much of its value as a comparative study and, moreover, is in danger of leaving the student with a confused rather than a clarified conception of ontogeny in general. Under these conditions it seems desirable to select for the routine course in embryology the fewest number of forms that will illustrate all the essential stages in mammalian development.

The problem of selecting these few forms for study is by no means an easy one. The value of the pig for the study of organogeny is generally recognized. Suitable pig material, too, may sometimes be had for the study of the somites and germ layers, but in general the chick has to serve for this part of the course. For the study of germ cells, fertilization and cleavage, various forms, chiefly invertebrates, are used. The purpose of this paper is to call attention to the availabiUty, and suitability, of mouse material for the study of all steps up to the end of the blastocyst stage.

The earliest stages in a considerable number of mammals have been studied in detail and there has been found to be a fair degree of resemblance between the different forms. For medical embryology, and for the more extended courses in comparative embryology, there can be no question as to the desirabiUty of showing the student these mammalian ova sectioned in situ within the ovary and uterine tube. The chief obstacle to their use no doubt has been their inaccessibiUty. In the case of the mouse, however, there is a considerable literature'

A good bibliography will be found in G. Carl Huberts paper on **The development of the Albino rat, Mus norvegicus albinus. I. From the pronuclear stage to the stage of mesoderm anlage; End of the first to end of the ninth day." Memoir of the Wistar Institute of Anatomy and Biology No. 5, 1915.

355

356 C. H. DANFORTH

relating to the early stages and we have found that suitable material for class work may be obtained with Uttle difficulty.

We first made the experiment of using early stages of the mouse for class study in 1912, with results so satisfactory that the material •has been used with the three succeeding classes. With the hope that our experience may be of some value to others, the following brief account of the method of handling the mice and the average cost of the sections is presented. This discussion may be prefaced by a description of the early part of our embryology coiwse as it is being given at the present time.

At the beginning of the course male and female mice are dissected sufficiently to show the form and relations of the genital glands and ducts. Living sperm cells are then studied in salt solution. The ovary is dissected and examined imder hand lens or binocular microscope. Next spermatogenesis is studied in sections of the testis. In sections of the ovary the' student finds and draws a series of stages showing the growth of the ovum and development of the follicle. The last stage in this series shows a follicle in which the corona radiata cells have assumed the appearance seen at ovulation and surround an ovum which has given off the first polar body and formed the second maturation spindle. From the uterine tube and uterus sections are studied and drawings made of the following stages: pronuclei and second polar body, two and four cell ova, late cleavage, morulae and stages in the formation of the blastocyst. Beyond this stage mouse embryos are not so satisfactory owing to the elongation of the blastodermic vesicle and the excessive development of entoderm. Some of the later stages of implantation on the other hand are very instructive.

The prime essential for securing this material is a stock of vigorous mice of good breeding age, the best season for the work being in the spring from February to May. In our experience young females from three to four months old have proved most satisfactory. While older females occasionally breed regularly they are much less dependable. Detailed accounts of the methods employed in securing the material will be found in several of the papers referred to in the footnotes, but it may be well to summarize briefly a few points in the breeding habits of the mouse and to indicate what seem to be the best methods of taking advantage of them.

Female mice of proper age tend to ovulate approximately every twenty-one days during the spring months. They almost invariably ovulate within a few hours after parturition. This latter fact is important since it enables one to know the approximate time of an ovulation without the necessity of keeping any special records. We have found it most convenient to keep the mice in small groups consisting of one or two males and six or eight females. They may be fed on dog biscuits, cracked corn and oats, with a little salt and occasionally a few sunflower seeds. Whenever a female is seen to be pregnant she is isolated, and kept till parturition occurs. Suckling the young may prolong the duration of the next pregnancy by as much as 50 per cent

USE OF THE MOUSE IN EMBRYOLOGY 357

but it has not been shown definitely that it has any eflfect on the progress of development during the first four days in which period most of the desired stages are to be found. Nevertheless, since the young must be sacrificed anyway, we have made it a practice to kill them at once.

When yoimg mice are expected we have found it best to look in the* cages early in the morning and again just before leaving at night. Young are more frequently found in the morning, but a considerable numbMBr are born during the day. Not infrequently a female will be found in the process of parturition. Mark and Long find that the first polar body is formed and ovulation occurs from 13f to 28J hours after parturition. In getting these stages for the class it is best to select females that are found with young at the night inspection. These need not be mated and should be killed diuing the following forenoon. It will probably be found advantageous to leave these stages till the last since some of the ova expected to show fertiUzation wfll be found in reaUty to be in earUer phases of maturation.

For all subsequent stages insemination is necessary. Artificial insemination was practiced by Mark and Long* but for the purposes under consideration it has few advantages. The method that has proved most satisfactory in this laboratory is as follows. A few mature males are isolated and kept in separate cages. When it is desired to mate a female, she is put in the cage with one of these males. It is essential that the male be left in his home cage, otherwise he will lose much time in exploring the new surroundings. This is not true of the female. If the female is in the proper condition successful coitus usually occurs within 20 minutes. Coitus has been described in detail by Sobotta.* Following the sperm the contents of the seminal vesicles are discharged into the vagina forming a large, whit6 plug which may be easily detected for several hours. Taking advantage of this fact, the female may be left in the cage for an hour or more at the end of which time it can be definitely determined whether or not coitus has occurred. If the female will not mate within an hour she is removed and given another trial from six to twelve hours later. During the active breeding season only a few females refuse to mate. This procedure gives a much higher percentage of successful seminations than is reported for the artificial method and, unless extreme accuracy is required, takes less time.

The best time for insemination is probably about twenty-four hours after parturition. Five or six hours after the insemination ova in the tube will usually be found to contain two pronuclei. This is one of the easiest stages to obtain. Specimens taken at twelve hour intervals for the next four days give a fairly complete series of stages up to the formation of the blastocyst.

Long, J. A. and Mark, E. L. "The maturation of the egg of the mouse/' Carnegie Institution of Washington Publication No. 149, 1911.

• Sobotta, J. "Die Befruchtung und Furchung des Eies der Maus." Arch, f. Mikr. Anat., Bd. 45, pp. 15-93, 1895.

358 C. H. DANFORTH

It will be seen from the foregoing account that the securing of these stages requires no unusual skill or special apparatus. . All of this work may easily be done by an intelligent technician. The method outlined here has an advantage in that it results in a greater variety of stages and a reduced expenditure of time. With the exception of an occasional individual in which for some reason ovulation or semination have failed to occur every mouse killed yields some interesting stage. Both sides show ova in the same state of development, so that two sets may be obtained from each mouse.

The mice are killed either by chloroform or by decapitation. The ovaries and uterine tubes are then fixed in ordinary Zenker's fluid or the Mark and Long modification and subsequently imbedded in the usual manner. It is foimd desirable to cut the sections 8 micra thick. An ovary and uterine tube yield sections enough for two 36 x 75 mm. slides. It would be an easy matter to separate the ovary and tube, in which case either one would go on a single slide. Kirkham* gives a method for locating the position of the ova before the slides are finished. By this means small slides could be used and occasionally several specimens could be made from one tube. Alum haematoxylin and eosin give a satisfactory stain.

It is in the small size of the parts to be sectioned, the possibility of getting any desired stage, and the ease with which the adults can be handled that the chief value of the mouse as a source of embryological material lies. The current price of adult mice is from 20 to 25 cents each and we find that it costs about 4 cents a month per mouse for maintenance. Including the cost of males and unproductive females our experience indicates that the average total cost of each usable ovary and tube, properly fixed, stained and mounted ready for study is 54 cents.* This figure includes the cost of all reagents, slides, covers, etc., but does not take account of the time required. This is perhaps rather more expensive than would be the corresponding stages of an echinoderm, for instance, but it is so much more valuable that the additional expense seems justifiable. It might be mentioned incidentally, that in our laboratory course one specimen of each stage for every five or six students has proved adequate. At this rate, provided ten stages are used, the intial cost will be seen to be about $1 per student. Except for breakage, of course, the slides may be used indefinitely.

Kirkham, W. B. "The maturation of the mouse egg." Biol. Bull., vol. 12, no. 4, pp. 259-265, 1907.

This statement is based on figures obtained in connection with the preparation of a series of mouse embryos and ova collected in the spring of 1915, at which time all the costs in any way incident to the work were tabulated as accurately as possible. The data are available to anyone who may wish to repeat the work.

BOOKS RECEIVED

The receipt of publications that may be seat to any of the five biologioal Journals published by The Wistor Institute will be acknowledged undw this heading. Short reviews of books that are of special interest to a large number of biologists will be published in this Journal from time to time.

THE RAT. Reference tables and data for the albino rat (Mus norvegicus albinus) and the Norway rat (Mus norvegicus) ; Compiled and edited by Henry H. Donaldson; Memoirs of the Wistar Institute of Anatomy and Biology, No. 6, Philadelphia, 1915.

This is primarily a reference work for those interested in the rat as a laboratory animal. It is based chiefly upon the extensive published investigations of Professor Donaldson and his colleagues, but includes some work hitherto unpublished, together with available data compiled from various sources. The introduction includes a discussion of the classification of the common rats, and a history of their migrations. The book proper is divided into two parts, the first dealing with the common albino rat, the second with the wild Norway rat (from which the albino has been derived). Various chapters deal briefly with the biological characters, heredity, anatomy (including histology and embryology), physiology and pathology. A convenient index is provided.

The main purpose of the work is to present those data which can be systematically arranged in quantitative form. The data are arranged chiefly in tables and graphs showing the growth in size and weight of the whole body and of the various constituent parts, systems and organs. Tables of growth in the chemical constituents are also included. The tables are conveniently arranged for reference, so that for a given animal the normal weight of any corresponding orgah is immediately obtained.

While these normal tables by no means eliminate the necessity of adequate controls in experiments upon the rat, they furnish a very useful and convenient basis whereby the deviations found in the various local strains used can be compared and measured. If the experimenters will utilize the tables in the manner recommended, it will make possible a standardization of their results upon a basis of comparison much better than any hitherto available.

The necessity for more complete data upon the normal variability of various biological characteristics is especially evident when we consider that without this knowledge it is impossible to draw trustworthy conclusions from the apparent results of experiments, or to judge as to whether the number of controls used is probably adequate. The apparent discrepancy in the variability of the body weight of the albino rat as found by Jackson (table 58) and by King (table 67) is probably due to the fact that the former is based upon a 'random sample' of the general population, whereas the latter is based upon a study of selected litters of strong and vigorous individuals (Anat. Rec, vol. 9, p. 751), among which less variability might be expected. The great advantage of controls from the same litter is emphasized by evidence that fraternal (intra-litter) variability is but little more than half that of the general population.

359

360 BOOKS RECEIVED

For data not included in the scope of the present work (which deals with the normal rat only), references are grouped at the ends of the various chapters. A general bibliography of 948 titles of works dealing wholly or partly, with the rat is also given. Even this large list is incomplete, since numerous bacteriological and descriptive zoological references were intentionally omitted.

The already large number of investigations based upon the rat is likely to increase greatly in the future, as the advantages of this animal for experimental work become better known. Such work will be greatly facilitated by Donaldson's manual. It is to be hoped that the more evident gaps in our knowledge of the rat, especially in its histology and embryology, will be speedily filled; so that for at least one laboratory type biometric norms throughout the life cycle will be available. An albino rat colony will thus perhaps in the near future become a recognized necessity in every well-rregulated biological laboratory.

CM. Jackson.

AMERICAN ILLUSTRATED MEDICAL DICTIONARY. A new and complete dictionary of terms used in Medicine, Surgery, Dentistry, Pharmacy, Chemistry, Veterinary Science, Nursing, Biology, and kindred branches; with new and elaborate tables. Originally published in 1900. Eighth Revised Edition. Edited by W. A. Newman Dorland, M.D. Large octavo of 1135 pages, with 331 illustrations, 119 in colors. Containing over 1,500 more terms than the previous edition. Flexible Leather, $4.50 net; thumb index, $5.00 net. Philadelphia and London; W. B. Saunders Company, 1915.

From the Preface. The aim of the author of this work has been to produce, in a volume of convenient size, an up-to-date medical dictionary, suflBciently full for the varied requirements of all classes of medical men. Physicians and students have long felt the need of such a work. The book does not claim to be an encyclypedia; is a dictionary, a concise and convenient word-book, aiming to furnish full definitions of the terms of medicine and kindred branches, and such collateral information as medical men generally would be likely to look for. The author has sought a middle course between the large, unwieldy lexicon and the abridged students' dictionary, avoiding the disadvantages of each.

The present edition has been carefully revised throughout, so thoroughly, in fact, that it was necessary to make entirely new plates for it. Several hundred new terms have been defined, and the text matter has been increased by 30 pages.

During the past two years a multitude of new tests, both clinical and laboratory, have been published, with the result that the list of tests in the Dictionary has been greatly increased. This list has grown from ten pages in the original edition to nineteen pages in the present one. This is only a single instance of the constant and rapid increase in the terminology of medical science.

STUDIES ON THE BLOOD VASCULAR TREE

I. A COMPOSITE STUDY OF THE FEMORAL ARTERY

BENJAMIN B. LIPSHUTZ

Daniel Baugh Institute of Anatomy and Biology of the Jefferson Medical College of

Philadelphia

FIVE FIGURES

The constant presence of variations in the origin of the branches of the larger arterial trunks of the body is evident from even general observations in the laboratory during regular class dissections. Hitzrot's study of the axillary artery and Bean's on the subclavian artery disclose the fact, that the variations in the origin of the branches of these vessels conformed to well defined anatomic types. At the suggestion of Prof. J. Parsons Schaeffer a study of the blood vascular tree was begun at the beginning of the college year, 1914. In this the first paper, a study of the femoral artery will be presented. I wish to take this opportunity of acknowledging my thanks and gratitude to Doctor Schaeflfer for the interest and attention he has given every detail of this study.

The records which underlie the present study were made from dissections by students and from personal dissections at the Daniel Baugh Institute of Anatomy of the Jeflferson Medical College. Dissections of fifty-three cadavers were recorded : twenty-eight male white, six female white, four female negro, and fifteen male negro. There were forty-nine dissections from the left side of the body and fifty from the right side, ninety-nine in all.

While many minor variations not entirely in accord with the described types were observed in this study, yet the cases permit of a classification into distinct types. The classification of the femoral artery into its types is based on the origin and distribution of the profunda femoris artery and the medial and lateral

361


THE ANATOMICAL RECORD, VOL. 10, NO. 5 B, 1916

362 BENJAMIN B. LIPSHUTZ

circumflex arteries. Section A of this paper describes the various types. Section B contains a description of the origin and distribution of the individual branches. Section C summarizes and discusses the results of the present study.

SECTION A. DESCRIPTION OF TYPES TYPE I

This type (fig. 1) is present in 40 per cent of the cases classified — 14 per cent on the left side of the body and 26 per cent on the right side In this type the medial and lateral circumflex femoral arteries both take origin from the profunda femoris artery. Each branch usually has a separate origin.

The lateral circumflex artery in 40 per cent of this type, takes the form of a double vessel. This additional vessel courses parallel to the descending branch of the lateral femoral circumflex. The external pudendal arteries arise from the femoral as separate branches. The superficial epigastric and the superficial circumflex iUac take origin from the femoral as a common trunk. The highest geniculate artery arises from the femoral cephaUc to the adductor opening, ventral to the adductor magnus muscle. There are in this type twenty-four male white, four female white, ten male negro, and two female negro subjects. This is the type described in the English, German and French anatomical text-books most universally used.

TYPE II

This type (fig. 2) occurs in 25 per cent of the cases classified — 19 per cent on the left side of the body and 6 per cent on the right side. The medial circumflex takes origin as a separate branch from the trunk of the femoral artery. The lateral circumflex arises from the profunda femoris artery. The lateral circumflex in 20 per cent of this type is represented by an additional descending branch as in Type I. The superficial epigastric and superficial circumflex iliac arteries arise from the femoral as separate branches. The external pudendal arteries take origin

STUDIES ON THE BLOOD VASCULAR TREE


363


from the femoral in a common trunk. The highest geniculate artery arises in the same manner as Type I. There are of this 1

Fig. 1 This arrangement of the branches occurs iii 40 per cent of the cases classified. The lettering is alike on all the figures and is as follows: 1, A. epigastrica superficialis; i8, A. circumflexa ilium superficialis ; 3j A. pudenda externa superficialis; 4, A. pudenda externa profundus; 5, A. circumflexa femoris lateralis; 6f A. circumflexa femoris medialis; 7, A. profunda femoris; 8, A. genu suprema; 9y A. femoralis.

Fig. 2 This type is found in 25 per cent of the extremities observed. For index to lettering refer to figure 1.

364 BENJAMIN B. LIPSHUTZ

type twenty-five subjects in all; twelve male white, two female white, ten male negro, and one female negro.

TYPE III

This type (fig. 3) occm^ with slight variations in 19 per cent of the subjects studied, nineteen in all. Seven on the right side of the body and twelve on the left. In this type the lateral circumflex arises from the femoral and the medial circumflex takes origin from the profunda femoris. The superficial epigastric, the superficial circumflex iUac and the external pudendal arteries arise, in 52 per cent of these subjects in a common trunk from the femoral. The remaining 48 per cent ramify as in Types I and II.

TYPE IV

This type (fig. 4) is found in 11 per cent of the cases classified — three on the left side of the body and eight on the right side. The lateral and medial circumflex both arise from the femoral. In four cases there is present an additional lateral circumflex as in Types I and II, arising three times from the femoral and once from .the profunda femoris. The superficial epigastric and the superficial circumflex iliac and the external pudendal vessels ramify as in Types I and II.

SECTION B. DESCRIPTION OF BRANCHES

A, epigdstrica super ficialis. This vessel arises in almost every case from the ventral aspect of the A. femoraUs. In eighty-two extremities studied in which this branch is followed out, it occurs forty-one times in a trunk common with the A. circumflexa superficialis, twenty-nine times as a separate branch, and twelve times in a trunk common with the A. pudenda externa superficialis and the A. circumflexa ilium superficiahs.

A. circumflexa ilium superficialis. This artery is worked out in eighty-two extremities. It occurs twenty-nine times as a separate branch, forty-one times in a trunk common with the A. epigastrica superficialis, and twelve times in a trunk common

STUDIES ON THE BLOOD VASCULAK TREE


365


with the A. epigastrica superficialis and the A. pudenda externa superficialis.

A. pudenda externa superficialis. This vessel arises in 28 per cent of the subjects studied in a common trunk with the A. pudenda externa profundus. This artery is absent nine times. It takes origin twelve times from the A. femoralis in a common trunk with the A. epigastrica superficiaUs and A. circumflexa



Fig. 3 This type occurs in 19 per cent of the vessels studied. For index to lettering see figure 1 .

Fig. 4 This type is present in 11 per cent of the vessels observed. For index to lettering see figure 1.

366 BENJAMIN B. LIP8HUTZ

ilium superficialis. Its most common origin is as a separate branch from the medial side of the A. femoralis.

A. piuienda externa profundus. It arises in 11 per cent of the extremities observed from the A. circumflexa femoris mediaUs. In 28 per cent it takes origin from the A. femoralis in a trunk common with the A. pudenda externa superficialis. In 52 per cent of the arteries observed, it takes origin as a single branch from the medial side of the A. femoralis. It is found absent seven times.

A. circumflexa femoris medialis. It arises from the A. femoralis in 36 per cent of the cases studied, 24 per cent on the left side of the body and 12 per cent on the right side. It takes origin from the A. femoralis cephalad of the A. profunda femoris, twentysix times as a single branch and three times in a trunk common with the A. circumflexa femoris lateralis. In two extremities observed (fig. 5) it arises from the lateral side of the A. iliaca externa, courses distalward and medialward over the femoral vein within 1.5 cm. of the inguinal ligament to gain its usual site and distribution. In one instance it takes origin from the A. iUaca externa in a common trunk with the A. obturatoria and the A. epigastrica inferior. This vessel is absent in three cases being replaced by the A. externa pudenda profundus.

A. circumflexa femoris lateralis. This vessel takes origin from the A. femoraUs in 30 per cent of the cases observed, arises nine times cephalad of the origin of the A. profunda femoris. It is represented by two vessels in 25 per cent of the extremities studied. In all but three cases this additional vessel courses parallel with the descending branch of the A. circumflexa femoris lateralis. It arises on the right side of the body five times from the A. femoraUs, eight times from the A. profunda femoris; and on the left side eight times from the A. profunda femoris and four times from the A. femoraUs. Both branches arise from the femoral six times and from the profunda eleven times. The descending rami outnumber both the transverse and ascending branches. In 8 per cent of the arteries classified, this vessel takes the form of an arterial arcade. It arises three times from the A. femoraUs in a trunk common with the A. circumflexa

STUDIES ON THE BLOOD VASCULAR TREE


367


femoris medisplis above the A. profunda femoris. It takes origin from the A. profunda femoris in 69 per cent of the eases classified. A. profunda femoris. Its point of origin from the A. femoralis is variable. It arises four times at the level of the inguinal Ugament (Poupart's ligament). It is distant from the inguinal ligament 1 to 2 cm. in 22 per cent of the extremities studied,



Fig. 5 This unusual arrangement, in which the A. circumflexa femoralis medialis takes origin from the A. iliaca externa and pursues a course medialward and caudad over the femoral vein and artery to gain its usual site is found in 2 per cent of the cases classified.

368 BENJAMIN B. LIPSHUTZ

2 to 3 cm. distant in 49 per cent, 3 to 5 cm. distant in 18 per cent, and in one instance 9.5 cm. distant. In two extremities observed the A. profunda femoris arises from the lateral side of the A. femoralis and courses cephalad and medialward over the A. and V. femoralis to gain its usual site. There are two Aa. perforantes in 12 per cent of the arteries observed, three in 38 per cent, four in 41 per cent, and five in 9 per cent. The profunda femoris perforates the adductor magnus muscle at its termination as two or three terminal branches, less frequently as a single branch. The profunda femoris is absent as such in one case observed, i.e., it shows complete dissociation. The lateral and medial circumflex femoral arteries take origin from the A. profunda femoris in 40 per cent of the extremities observed. They arise in a trunk common to both in 9 per cent of the cases classified. In 36 per cent of the subjects studied, the A. profunda femoris does not give origin to the A. circumflexa medialis and fails to give oflf the A. circumflexa lateraUs in 30 per cent of the cases observed.

A, genu suprema. This artery is found to take origin most frequently from the A. femoraUs close to the opening in. the adductor magnus muscle. In 8 per cent of the cases observed, it occurs as a branch from the upper part of the A. poplitea. Its branches, R. saphenous and R. musculo-articularis arise separately from the A. femoralis in 12 per cent of the cases classified.

SECTION C. SUMMARY AND DISCUSSION

1. A comparison of the branches of both sides of the body demonstrates the predominance of Type I (fig. 1) on the right side of the body and Type II (fig. 2) on the left side. Cunningham, Piersol, Quain, Testut and other standard anatomical treatises give Type I as the most frequent. A similar arrangement of the branches on each side of the body is present in 40 per cent of the subjects studied.

2. There are twenty-eight male white, six female white, three female negro, and sixteen male negro subjects included in this study. No relation of the branches to age could be drawn, as

STUDIES ON THE BLOOD VASCULAR TREE 369

there are only adults in this series. Thirty per cent of the dissections were made on negro subjects. The latter showed an unusually large number of variations and abnormalities. The negro subjects presented a greater proportionate niunber of variations and anomahes than the white.

3. The branches of the femoral artery differ in their origin on each side of the body in 60 per cent of the extremities studied. The representation of the lateral circumflex femoral artery as a twin vessel is of more frequent occiArence on the right side of the body. The medial circumflex femoral artery arises more frequently from the femoral artery on the left side of the body. The origin of the lateral circiunflex artery from the femoral, either as a single or double vessel is found to occur with more frequency on the right side of the body.

4. Anomalies when present are not found as a rule on both sides of the body. The origin of the medial circiunflex femoral artery from the external iliac taking the course medialward and caudad over the femoral vein to gain its usual site is found in 2 per cent of the cases observed. Another anomaly is found in connection with the medial circumflex, the latter arising from the external iliac in a tnmk conmion with the obturator and inferior epigastric arteries. This arrangement is found in one subject and on the right side of the body. The origin of the lateral and medial circumflex arteries in a conmion trunk from the femoral is foimd in 3 per cent of the cases classified. The lateral circumflex is present as a twin vessel in 25 per cent of the cases observed. The profunda femoris artery is found absent in one case; in this instance, the lateral and medial circumflex in the femoral give origin to the perforating branches.

An anomaly not observed in this series is the presence of a double femoral artery. The femoral artery after giving off the profunda divides into two stems which reunite at a variable distance above the adductor opening so as to form a single popUteal artery. Quain reports one in twelve hundred bodies. Musgrove reviews double femoral arteries and found six reported in the literature. Bianchi and Chretien report cases in which this rare anomaly is found.

370 BENJAMIN B. LIP8HUTZ

A very rare and interesting anomaly is the persistence of the A. comes nervi ischiadici as the principal vessel of the thigh — a condition which is found in all lower vertebrates. A. Manno reviews this anomaly. This unusual variation represents a cessation in the development of the femoral artery. Primarily the A. glutea inferior (sciatic artery) is the main arterial stem of the lower extremity, extending the entire length of the dorsal surface of the Umb to the plantar surface, where it divides into the digital branches. The external iliac at this stage is small and terminates as the profunda femoris. In the lower vertebrates the inferior gluteal (sciatic) is the principal artery of the thigh and becomes continuous with the popliteal, the femoral being relatively insignificant, terminates as the profunda femoris.

LITERATURE CITED

Bean, R. B. 1904 A composite study of the subclavian artery in man. Amer.

Jour, of Anatomy, vol. 4. BiANCHi, S. 1889 Sopra un rarissimo caso di arteria cruralis bifida. Sper mentale, Firenze, 63. BiMAR. 1882 Sur une anomalie de 1 artere femorale. Gaz. Hebd. s.c. med. de

Montpel., 4. Chretien. 1880 Mem. soc. de med. de Nancy, pp. 53-55. HiTZROT, J.M. 1901 A composite study of the axillary artery in man. Johns

.Hopkins Hospital Bulletin, vol. 11. Kreibel and Mall, 1910 Manual of human embryology, Philadelphia, Pa. Manno, A. 1906 Sopra una di arteria ischiadica. Neir uomo-Estratto dagli

studi Saresi. MusGROVE, J. 1892 Bifurcation of the femoral artery with subsequent reunion. Jour, of Anat. and Physiol., London, vol. 26. QuAiN, R. 1844 Commentaries on the arteries, London. RuGE, G. 1894 Varietaten im Gebieteder Arteria FemoralisdesMenchen, dee

Gefftsskanal im Adductor Magnus. Morphol. Jahrb., Leipz. 22. Zaaiger, T. 1894 Seltene Abweichung der Arteria Profunda Femoris. Anat.

Anz., Jena, 9.

THREE CASES OF THE PERSISTENCE OF THE LEFT SUPERIOR VENA CAVA

ROLLO E. McCOTTER From the Department of Anatomy y University of Michigan

THREE FIGUBES

Each of the cases of persistent left superior vena cava about to be described is of special interest from the fact that each presents an interesting and somewhat rare condition of the relations of the left superior vena cava to the other venous systems and to the heart. In the first case there is a persistence of the primitive venous system — the cardinal veins — without an apparent attempt at the metamorphosis to the adult condition. In the second case the left superior vena cava not only has the usual origin, course, and termination of this vein but also communicates by a large oval foramen with the left atrium. The left superior vena cava in case three terminates in the left superior pulmonary vein.

Upon reviewing the literature for the reported cases of double superior vena cava the writer has had the opportunity to consult the excellent papers on this subject by Marshall C50), Gruber ('64), Bauer ('96) and Ancel and Villemih ('08) from whose pubUcations he has freely drawn for certain cited cases, for which the original account was inaccessible.

The above mentioned authors have collected and reported 91 cases of persistent left superior vena cava. To this number the writer will add 29 older and more recent observations. For convenience they have been placed in tabulated form.

371

372


ROLLO E. McCOTTER


Summary of reported cases of double superior vena cava ADULTS


CHILD RKN 1-11 YR.


NEWBORN


FETUSSS


UNCLAS8IFIKD


TOTAL


Double superior vena cava

without«anastomQsis

Double superior vena cava

with small anastomosis . . . Double superior vena cava

with normal anastomosis Left superior vena cava

without right


17

10

9

6

2


5

2


10

3 1 2


19

o 4 3 2


13

2 2 6



Persistent left superior vena cava unclassiJBed... .



Total


44


7


16


30


23


120

V.Subc V.Ca>


V.Subc. D. raSup.D.


Hemi A

Vv.Pu


II.R.Q


ul. D.


Inf.


Fig. 1. The dorsal aspect of the heart and great vessels of an adult female showing the origin, course and tributaries of the right and the left superior venae cavae. One-third natural size.

(,'ase 1 was a well nourished female subject of about 35 years of age. The clinical history was not obtainable. The heart shown in figure 1 was of normal size. The grooves on the surfaces were well marked. There were no defects in the interventricular or interatrial septa. The foramen ovale is completely closed. The systemic and pulmonary aortae take origin from the base of the heart and follow the normal course of these vessels. The right superior vena cava

PERSISTENCE OF LEFT SUPERIOB VENA tAVA 373

is formed in the usual way by the union of the internal jugular and subclavian veins. It courses downward in front of the root of the right lung, to the right and posterior to the ascending aorta and terminates in the superior portion of the right atrium. At about 5 cm. below its formation the superior vena cava receives the azygos vein on its posterior wall. The left superior vena cava like the right is formed by the confluence of the internal jugular and subclavian veins and courses downward anterior and to the left of the aortic arch, anterior to the left pulmonary veins, then passes over the surface of the left atrium to enter the coronary sulcus. It courses to the right aroimd the base of the heart in this groove and terminates in the right atrium anterior and to the left of the opening of the inferior vena cava. At about 5 cm. below its formation it receives the left azygos vein. There is no cross branch uniting the two superior venae cavae, which are of equal size.

By referring to the table it will be seen that there have been 64 cases of this type of duplication of the superior vena cava reported, only 17 of which could be definitely determined as having been observed in adults. Of the remainder 13 could not be classified with regard to the age of the subject.

Case 2 was a well developed male subject about 45 years old. The heart from this subject is very much enlarged. The atria are equal in volume to the ventricles. The left ventricle shares equally with the right in forming the anterior surface. The auricles are much elongated and have fimbriated margins. The aortae have the usual position and relations. The right superior vena cava is formed as usual and takes the ordinary course downward to the right atrium. At about 2.5 cm. below its formation it receives a cross branch (innominate) from the left superior vena cava. 7 cm. below its formation it receives the azygos vein.

The left superior vena cava originates from the union of the left internal jugular and subclavian veins. It passes downward for* 2.5 cm. where it sends a branch to the right superior cava. Below this point it becomes reduced to about one-half its former diameter and courses downward anterior to the left pulmonary artery and left superior pulmonary vein and onto the surface of the left atrium. Here it becomes dilated to about four times its former diameter and crosses the surface of the left atrium to reach the atrio-ventricular groove. It courses in this groove to the right around the base of the heart and terminates in the right atrium anterior and to the left of the termination of the inferior vena cava. At the level of the inferior pulmonary vein in its course over the surface of the left atrium in the terminal dilated portion of the left superior vena cava is a large oval foramen, 3.5 cm. in a vertical diameter and 1.5 cm. in a horizontal diameter, which communicates vnth the left atrium.

374 ROLLO E. McCOTTER

Although the duplication of the superior vena cava has been observed to be relatively frequent in man the writer has been unable to find a reported case where the left superior vena cava communicated with the left atrium. However, of interest in this connection is the case of Ring (1805), in which the left superior vena cava terminated in the left atrium together with the inferior vena cava. Biittner-Weese (1769-1819) described one case and Breschet ('26) two cases where the left superior vena


bcD. I.D.

Sup.D. \z.

=»ul. R. D. il.S.

»ul. D.

If. Os.>

OsV.C.Sup.S.etAtr.S.

Fig. 2. The heart and great vessels of an adult male viewed from the left and dorsally. It shows the origin and course of the left superior vena cava and its Communication with the right and the left atrium. One-third natural size.

cava terminated in the left portion of a common atrium in the newborn. Martin ('26) states that he saw, in a 1.5 months old child, the left superior cava ending in the left atrium. There were also many other disturbances of development. In Gruber's ('46) case the left superior vena cava terminated in the highest portion of the left atrium. Luschka ('62) described the left superior vena cava in a newborn child that terminated in the left atrium just anterior to entrance of the left pulmonary veins. It will be seen that the reported cases of left superior vena cava

PERSISTENCE OF LEFT SUPERIOR VENA CAVA 375

have been observed only in very young infants and in the majority of them this anomaly was accompanied by other disturbances of development. It is questionable if Gruber's case should be included in this group because this anomaly might be produced by a union between the left superior pulmonary vein and the left superior vena cava, the union between the former and the lung having been lost early.

In this catagory may be included, also, those cases where the coronary sinus communicated with the left atrium but where the left primitive anterior veins have passed through the usual metamorphosis to the adult human condition. Four of these cases have been reported. Lindner (1787) observed an instance where the coronary sinus terminated in the left atrium. Jeffray describes the same condition in the heart of a fetus. Meckel (1816) reports a similar condition in his text-book. A case was seen by Bauer ('96) i^i which he describes very carefully a coronary sinus that communicated with the left atrium by an oval foramen 14 mm. and 11 mm. itf transverse and antero-posterior diameter respectively. The usual communication between the coronary sinus and right atrium was completely closed.

Case 3 is a well developed male subject 57 years of age. The heart as shown in figure 3 is apparently normal. The aortae and the right superior vena cava have their usual relations. The left superior vena cava is formed by a very large subclavian vein uniting with a small internal jugular. It courses obliquely downward and to the right for 3 cm. where a large branch passes across the mid-line to the right superior cava. Below the point where the branch is given off to the right side the left superior vena cava is about one-third its original diameter. After coursing downwards for 4 cm. it terminates in the left superior pulmonary vein. The pulmonary vein, which appears to be situated higher in the hilum than usual, is formed by the confluence of three veins from the upper lobe of the left lung. After coursing downward and to the right it terminates in the upper part of the posterior wall of the left atrium. The left inferior pulmonary vein has the usual course and termination.

The somewhat rare condition of a communication between the left pulmonary veins and the persistent left superior vena cava or with a persisting portion of this channel has been reported eleven times. These cases may be separated into four groups

376 ROLLO E. McCOTTER

according to the portion of the anterior cardinal vein that remains patent and offers a drainage channel for a portion of the left lung either into the right atrium by way of the innominate vein or the coronary sinus or as a channel by which a large part of the venous blood from the upper part of the left side of the body reaches the left atrium.

In those cases where the left superior vena cava has the usual origin, course and termination, Wilson (1798) was the first to report a case where left pulmonary veins terminated in the


Subc. P

^on D. ava Sup. D. Azy

Pul. R. D =>ulD


Fig. 3. The heart and great vessels of an adult male viewed from the left and dorsally. It shows the origin and course of the left superior vena cava and its termination in the left superior pulmonary vein. One-third natural size.

left superior vena cava. His case was a malformed child. Theremin ('95) described a similar condition in a child two months old. In the second class of cases the left superior vena cava has the usual origin of this vein; it courses downward and terminates in the pulmonary vein which terminates as usual. In these cases there is a well formed coronary sinus. Case 3 as described in this communication is of this type. Hyrtl ('39) observed 2 cases, one in a newborn female child the other in a 60-year-old male, where the left superior vena cana joined the left superior pulmonary vein which terminated as usual in the

PERSISTENCE OF LEFT SUPERIOR VENA CAVA 377

left atrium. Somewhat more recently Revilliod ('89) described a condition where the upper persisting portion of the aixterior cardinal veins entered the left atrium together with the left puhnonary veins. This anomaly occurred in a 3 months old female child.

The third group of cases presents the proximal portion of the anterior cardinal veins alone persisting and gives the appearance of the coronary sinus originating in the pulmonary veins. The pulmonary veins have lost the usual connection with the left atrium. Hickman ('69) states that in a female 28 years of age the pulmonary veins from the left lung enter the right atrium. This interesting condition is produced by the union of the pulmonary veins with the coronary sinus through a persistent portion of the duct of Cuvier. Somewhat more clearly Nabarro ('03) describes this condition in a 5^ months old child. In this case all of the pulmonary veins opened into the right atrium by way of a large coronary sinus.

Finally, in the fourth group the persistent portion of the left anterior cardinal has lost its connection with the left duct of Cuvier (coronary sinus) which gives the appearance of the pulmonary veins terminating in the left innominate vein. The pulmonary veins in these cases have lost the connection with the left atrium. Blair ('02) claims that a pulmonary vein, normal in size, originating in the superior lobe of the left lung terminates in the left innominate vein. Somewhat similar was the case of Thane ('06) where a superior pulmonary vein entered the left innominate in an adult. Patterson ('13) discovered a case where the upper lobe and the superior half of the inferior lobe drained into the left innominate vein. Niitzel ('14) states that Ramsbotham observed a case where the left superior pulmonary vein terminated in the left subclavian vein. Johnston ('15) very clearly describes a case where the left superior pulmonary vein joins the upper persisting portion of the left superior vena cava, the left superior vena cava terminated in the left innominate vein and the coronary sinus was completely formed.

Under this general grouping may also be included those cases reported where there is a communication between the right

THE ANATOMICAL BECORD, VOL. 10, NO. 5

378 ROLLO E. McCOTTER

superior pulmonary veins and the normal superior vena cava. The causative factors are the same in either case. Meckel ('20) observed the right superior pulmonary vein terminating in the superior cava in an adult. In his short notes Otto ('30) shows the pulmonary vein ending in the right atriimi. Cooper ('36) and Chassinat ('36) each describe a case where the right pulmonary vein ended in the superior cava. Somewhat more clearly Lambl ('60) describes this abnormality. He reports the communication of the right pulmonary vein with the right atrium. Torster ('61) presents in his work on the anomalies of the human body a note on the origin or the termination of the pulmonary veins in the superior or the inferior vena cava without giving the details in these cases. Somewhat indefinitely Duchek ('62) states that he observed the left superior vena cava arising in the right superior pulmonary vein, in a male adult. Gruber ('76) states that he saw the right superior pulmonary vein communicating with the superior vena cava. Gegenbauer ('60) describes a case in a 2 year old child where the right superior pulmonary vein ends in the superior vena cava. Chiari ('80) observed the abnormal termination of the right pulmonary vein in the right atrium. Gruber ('80) described a case where the right pulmonary vein terminated in the superior vena cava in an adult female. Topley ('82) observed this interesting abnormality: in a 20-year old male subject the superior vena cava is formed as usual; shortly after receiving the azygos vein it divides into two limbs, the ventral terminating as the ordinary vena cava superior and the posterior limb, after passing between the branches of the right pulmonary artery and bronchus, enters the left atrium. In a case having anomaUes of many organs Epstein ('86) described the right and left pulmonary veins uniting to form a common trunk which courses behind the heart and terminates in the superior vena cava. Rokitansky ('86) mentions in his text-book on pathological anatomy a case of abnormal termination of the right pulmonary vein in the right superior cava. He also describes a case where the right pulmonary veins terminated in the right atrium. In his description of a double superior vena cava Hepburn ('87) has shown

PERSISTENCE OF LEFT SUPERIOR VENA CAVA 379

that the right puhnonary vein opened into the right atrium. Miura ('89) describes the finding at autopsy on a 6 months old child the two right and the two left pulmonary veins united into a small vein on each side and these finally uniting to form a single trunk before terminating in the azygos vein. The azygos vein communicates with the upper part of the right atrium. Shepherd ('90) observed a case where the single right pulmonary vein terminated in the azygos vein just before it arched over the root of the lung. Audry and Lacroix ('90) mention a case where the right pulmonary veins joined the right atrial wall. Etlinger ('91) briefly states that he observed a similar case where the right pulmonary vein entered the right atrium. Ingalls ('07) states that two right pulmonary veins communicated with the superior cava just before it terminated in the right atrium in his case. A third pulmonary vein terminated as usual. Stoeber ('08) reported a case of 'cor triatriatum' where the pulmonary veins from the right and left lower lobes of the lungs terminated in the left atrium. The pulmonary veins from the upper lobes terminated in the right atrium. Schroeder ('11) described a case which he observed in a 2e-day old child where the left superior vena cava terminates in the left atrium and the right pulmonary veins communicate with the right innominate vein. Merkel ('12) has observed a case in a 70-year old male subject in which the right pulmonary vein communicated with the superior vena cava and also with the left atrium. Brown ('13) in describing Doctor Park's case states that the right pulmonary veins joined the superior cava. There were no right pulmonary veins that entered the left atrium. Gerard ('14) interestingly describes a case where the right superior pulmonary vein entered the superior cava. Niitzel ('14) also describes a similar condition observed in a 47-year old subject.

The embryological significance of the persistence of the left superior vena cava as shown in figure 1 has been so frequently described that a detailed discussion of this unusual condition is deemed unnecessary at this time. Case 2, however, in which in addition to the complete persistence of the left anterior car 380 ROLLO E. McCOTTER

dinal vein and duct of Cuvier there is a communication between this venous channel and the left atrium deserves further consideration. In the text-book of human embryology edited by Keibel and Mall it is stated that there develops in the early fetal heart, beside the atrial and ventricular chambers, a third chamber, known as the sinus venosus. The sinus venosus becomes elongated laterally into the right and left sinus horns. The sinus communicates with the common atrial chamber by a large oval foramen with its long axis placed transversely. During the normal course of development the left sinus horn becomes reduced in size, and finally loses its connection with the left cardinal vein. At about the same time the left half of the large oval foramen connecting these two chambers becomes constricted so that the opening becomes reduced to less than half its original diameter. The interatrial septum develops just to the left of this constricted opening. It appears quite probable that in Case 2 the left sinus horn and the oval foramen uniting the sinus venosus and the common atrial chamber did not undergo the usual changes as very briefly described above but developed proportionately with these structures on the right side. With the development of the interatrial septum, dividing the common atrium into right and left portions, the large oval foramen uniting the sinus venosus and common atrium, and which remained undiminished in size, also became divided into right and left portions and maintained a communication with the right and left atrial chambers respectively.

In his description of the development of the pulmonary veins for the cat Brown ('13) has shown in 4.5 mm. embryos and in his figure 1 that the anlage of the pulmonary veins and the anterior cardinal veins are connected with an indifferent capillary plexus surrounding the foregut and lung anlage. Undoubtedly, then, the communication of the pulmonary veins with the left superior vena cava as is shown in figure 3 is the persistence of the primary condition described above. In the writer's case that portion of the left anterior cardinal vein between the level of the superior pulmonary vein and the heart has undergone the usual changes that are found in man. The

PERSISTENCE OF LEFT SUPERIOR VENA CAVA 381

left superior vena cava in this case gives the appearance of terminating directly in the superior pulmonary vein.

In conclusion I beg to thank Mr. Atwell for the execution of the drawings which were prepared according to the methods he described in volume 10 of the Anatomical Record.

LITERATURE CITED

AuDRY, C. ET Lacroix, E. 1890 Lyon M6d.

Ancel, p. et Villemin, F. 1908 Sur la persistance de la veine cave superieure

gauohe chez I'homme. Jour, de I'anat., vol. 44, pp. 46-62. Bauer, K. 1896 Ein Fall von Verdoppelung der oberen Hohlvene und ein Fall

von EinmUndung des Sinus coronarius in den linken Vorhof. Morph.

Arbeiten, vol. 6, pp. 221-242. Beyerlein, K. 1914 Die persistierendeVena cava superior sinistra als Abfluss rohr fur das Coronarvenenblut. Frankfurter Zeit. f. Path., vol.

15, pp. 327-332. Birmingham, A. 1893 Extreme anomaly of the heart and great vessels. Jour.

Anat. Physiol,, vol. 27, pp. 139-150. Blair, V. P. 1901 Three anomalies of the thoracic blood vessels. Am. Jour.

Anat., vol. 1, p. 513. Breschet, G. 1826 M6m. sur T ectopic de I'appariel de la circulation et par ticuli6rment sur celle du coeur. R6pert. g6n6r. d'anat. et Physiol.

Path, et Clinique Chirurg., vol. 2, p. 1. Brown, A. J. 1913 Development of the pulmonary veins in the domestic cat.

Anat. Rec, vol. 7, pp. 299-315. BtJTTNER-WEESE, 1769-1819 Anat. Wahrnehmungen . Konigsberg u. Leipsig,

No. 5, p. 36. Cameron, J. 1915 A specimen showing complete remains of the left superior

vena cava with a description of a venule which represents the 'lost'

intrapericardial portion of the duct of Cuvier. Jour. Anat. Physiol.,

vol. 49, pp. 140-147. Chassinat. 1836 Archiv. Generales de Medicine. Jour, complem. Des science

M6d., Paris., Ser. II, vol. 41, p. 80. Chiari, H. 1880 Aus der ProsecturdesSt. Anna-KinderspitalinWien. Jahrb.

f. Kinderheilkunde, Bd. 15, S. 319-323. Cooper. 1836 London Med. Gazette, vol. 18, p. 600. Dietrich, A. 1913 Uber ein Fibroanthrosarkom mit eigenartiger Ausbreitung

und uber eine Vena cava superior sinistra bei dem gleiden Fall. Archiv

f. Path. Anat., Bd. 212, S. 119-139. DucHEK. Die Krankheiten des Herzens. Erlangen 1862, S. 67. Epstein, A. 1886 Beitrag zu den Bildungsfehlern des Herzens. Zeitschr.

f. Heilkunde, Bd. 7, S. 308-322. Etlinger, N. 1891 Zur Causistik der angeborenen Herzfehler. Archiv f.

Kinderheilkunde, Bd. 12, S. 348-359.

382 ROLLO E. McCOTTER

Fitzgerald, D. P. 1909 The study of developmental abnormalities, etc. Dublin Jour. Med. Sci., vol. 127, p. 14.

GERARD, G. 1914 Anomalie vasculaire rare. Soc. de Biol., vol. 77, pp. 131-133.

GoLUBEW, W. W. 1895 Ein Fall von Vena cava superior duplex. Whatsch., No. 37.

Gruber, W. 1846 Anatomische Mittheilungen. Vierteljahr. f. prakts. Heilkunde., Bd. 9, S. 78.

1864 Duplicitat der Vena cava superior. M^m. de I'Acad. Imp. des sc. de St. Petersbourg, Ser. 7. Tom. 7, No. 2, S. 45. 1876 Ein Fall von Einmiindung der Vena pulmonalis dextra superior in die Vena cava superior. Archiv f . Path. Anat,, Bd. 68, S. 284-287. 1880 a Duplicitat der Vena cava superior, mit Vorkommen zwier Venae azygae und einer sufficienten Valvula an der MUndung der Vena azygos sinistra. Archiv f. path. Anat., Bd. 81, S. 462-465. 1880 b Vorkommen einer Vena cava superior sinistra. Archiv f . path. Anat., Bd. 81, S. 458.

Gegenbauer, C. 1880 Ein Fall von Einmiindung der oberen rechten Lungenvene in die obere Hohlvene. Morph. Jahrb., Bd. 6, S. 315.

Hepburn, D. 1887 Double superior vena cava, right pulmonary veins opening into the right auricle and a special interauricular foramen. Jour. Anat and Physiol., vol. 21, pp. 438-443.

Hickman, W. 1869 Transposition of viscera; malformation of heart, pulmonary veins from the right lung entering the left auricle, and from the left lung entering right auricle. Tr. Path. Soc, vol. 20, p. 93.

HuTTON, W. K. 1915 An anomalous coronary sinus. Jour. Anat, and Physiol., vol. 49, pp. 407-413.

Hyrtl, J. 1839 Venen varietaten. Med. Jahrb. d. K. K.osterreichenStaates, Bd. 18.

Ingalls, N. W. 1907 Communication between the right pulmonary veins and the superior vena cava. Anat. Rec, vol. 1, p. 14.

Jeffray, J. 1835 Observations on the heart of a fetus. Quoted by Marshall.

Johnston, T. B. 1860 A rare vascular anomaly; opening of the upper left pulmonary vein into a persistant left superior vena cava. Jour. Anat. Physiol., vol. 49, pp. 182-186.

Lambl, D. 1860 Aus dem Franz Joseph Kinderspital in Prag., I Teil, S. 132.

LeBlanc, R. 1915 A left superior vena cava. Guy's Hospital Gaz., vol.29, p. 52.

LeCat, C. 1738 Hist, de I'Acad. roy. des sc. Paris 1740, 4°, p. 44.

LuscHKA, H. 1862 Anat. des Mensch., Bd. 1, 2 Abth., S., 439.

Martin, G. 1826 Bull, de la soc. Anat. de Paris., 2 edit. Paris 1841, pp. 39-43.

Meckel J. F. 1816 Handb. der Mensch. Anat., Bd. 3, S. 67.

1818 tiber einige seltene Bildungsabweichungen. Deutsch. Archiv f. d. Physiol., Bd. 4, S. 479-480.

1820 Einmiindung der Vena pulmonalis dextra superior in die Vena cava superior. Tab. Anat. Path., Fasc. 2, Leipsig.

Merkel, H. E. 1912 a Missbildung im Bereich der oberen Hohlvene. Muenchener medizinische Wochenschrift., Bd. 59, S. 615. 1912 b Missbildung der oberen Hohlvene. Muenchener Medizinische Wochenschrift., Bd. 59, S. 110.

PERSISTENCE OF LEFT SUPERIOR VENA CAVA 383

MiuRA, M. 1889 Ein Fall mit angebornen Herzanomalien. Archiv f. path.

Anat., Bd. 115, S. 353. Nabarro, D. 1903 Two hearts showing peculiarities of the great veins. Jour.

Anat. Physiol., vol. 37, pp. 382-391. Neuberqer, H. 1913 Ein Fall von vollkommener Persistenz der linken Vena

cardinalis posterior bei felender Vena cava superior. Anat. Anz.,

Bd. 43, S. 65-80. NCtzel, H. 1914 Beitrag zur Kenntnis der Missbildungen im Bereiche der

oberen Hohlvene. Zeitschr. f. Path., Bd. 15, S. 1-19. Otto, A. 1830 Lehrbuch der path. Anatomie. Patterson, J. 1913 An unusual anomaly of the left pulmonary vein. Jour.

Amer. Med. Assn., vol. 61, p. 1898. Revilliod, £. 1889 Anomalie du coeur .... Anomalies arterielles et

veineuses. Revue m^dic. de la Suirre Romande Gen6va, no. 3, p. 159. Ring, R. 1805 Med. and Phys. Jour., London, vol. 13, p. 120. Robertson, J. I. 1914 Cardiac malformations in which the great efferent

vessels arise from the right ventricle. Heart, London, vol. 6, pp.

99-104. Rochevalier, M. 1909 Sur la persistance de la veine cave sup^rieure gauche

chez rhomme. Th^se., Montpellier. Quoted in Jahresberichte der

Anat. u. Entwicklung., Bd. 15, Abt. 3. RocKiTANSKY, C. 1886 Lehrbuch der path. Anat., Bd. 2, S. 248. ScHRdEDER, R. 1911 t)ber anomalien der pulmonalvene zugleich im beitrag

zum Cor biloculare. Archiv f. path. Anat., Bd. 205, S 122. Shepherd, F.J. 1890 Right pulmonary vein emptying into vena azygos major.

Jour. Anat. Phsyiol., vol. 24, pp. 69-70. Stoeber, H. 1908 Ein weiterer Fall von Cor triatriatum mit eigenartig ge kreuzter Mundung der Lungen-venen. Archiv f. path. Anat., Bd.

193, S. 252. Thane, G. D. 1906 A specimen in which the upper pulmonary vein of the left

side opens into the innominate vein. Jour. Anat. Physiol., vol. 40,

p. xi. Theremin, E. 1895 Etudes sur les affections congenitales du coeur. Paris. Tichomiroff, M. 1895 Ein Fall von conginitalen Mangel der linken Lunge

mit Persistenz der linken oberen Hohlvene bei einem erwachsenen.

Mensch. Internat. Monatssch. f. Anat., Bd. 12, S. 37. TOpley, R. 1882 Eine neue varietat der oberen Hohlvene. Prag. Mediz.

Wochenschr., Bd. 7, S. 223-234. TOrster. 1861 Missbildungs des Mensch. Jena., S. 145. Walsh AM, W. J. 1880 Anatomical variations. Bartholom. Hosp. Reports,

vol. 16, p. 93. Wilson, J. 1798 On a very unusual formation of the human heart. Philos.

Trans. Abridg., London, pt. 1, pp. 332-333 THE IMPLANTATION AND EARLY SEGMENTATION OF THE OVUM OF DIDELPHIS VIRGINIANA

CHARLES H. SPURGEON AND RALPH J. BROOKS Department of Biology ^ Drury College

FIFTEEN FIGURES

Since few investigators have described the development of the opossum and since there is rather incomplete information at hand concerning the early stages of its development, it seems desirable that the subject be re-investigated. The following results will serve as a preliminary account of a more extensive work which will appear later. During the last five years we have collected and examined about fifty female opossums for embryos. We were unable to find any early stages of development until March 10, 1914.

November 2, 1913, six opossums, three males and three females were collected at Graydon Springs, Missouri. They were taken to Springfield, Missouri, and kept in a cage, made of chicken-wire netting, in which a large box was placed which served as a bed and hiding place. During February, 1914, the females were examined from time to time to see if the external genitals and marsupium, including the mammary glands, showed signs of oestrus such as congestion and enlargement of the external genitals and mammary glands and the moistening of the marsupium.

These conditions were observed February 12, 1914, in one large, adult female. This specimen was killed and the ovaries and the uteri were examined. The uteri were not enlarged and ovulation had not taken place as sections made later of the ovaries showed. On February 23, 1914 another large female was killed and examined when the above mentioned conditions

385

394 CHARLES H. SPURGEON AND RALPH J. BROOKS

IMPLANTATION, OVUM OF DIDELPHIS VIRGINIANA 389



9


6

BOOKS RECEIVED

The receipt of publications that mny be sent to any of the five biological Journals published by The Wistar Institute will be acknowledged under this heading. Short reviews of books that are of special interest to a large number of biologists will be published in this Journal from time to time.

THE ALLIGATOR AND ITS ALLIES. Albert M. Reese, Ph.D., Professor of Zoology in West Virginia University (Author of "An Introduction to Vertebrate Embryology*'), with 62 figures and 28 plates, 1915, $2.50. New York: G. P. Putnam's Sons.

From the Preface: The purpose of this volume is to bring together in convenient form for the use of students of zoology some of the more important details of the biology, anatomy and development of the Crocodilia. For obvious reasons the American Alligator is the species chiefly used.

In the first chapter the discussion of the alligator is largely the result of the personal observations of the author; the facts in regard ta the less familiar forms are taken from Ditmars and others. The description of the skeleton, with the exception of short quotations from Reynolds, is the author's.

The chapter on the muscular system is a translation from Bronn's Thierreich, and the author has not verified the descriptions of that writer.

The description of the nervous system is partly the author's and partly taken from Bronn and others.

The chapters on the digestive, uropenital, respiratory, and vascular systems are practically all from descriptions by the author.

The chapter on "The Development of the Alligator" is a reprint, with slight alterations, of the paper of that title published for the author by the Smithsonian Institution.

The bibliography, while not complete, will be found to contain most of the important works dealing with this group of reptiles.


396

ORIGIN OF THE BLOOD CELLS. DEVELOPMENT OF THE HAEMATOPOETIC ORGANS AND REGENERATION OF THE BLOOD CELLS FROM THE STANDPOINT OF THE MONOPHYLETIC SCHOOL'

VERA DANCHAKOFF The Rockefeller Institute for Medical Research

ONE PLATE

The last century was marked by very intensive work in the field of haematology, including, as it does, erythropoesis, lymphoand leucopoesis. Many investigators have endeavored to throw light on such obscure problems as the embryonic and phylogenetic development of the blood, the structure of the haematopoetic organs, and the development of the vessels in which the differentiated products are conveyed into body tissues. Finally, the function of the different white blood cells has formed a new and interesting problem.

International cooperation has contributed certain results to this field of research, a part of which will have permanent value and form the basis for further investigations. I do not need to emphasize the merits of the investigators, to whom we owe an elucidation of the development of the lymphatics. Most of the students of blood embryogeny and blood phylogeny made their investigations in Europe. From what I can judge, America presents good opportunities for a rational understanding of the function of the lymphatic tissue, as is exhibited in some of the work now being done at the Rockefeller Institute.

The student of biology is engaged in a study of the development and function of living matter, but function is inseparably connected with structure. A correct understanding of the fimction of the different blood cells could not even be approached

Lecture delivered before the College of Physicians and Surgeons, Columbia University, New York, on November 24, 1915.

397

398 VERA DANCHAKOFF

some ten years ago. At that time the mutual relationship of the different blood cells was not established and there existed very few indications concerning the structure and localization of the haematopoetic organs. It is my intention to present here the results of those investigations which are principally concerned with the development and structure of the haematopoetic organs and of the elements which are differentiated in these organs.

A brief r6siun6 of the knowledge of haematopoesis during the last century may be permitted. The zoologists, embryologists and histologists considered the blood islands of the area opaca as the source from whence arose the first red blood cells. The concurrence of views disappeared, however, as soon as the question of the origin of the blood islands was considered.

Very definite indications have shown that the blood development in manmials is transferred in early embryonic life from the area vasculosa to the liver and finally to the bone marrow; but the search of a haematopoetic organ, homologous to the mammalian liver, was fruitless in the meroblastic eggs of the birds and reptiles.

The problem of the genesis of a new haematopoetic organ was readily answered. Blood cells, capable of mitosis, were ejected at certain times from the existing and functioning organ, as for instance, from the area vasculosa into the liver. Here they found favorable conditions for their development, multiplication and differentiation, and the new haematopoetic organ began to function. How radically this interpretation has changed will be shown in this commimication.

The facts, reported above, concerned chiefly the erythropoesis. The localization, the time and the source of the development of the white blood corpuscles were sometimes accoimted for in a very peculiar way. The marked differences in the structure of the red and white blood corpuscles led to the assertion that these different cells must have a separate origin. The fact that white blood cells were not found in the vessels of young embryos and the presumably short duration of the haematopoetic function of the area vasculosa led many investigators to look for a development of leucocytes within the embryo body. Of course a separate

ORIGIN OF THE BLOOD CELLS 399

genesis of the red and white blood cells under such circumstances was taken for granted. The presumption that the erythrocytes developed exclusively in the blood islands and that the leucocytes differentiated later in the embryo body itself seemed to be unshakable. The observations of V. der Stricht, describing the presence of leucocytes in the area vasculosa of the chick embryo with two segments, stood isolated. The opinion of Saxer and Bryce recognizing a conunon stem-cell for the white and the red blood corpuscles, seemed to be in contradiction to all known facts.

The conception of the lymphatic cells of the thymus, as deriving from the epithelium, shows how paradoxical was the judgment about certain forms of the white blood corpuscles. The same origin was conceived for the spleen cells in the reptiles. This latter assumption again prompted the admission of different stemcells in the case of the lymphocytes and the polymorphonuclear leucocytes.

In speaking of definite stem-cells I have in mind such kinds of cells generally, as may be differentiated and identified morphologically. Up to the present, a definite morphological structure has been required as indispensable for an admission of differences in the stem-cells. One has but to remember the elaborate, but vain efforts of Schridde to put in evidence a difference between the leucoblasts and the lymphoblasts.

In order to avoid somewhat irrelevant details, I diall take as support for my position the meroblastic eggs of reptiles and birds. All the blood cells of these animals are real cells, containing both cytoplasm and nucleus. This fact is not unimportant for a true conception of the principle of haematopoesis. The statement that the erythrocytes arise in the early stages of embryonic development from the blood-islands cells in the area opaca has remained unshaken. Recent studies of the origin of the blood islands has established definitely their mesodermic origin. The mesoderm contributes to the development of both, — the bloodisland cells and the endothelium. The mesenchymal cells of the mesoderm (MSCH.) appear to be a source from which develop various specific differentiation products depending on the gradual assertions of the environment.

400 VERA DANCHAKOFF

We may, with our present knowledge, define at least some of the conditions necessary for an arrangement and differentiation of the mesenchyme into haematopoetic organs (namely, those comprising the differentiation of red and granular cells). The area opaca, as is known, lies above the yolk, and the connection between the development of a haematopoetic organ and an abundance of nutritive material is still more obvious if the results of studies on the development of other haematopoetic organs are compared therewith.

Still another condition seems to be of equal value to the above. This consists of the localization of the developing haematopoetic organ in a place, where neither extensive growth, replacement of different organs nor muscle contraction takes place. Only in such regions of the embryo body will the mesenchyme imder normal conditions develop into a haematopoetic organ, alwajrs characterized by a highly developed network of capillary veins.

The formation of the blood-islands is accompanied by the development of an endothelial membrane. A vascular wall surrounds the blood-islands, regardless of the fact that a communication with the heart may or may not be effected (Loeb, Stockard). I must emphasize here that the endothelial capillaries develop not only around the blood-islands, but also in regions where these are lacking, and that some of the bloodislands remain outside of the haematopoetic capillaries.

Just before the differentiation of the endothelial membrane takes place, all the cells of the blood-islands have a similar structure (BLISC. {HBL.L.LMC.) ), regardless of whether they appear as aggregates of numerous cells, merely as small groups or even as isolated cells. These cells possess an intensely basophilic, slightly vacuolized cytoplasm, a spherical nucleus with a well pronoimced nucleolus, and they are potentially spherical, but very amoeboid in character. This structure, characterizing, as it does, the stem-cells of the blood elements from the earliest stages of their appearance, remains unchanged in the development of new haematopoetic organs (compare EV.HBL., IV.HBL. and HBL. in I, II and III).

ORIGIN OF THE BLOOD CELLS 401

The differentiation of the endothelial membrane (I, ENDC.) separates the major portion of the blood-islands from some of these cells, as well as from other amoeboid cells, which still continue to split off from the mesenchyme (I, EV.HBL.). This separation of similar elements establishes very different environments for different groups of cells. The physical and chemical conditions within the vessels, which are lined with a continuous endothelial membrane, must differ largely from the conditions offered by the tissue outside the vessels. This difference in environmental conditions necessarily affects the further development of previously like stem-cells. However, the reproduction of the stem-cells is evidently not influenced by the different environmental conditions, for the mother cell multiplies within as well as outside the vessels (I, EV.HBL., IV.HBL.). The differentiation, on the contrary, is subject to strong influences of the environment and manifests itself in very different ways. Within the vessels the stem-cells produce in their cytoplasm haemoglobin (I, ERBL.), outside of the vessels the same young undifferentiated cells give rise to acidophilic granules (I, GRBL.). Changes in the nuclear structure keep pace with the differentiation in the cytoplasm.

In the early development of the blood cells in reptiles and birds (Danchakoff), and partly in fishes (Maximoff), many similar features may be noticed during the simultaneous differentiation of the granuloblasts and erythroblasts from a morphologically and genetically identical mother-cell. This stem-cell maintains its own existence (I, EV.HBL., IV.HBL.) by uiynterrupted multiplication, on the one hand, and on the other it differentiates into red cells (ERC.) within the vessels and into granular leucocytes (LCC.) outside the vessels.

The first development, differentiation and multiplication of the blood cells occurs in mammals (Maximoff), birds, reptiles and in some fishes on the surface of the yolk. As is known the fundamental difference existing between the eggs of reptiles and birds and those of mammals, consists in the fact that the latter have lost the yolk so characteristic of the eggs of reptiles and birds. This fundamental difference necessarily requires a difference in

402 VERA DANCHAKOFF

the development of the haematopoetic organs in these groups of anhnals.

The rapid appearance of anlagen and the development of different organs in the embryo body naturally attracted the attention of the embryologists to the body of the embryo itself. This fact accoimts for a comparatively early recognition of the haematopoetic function of the liver. The first development of the blood cells in the area vasculosa, the embryonic localization of the haematopoesis in the liver and its final establishment in the bone marrow, — such are the data fixed for the erythro- and granulopoesis in the manmials.

The most minute and laborious study of the whole embryo body in birds has failed to show the localization of the erythropoesis before a comparatively late stage. By the 13-14 days of incubation it has started in the bone marrow. A cursory glance at the liver of the meroblastic eggs was sufficient to establish an absence of any noticeable blood development in this organ. Neither the thymus, the spleen, nor the tissue of the WolflSan body (mesonephros) shows any indication of erythropoesis. All these organs contain vessels and capillary nets but they are all merely thoroughfares.

Although in regions far removed from the embryo body in the annexes of the yolk-sac there was demonstrated by injection (PopofT) an amazingly extensive and exuberant net-work of large venous capillaries, its development and existence seemed to be fully explained by the resorption of the enormous quantity of yolk, and qo one thought of thoroughly analyzing the cell elements present within the capillaries. Whereas a closer study (Danchakoff ) of this capillary net and of its contents would show that the large meshes of this net are distended by young undifferentiated blood cells, and it would be easy to observe that outside of the capillaries the basophilic amoeboid cells are also rather numerous. The structure of all these cells, both within and without the capillaries would permit of ascribing to this capillary net a real haematopoetic (erythropoetic and granulopoetic) function, homologous to that of the mammalian liver.

ORIGIN OF THE BLOOD CELLS 403

Both within and without the vessels the young mother-cells bear the same structure as the blood-island cells after their isolation (I, EV.HBL,, IV.HBL.). This morphological cell unit was most unhappily termed the large lymphocyte. The efforts of the last 7 to 8 years have not yet been able to render conclusively the dictum, that the morphological cell unit, formerly known as the large lymphocyte, appears first by the dissociation of the bloodislands and is a young, undifferentiated cell. This cell differentiates in accordance with given environmental conditions. Briefly, it corresponds morphologically to the large lymphocyte and is physiologically the haemoblast.

It appears only natural that the young stem-cells, observed in the haematopoetic net of the yolk sac annexes, corresponds exactly to the stem-cells which were first found in the bloodislands; for they appear to be the direct result of an uninterrupted reproduction of the latter.

The erythropoesis and the granulopoesis now remain distinctly separated, as in the first stages of their development. The distribution of the different cell elements within the haematopoetic capillary net in the annexes is always very characteristic. Every cell is free and does not manifest any continuity with its neighbor. The cells he very closely together in the zones adjacent to the endotheUal membrane, and their reciprocal pressure flattens them and renders them polygonal. Even a slight fluid current could hardly be supposed to be present in these parts of the vessels. In the centre of the vascular lumen the cells are arranged more loosely and are free from pressure. The position of the cells shows clearly, that they are removed from here and directed by the current into the body of the embryo.

It is easy to notice, that the most highly differentiated cells, rich in haemoglobin, lie in the centre of the vascular lumen, just where the current is strongest. Besides these already differentiated cells the capillaries include numerous cells in various stages of differentiation, beginning with the youngest haemoglobin-free stem-cells, and including the whole range of cells which gradually accumulate haemoglobin in their cytoplasm and equally

404 VERA DANCHAKOFF

gradually change their structure into that characteristic of an erythrocyte.

The youngest stem-cells, which I will call lymphoid haemccytoblasts, are found in the zones inmiediately adjacent to the endothelial membrane, and these young blood cells within the vessels are so much like the white blood cells lying outside the vessels, that Bizzozero regarded them in the bone marrow as a layer of white blood corpuscles. In the embryonic haematopoetic organs these cells are very numerous and sometimes form a continuous layer of basophilic cells, becoming gradually substituted towards the centre by cells, which accumulate haemoglobin in their cytoplasm.

The structure of the haematopoetic net and the distribution of the cells in their different stages of development reminds one strongly of the conditions under which spermatogenesis takes place. Here is seen the same uninterrupted differentiation of the younger cells into highly differentiated cells, which thereby lose much of their usual cell appearance. We also find the same location of the young imdifferentiated elements, which secures their existence by uninterrupted multiplication and continuous splitting off of cell generations which undergo further differentiation. Finally, we see the presence of a current in the centre of the vessels, which removes the differentiated products.

As mentioned above, the area vasculosa of birds and reptiles contains outside the vessels numerous cells, which give rise to the granular leucopoesis. Both in the earliest stages and now in the yolk sac annexes the leucocytes develop extravascularly. They nevertheless show very close connection with the vessels, occasionally surrounding them by a continuous layer. Their differentiation here follows the same principles as elsewhere and from a common morphologically and genetically identical stemcell (I, EV.HBL.) leads to the development of numerous special (acidophiUc) leucocytes (I, LCC). The differentiation of blood cells in the area vasculosa and in the annexes of the yolk sac is completed by the development of the cell elements described.

Another question now arises, viz., the question of how a new haematopoetic organ originates, whether it is the mammalian

ORIGIN OF THE BLOOD CELLS 405

liver or the definitive haematopoetic organ — the bone marrow, or finally, one of the organs assigned to the lymphopoesis.

I have mentioned above how easily this problem was solved not long ago. The first red cell (the erythroblast) differentiated from the blood-island cell; it supplied the organism, by multiplication and differentiation, with erythrocytes. At a given time the current transported the erythroblasts into definite regions of the embryo body. They stopped there, multiplied and differentiated further. Shortly the development of a new haematopoetic organ appeared as a strictly localized metastasis. It was difficult even to expect any other conclusion from studies made upon haematopoetic organs, the development of which was more or less advanced. Indeed, such organs offer a complete parallel between the differentiating blood cells in the new organ and those, which were observed in the earlier organ. Only studies of the very earUest stages in the development of a new haematopoetic organ will show by definite evidence, that the development of a new organ is due, not to metastasis from a former existing organ, but to a local differentiation and multiplication of the mesenchymal cells, still preserving their faculty of poljrvalent differentiation (see II and III).

The young undifferentiated mesenchymal cell (MSCH.) maintains itself for many days, if not for the whole duration of the life of the organism, and evidently retains its potency for multiplication and differentiation. Similar mesenchymal cells give rise to accumulations of amoeboid cells around the embryonic double aorta, described by Professor Huntington. The blood-island-like agglomerations of similar cells in the development of the bone marrow arise also by a splitting off from the mesenchyme. An endotheUal vascular membrane, partly formed in locoy partly growing from outside the bone marrow cavity, surrounds some of these cell groups and marks them at the same time as erythroblasts. Similar cells, remaining between the capillaries, become the source of the granulopoesis (Maximoff, Danchakoff).

That being the case, we have to conclude, that the whole development of the new haematopoetic organ f. ex. of the bone marrow (see plate III) takes place locally and leads to the double

406 VERA DANCHAKOFF

differentiation of a common mother-cell into red and granular cells, the young stem-cell itself being most tenaciously retained in the precincts of the haematopoetic organ.

Nevertheless, it is possible to notice certain features of the haematopoesis in the bone marrow, characteristic for this organ, which appear already in the embryo and are very conspicuous in the adult animal. This is a reduction in number of the young undifferentiated stem-cells, both in the erythropoesis and the leucopoesis regions. The development of the highly differentiated erythrocytes and leucocytes is effected by the further development of cells which already contain respectively haemoglobin (erythroblasts) or acidophilic granules (granulocytoblasts). The haematopoesis now develops in a more homoplastic way, differing from that seen in the early embryonic stages in which the haematopoesis develops principally in a heteroplastic way. This difference is, however, not essential; one has but to bleed the animal, whereupon the stem-cell promptly produces by multipUcation a whole generation of similar stem-cells (Danchakoff), which are very numerous in the haematopoetic tissue of the embryonic organism.

Some essential differences may still be observed in the structure of the bone marrow, in comparison with the former haematopoetic organ, namely new directions in the differentiation of the blood cells, leading to the development of small lymphocytes and of Mast cells. Since the latter find their origin on a larger scale in the connective tissue and in special organs, lymph glands, thymus and spleen, the mode of their differentiation is more conveniently studied in the latter organs.

Finally, I have to emphasize the striking analogy seen in the general conditions which accompany the development of every new erythro-granulopoetic organ : an abundance of food supply and the localization distant from any rapidly growing organs; these conditions are met with both in the yolk sac and in the cavities of the bones and seem to be so essential that. in reptiles which have no extremities, haematopoesis finds its localization in the cavities of the vertebra, thus developing a haematopoetic organ in the form of a whole series of isolated centres

ORIGIN OF THE BLOOD CELLS 407

(Danchakoff). Even the localization of haematopoesis in the liver of mammals, which have lost the yolk in their eggs, correspond in the highest degree to the conditions cited and which, at this time, the embryo offers.

Some of the environmental conditions for the differentiation of the mesenchyme into an erythro-leucopoetic organ, as well as those for the development of a haemocytoblast into an erythrocyte or into a leucocyte could be, at least partly, explained. However, the differentiation of the blood stem-cells into erythrocytes and leucocytes does not present all the possible inherent potentialities of the haemocytoblast. New differentiation products result under new conditions in the mesenchyme of the embryo body (see plate II).

The mesenchyme cells are disseminated through the whole organism and occupy all the interspaces between various organs. It is diflScult at present to define the conditions under which the mesenchymal cell imdergoes differentiation into a fibroblast. This ubiquitous differentiation has for long overshadowed, by its omnipresence and its conspicuousness, all the other Unes of development which find place in the so-called connective tissue. Meanwhile certain other directions in the differentiation of the mesenchymal cells are almost as general as the differentiation into the fibroblasts; they convert the connective tissue into a seat of most interesting diffuse lymphopoetic, and, in early embryonic stages and in lower animals, into granulopoetic differentiation. The development of the lymphatic glands, of the thymus and of the spleen thus becomes merely localized hypertrophic centres of a similar diffuse differentiation, spread out through the loose connective tissue.

In speaking of the lympho-granulopoesis, I therefore associate the differentiation phenomena in all the regions above mentioned in one common account. These lines of differentiation are very simple and similar. The first isolation of the mesenchymal cells from the general ties takes place as a rule in the form of large lymphoid haemocytoblasts (II, HBL., L.LilfC), which sometimes arrange themselves as blood-island-like groups of cells. Their rapid, iminterrupted multiplication produces numerous smaller

408 VERA DANCHAKOFF

cells (II, SM.HBL.), These cells become a source of the true small lymphocytes (II, SM.LMC) and of nmnerous wandering cells (II, TT.C), so characteristic of the loose connective tissue. The latter may also arise directly from the mesenchymal cells, especially in later embryonic stages. With the appearance of the small lymphocytes and wandering cells the differentiation possibilities of the lymphoid-haemocytoblast in the connective tissue are still not exhausted. Throughout the regions of the embryo body in birds and especially in reptiles, but less evidently in mammals, the splitting off of granuloblasts is very conspicuous (II, GRBL,). Most of them contain acidophilic granules. The differentiation of the mast-cells with basophilic, metachromatic granules at the expense of the mesenchyme cells takes place in the later embryonic stages.

For a long time the ability of the small lymphocytes to multiply by mitosis was denied; at the present their reproduction by mitosis is fully admitted, as well as their faculty of further differentiation into mast-leucocytes and into specific granulocytes with spherical acidophylic granules, very numerous in the connective tissue of birds (II, GR.LMC). Likewise they may differentiate into plasma cells in the adult organism (II, PLC.)

The question of regeneration of the blood cells in an adult animal is a very important problem and the embryological data above described may contribute directly to its elucidation. The question of blood regeneration has interested most keenly the pathologist-clinicians. Facing the various highly differentiated products {LCCy ERC.y PLC. J mast-cells, etc.) which, as above mentioned, develop in the adult animal chiefly at the expense of cells in intermediate stages of differentiation, the pathologist-clinicians could not but admit a polyphyletic origin for various blood cells. The red cells possessed their own stem-cells, as also the different leucocytes and lymphocytes.

This statement is not altogether wrong; inasmuch as referring to haematopoetic organs in an adult organism, it solves practically the problem of the usual haematopoesis. But this statement does not cover all the eventuaUties. Regeneration after bleedings, as well as the excessive increase in number of white

ORIGIN OF THE BLOOD CELLS 409

blood corpuscles in leukemias, takes place not at the expense of specific granulo-lympho- or erythroblasts. The monophyletic school has the credit of demonstrating in all the haematopoetic organs, in the bone marrow, in the spleen, in the lymph glands and in the loose connective tissue, the presence of a similar morphologically and genetically young and imdifferentiated stem-cell, at the expense of which the regeneration really takes place, especially after great loss (Pappenheim, Weidenreich, Maximoff, Danchakoff). This young stem-cell bears the same structure as the morphological cell unit, which was recognized by the monophyletic school as being the mother-cell of all the different blood cells in the development of every haematopoetic organ.

I am afraid to be too positive, but there are some indications that after extensive destruction of the haematopoetic tissue regeneration occurs, at least in some cases, not only at the expense of haemocytoblasts, but also at the expense of imdifferentiated mesenchymal cells, retaining from the very beginning of their development all their polyralent potentialities. This would mean that the adult organism retains, from the remote period of its embryonic life, a stock of undifferentiated mesenchymal cells which begin to multiply and to differentiate in certain conditions of a disturbed equilibrium. This latter question is in a stage of development. No matter how this question may be solved, the similarity of the morphological structure of the stem-cell in all the haematopoetic organs and its common origin from the mesenchymal cells of the mesoderm in embryonic life demonstrates the close relations existing between different blood elements.

It would not follow that the various differentiation products are not different, or that they had no different functions. The erythrocytes, the small lymphocytes, the different leucocytes, the wandering cells of the connective tissue, the mast-cells and the plasma cells, — all these cells are different cell units, morphologically as well as physiologically. But in the early embryonic stages they all had a common mother-cell, and this mother-cell is preserved in the adult organism and becomes the source of differentiation and regeneration and most probably also the source of pathological proliferation.

THE ANATOMICAL HECORD, VOL. 10, NO. 5

410 VERA DANCHAKOFF

The conception of the monophyletic school has recently been subjected to searchmg criticism. As I, in great measure, uphold these conceptions, I feel obliged to plead their cause.

Embryos without blood circulation were obtained by means of the influence of alcohol on the fertilized eggs of Fundulus (Stockard).* Blood-islands developed on the yolk surface, and endothelial membranes surrounded them; the blood-island cells developed into haemoglobinic cells; but they were never carried into the embryo body, because the connection between the heart and these vessels failed to become established. During the summer of 1914 I personally observed precisely the same conditions in hybrids of Fundulus and Menidia, which were obtained for this purpose at the kind suggestion of Dr. Loeb.

The fact, that the blood-island cells develop into erythrocytes in embryos without circulation, is accounted for by a specific character of the mesenchymal cells, which differentiated only in haemoglobinic cells. But it is stated that no leucocytes are developed on the yolk sac in normal Fundulus embryos.

The specificity of the stem-cells of the white blood corpuscles is based upon observations of small groups of cells which resemble lymphocytes or leucocytes;" it is, moreover, stated that '* these embryonic cells all show a more or less degenerate appearance, but if they can be classed as any type of white blood cell, their origin is definitely removed and entirely distinct from that of the red blood cells."

These are some of the data, upon which an attempt was made to build a new embryopolyphyletic school.

The experimental analysis of the problem of haematopoesis is undoubtedly of very great importance. I am fully conscious, how important is the study of embryos without circulation and ^^ath disconnected vessels for a true conception of the development of vessels. But I fail to understand how the study of these embryos, on which the polyphyletic origin is now based, can help us in understanding the development of different blood cells and of haematopoetic organs.

The origin of blood and vascular endothelium in embryos without a circulation of the blood and in the normal embryo. Amer. Jour. Anat., vol. 18, no. 2, Sept., 1915.

ORIGIN OF THE BLOOD CELLS 411

Whether the circulation is established or not, the differentiation of the blood cells follows the same principles. How can the presence of the circulation embarrass the study of the development and differentiation of the blood cells in the haematopoetic organs, if only more or less differentiated cells are withdrawn by the circulation? It seems to me that the absence of circulation in itself must greatly obscure the study and hinder the true understanding of the processes. Indeed, the differentiated products are not withdrawn in embryos without circulation, they remain in loco and evidently influence very strongly the further reproduction and differentiation of young cells, for finally they all disintegrate.'

The environmental conditions are also taken into account by the supporters of the theory of the polyphyletic origin of the blood cells; but only for the multiplication of the erythroblasts, not for their differentiation. In spaces, Uned by endothelium, the erythroblasts do not multiply in the Fundulus embryos. If this statement, drawn from the study of Fundulus, was not generalized, we had to believe, until further proof, that the erythroblastic tissue of Fundulus is a very peculiar tissue, refusing to multiply inside the vessels. In birds and reptiles and in some cartilaginous fishes (Selachians) the erythropoesis takes place exclusively inside the vessels. Mitosis in erythroblasts inside the vessels are as numerous, as mitoses in granuloblasts outside the vessels. On the contrary, the differentiation of the cells is strongly influenced by the fact, whether the cells lie inside or outside the vessels.

But let us consider some of the arguments against the monophyletic school. A special value is evidently assigned to the following argument, for I find it quoted in different places in the paper. If all the types of blood cells did arise from a common mother mesenchymal cell, they should then be found in intimate association throughout all blood-forming regions.

« Analogous results of disintegration of spermatogenetic cells are offered by the interesting studies of B. D. Myers. The disintegration of the undifferentiated spermatogenetic cells is caused in his experiments also by a lack of withdrawing of the differentiated cells. See: Histological changes in testes following vasectomy. Proceedings of the Am. Assoc, of Anat., 1916.

412 VERA DANCHAKOFF

The conclusion cited can only appear as a sequence to an assertion, stating that various differentiation products, developing at the expense of a common stem-cell, must always be foimd in intimate association. But this assertion is not correct.

If various products of differentiation have their origin in a common stem-cell, they must be found under the influence of different environmental conditions which separate them. If various products of differentiation are found in absolutely identical environments the difference between them may be explained only by differences in their stem-cell.

Imagine in the remote past a heap of similar tree seeds. These seeds develop in our moderate climate into tall and many branched trees. Suppose the wind bears a part of the seeds away and brings them to a land possessing different environmental conditions, we will say to arctic lands. There the seeds may develop but they may produce trees no higher, than our moss (willow-salix in Spitzbergen). Have we the right to say that the difference between the various products of seed development are caused by differences in the seeds? They may be different or similar; morphologically they were similar.

How would it be possible to know if there were differences in the seeds? The only possibility of solving this problem would consist in sowing the seeds of arctic trees in our climate and vice versa. If the seeds of our tall trees again produce dwarf trees in the arctic lands, the seeds of the different products of development will have been shown to have been identical.

Similar experiments may be carried out with the haematopoetic tissue. The experiments are made the more possible, because of the short time periods, needed for the cell differentiations, and fully controlled by us.

It is difficult to assert at present, whether or not the monophyletic school will contribute the last word in the establishment of the mutual relationship and of the origin of the blood cells. The possibility is not excluded that perhaps some time we will find direct or indirect means of distinguishing between different mesenchyme cells, but there must be other arguments than those cited above to convince one of the truth of the position taken by

ORIGIN OF THE BLOOD CELLS 413

the polyphyletic school. However, the fact that the stem-cells, as stem-cells, keep so tenaciously under the most varied conditions their morphological structure and that only their products differ, seems to speak strongly for the existence of a single stem-cell, identical in the different haematopoetic organs.

Through the kindness of the Rockefeller Institute for Medical Research I have been enabled to include a colored plate, illustrating the course of the gradual differentiation of various cells in the haematopoetic organs.

PLATE 1


N \


EXPLANATION OF FIGURES

The plate shows the development and the gradual differentiation of the blood cells.

I Erythro- and granulopoesis in the yolk-sac of birds.

II L3rmpho- and granulopoesis in the special lymphatic glands and in the connective tissue.

Ill Starting point of the development of the erythro-granulopoesis in the bone-marrow, which proceeds, as is shown in /.

Arrow — denotes differentiation. Simple line — denotes multiplication. Cross — indicates cells, capable of mitosis.

ABBREVIATIONS


BLISC. (HBL.LXMCX blood island cell (haemoblast) large lymphocyte ENDC, endothelial cell ERBL,y erythroblast ERC.f erythrocyte EV.HBL.j extravascular haemoblast GRBL,y granuloblast GR.LMC.f granular lymphocyte


HBL,, haemoblast

IV.HBL.f intravascular haemoblast

LCC.f leucocyte

MSCH,f mesenchyme

PLC.f plasma cells

SMMBL,, small haemoblast

SM.LMCf small lymphocyte 414



CONCERNING THE CONCEPTION OF POTENTIALITIES IN THE EMBRYONIC CELLS

VERA DANCHAKOFF

The Rockefeller Institute for Medical Research

The study of a complex and peculiar tissue, such as blood, requires great care in the methods used and in the conclusions drawn. It seems especially desirable to establish the limits to our knowledge imposed by the actual methods.

The interesting article of Dr. Stockard, appearing in Science October 15, seems to reject the limitations of morphological study.

If two tissues behave differently in their development, the cause thereof, according to Dr. Stockard, lies in the difference of their anlagen, and even if no morphological differences can be traced in these anlagen, they exist potentially. These anlagen must be different, since the products of their development are different and the conditions of their development do not differ enough to account for the various products of the development.

Dr. Stockard discusses in his article the development of erythrocytes, endothelial cells and two kinds of chromatophores found in the yolk sac of the bony fish Fimdulus; though he admits that they develop from "apparently similar mesenchymal cells," yet he concludes, that "these four types of cells" must be considered "as being polyphyletic in their origin."

The conclusion, concerning the potential difference of the mesenchjmaal ceUs, is based upon statements: 1) that similar mesenchymal cells develop in different ways; 2) that the four types of cells, resulting from the differentiation of the mesenchyme cells in the yolk sac cannot 'intergrade^ nor 'transmutate.'

As to the statement of the non-transmutation of the differentiated cells, it is fully supported by facts, but is useless as an argument, for no one has endeavored to affirm that the chromatophores may differentiate into any other type of cells: they may only multiply. They have lost the faculty of splitting off undifferentiated cell generations, by reason of their highly speci^zed nature; they are merely able to repeat their life cycle between each two mitoses.

The life history of the endothelial cells is somewhat more complex. These cells, at least in the very early embryonic stages, are only slightly differentiated in birds and, according to Maximoff, in mammals; therefore they in part keep their potentiality for polyvalent development. One may contradict this fact But even if the mesenchymal cell as an endothelial cell really loses its faculty of independent existence and further development, it does not necessarily follow, that the mother cell of the endothelium must bear in its structure a constitution, which will with absolute necessity make it only an endothelial cell and not an embryonic connective cell nor even an imdifferentiated blood cell. The conditions, in which the one or the other mesenchyme cell may be found, cannot be immaterial in directing their development; — and the data foimd by the study of the yolk-sac of the birds and the reptiles speak strongly for a dissimilarity of conditions.

The non-transmutation is not a proof of a polyphyletic origin: a and b may never transmutate, but may present two different offshoots of a common stem. Dr. Stockard is inclined to see this common stem one generation back, without giving any material basis to support this view.

Moreover if the mere differentiation of cells leads one to admit a difference in the stem cells, the admitted differences in the stem cells must necessarily compel a conception of differences in the ancestors of the mother cells, and so on

All these assumptions have logical evidences only in case the different products are obtained ceteris paribus. The conditions of the environment in the yolk sac of fishes are supposed by Dr. Stockard to be identical for all the cells. A certain degree of uniformity (or an absence of special conditions) in the yolk sac of fishes may account for the absence there of leucocytic development. (Dr. Stockard did not find leucocytes in the yolk sac of Fimdulus, while in the yolk sac of reptiles and birds the leucocytes and their stem cells are very numerous.)

Very definite data obtained from a study of the development of the blood in the yolk sac of the reptiles and birds, urge us to postulate a potent influence of different conditions upon the differentiation of the primitive blood cells in various directions. The peripheral cells of the larger blood islands invariably differentiate into endothelial cells; the basophilic amoeboid cells of the blood islands invariably differentiatate within the vessels into red blood corpuscles, preserving at the same time their stock by continuous multiplication. Some of the smaller blood islands between the vessels, as well as niunerous other cells, isolated later from the mesenchyme, differentiate only iiito granular leucocytes.

The reference of various processes of differentiation to potential morphologically indefinable differences in the stem cells seems to me a source of danger, because similar assumptions are apt to impede further investigations. They put aside the important question, — "What are the conditions and the causes for the development of certain groups of morphologically identical mesenchyme cells into morphologically different products of differentiation?" Until our resources in methods of investigation are exhausted, a reference to invisible and indeterminable potential differences, as bases for cell differentiation, would seem to be illogical.

EVIDENCE OF HEMOGENIC CAPACITY OF ENDOTHELIUM

H. E. JORDAN Department of Anatomy , University of Virginia

The yolk-sac of the 10 mm. pig embryo is an active source of hemoblast origin from endotheUmn. The evidence of this process consists in a complete series of transition stages from primitive endothelium to definitive erythrocytes (Anat. Rec, vol 9, p. 92). Similar evidence accrues from a study of the capillaries and smaller blood vessels immediately next the embryoAic brain and spinal cord, and of the hepatic and mesonephric sinusoids. Certain objections can be made to the interpretation of appearances here given, most forceful of which is the one that hemoblasts of various stages of development at various phases of transit through an incomplete endotheUal wall of irregular contour give the deceptive appearance of endothelial origin. (Stockard, Am. Jour. Anat., vol. 18, p. 592). To meet this objection is the main purpose of this paper, other objections as concerns the yolk-sac vessete being elsewhere considered.

In searching through the 10 mm. pig embryo for intrasomatic evidence of giant cells in cormection with my study of these cells in the yolk-sac, my attention was arrested by the presence of peculiar cell clusters in the aorta. Brief attention has been called to these also by Emmel (Anat. Rec, vol. 9, p. 77). He describes them for pig embryos between 6 and 15 nmi. length and in rabbit and mouse; and I have seen them also in mongoose and turtle embryos. Their occurrence would seem to be quite general in young vertebrate embryos. It is from a study of these clusters that I believe the most cogent evidence accrues of intrasomatic hemogenic capacity of young endothelium; there is here no question of a deceptive appearance due to lodgment in an endothelial bay or lacima, and a pecuUar plane of section.

Here we may note the difficulties of presenting decisive proof that endothelimn transforms into blood cells. The morphologic evidence must consist in a complete series of developmental stages between an endotheUal cell and a separating primitive blood cell. But the critic can always object up to a certain point that the particular cell in question is not a blood cell; at a later point he can object that the cell, apparently separating from the endothelium, is simply a hemoblast in intimate contact with the endotheUmn due to pressure and the adhesive properties of protoplasm. This is why Stockard (Am. Jour, Anat., vol. 18, p. 229) can brush aside all the morphologic data as unsatisfactory, in

417

418 H. E. JORDAN

the light of his experimental findings in the narcotized Fimdulus embryo where the endothelium is proved to have no hemogenic f miction. In the case of individual cells the supporter of endotheUal hemogenesis is apparently helpless, even with a complete series of drawings, at the hands of the dissenter. The cell clusters of the aorta appear to be ideally constituted to meet all possible objections as to the hemogenic capacity of primitive endothelium.

These cell clusters appear in the aorta only throughout the region of the mesonephroi. They are limited to the ventral portion of the wall. They may consist of few or of many cells. At certain levels two clusters appear, one on either side of the mid-Une. Proximally they are intimately associated with the endothehum; in certain clusters true endothelium appears to be entirely lacking beneath the mass of primitive blood cells. Passing distally, transition stages appear between endothehum and hemoblasts. Occasionally cells show mitotic and amitotic division phenomena. The following gives a r6simi6 of the occurrence of aortic blood cell clusters in a 10 mm. pig embryo:

Scattered cells first appear in the mid-ventral portion of the aorta in slide no. 22; this is 5 slides caudal to the cephaUc tip of the mesonephros, at the point of division of the dorsal aorta into the paired aortae. Slide no. 26. . . .4 groups of 3 or 4 sections (10 microns) each.

27 .... a) cluster, passing through 10 sections. b) a double group of 4 sections.

28. . . .cluster of 4 sections.

(also scattered cells in a ventral branch — coeUac axis)

29 ... . cluster of 8 sections.

30 cluster of 4 sections.

31 and 32. . . .cluster of 13 sections. (130 microns, including several hundred cells: also scattered cells in ventral branch — superior mesenteric artery).

33 cluster of 11 sections.

34 3 groups of 4, 5 and 4 sections respectively.

35 .... 2 groups of 3 sections each.

37 a group of 3 sections.

Here the following points must be emphasized; 1) similar clusters are found nowhere else, either in the yolk-sac or the embryonic vessels or sinusoids, not even in the aorta or its branches, outside of the mesonephric area; 2) just cephalad of the first cluster the endothelial cells of the ventral portion of the aorta for some distance are short stout spheroidal elements, with early hemoblast cytoplasmic and nuclear characteristics, similar to the hemoblast transition stages described for the yolk-sac vessels; 3) from the ventral surface of the aorta, where the clusters are located, numerous median and ventro-lateral (mesonephric) branches arise, an occasional endothelial cell of which has the same spheroidal shape and early hemoblast characters; 4) the clusters are not caught in the mouths of these vessels but generally lie between the openings of such vessels; occasionally also a small cluster hes freely suspended attached to the side (outer) of the ventro-lateral branch.

HEMOGENIC CAPACITY OF ENDOTHELIUM 419

The interpretation I wish to maintain regarding these cell clusters, to which also Emmel seems to incline, is that they arise from the endothelium by process of proliferation and differentiation. Two main objections must be met: 1) that these cell clusters, presumably derived from endothelium, do not consist of primitive blood cells; 2) that as clusters of hemoblasts they are only spatially not genetically related to the endothelimn; that they are groups of developing blood cells swept toward the ventral Wall of the aorta by the stronger currients towards the numerous ventral branches and caused by pressure and their adhesive properties to adhere to the ventral wall of the aorta.

To the first objection the countervailing fact may be stated that according to all the criteria both cytopla^oic and nuclear, gathered from a study of isolated hemoblasts and erythroblasts in the yolk-sac vessels, liver and heart of the embryo, the majority of these cells are either hemoblasts or erythroblasts.

Against the second objection the following facts may be cited: 1) The cells frequently show a series of transition stages from endotheUum to hemoblasts in passing from the attached pole to the free periphery; 2) the fairly regular spheroidal shape of the clusters, indicating a imiform centrifugal growth; 3) similar clusters are found nowhere else, either in the yolk-sac or the body proper; if these clusters have been simply swept to their definitive locations along the ventral wall of the aorta by the blood current, then such clusters should be foimd also elsewhere, from whence they might be carried; the sinusoids of the liver, of the yolk-sac, of the heart, and the jugular veins, and the inferior vena cava, and the hepatic vein would seem to be equally favorable locations for their residence; 4) occasionally small clusters are attached to the outer sides of oblique ventral (mesonephric) vessels where these would not be expected to adhere if carried by the cmrent and pressed against the wall; 5) if carried here by the blood, we should expect to find certain clusters elsewhere in the aorta except attached to its ventral wall; in certain portions where clusters appear on the ventral wall the aorta is packed with late erythrocytes, but no hemoblast clusters are foimd among them; 6) the blood stream also moves toward the dorso-lateral branches, but such never contain cell clusters; 7) the strongest current of the blood in the aorta would seem to be in the direction of the long axis of the aorta, that is caudad rather than ventrad, causing a jamming of clusters (if originally free) in the terminal portion and branches, but here clusters are entirely lacking; 8) if, as originally free clusters, they were simply carried by the current, they would be expected to lodge in the mouths of the ventraJ aortic branches, which is not the case; 9) the clusters show a progressive increase in size corresponding with the age of the embryos, between 5 and 10 mm., indicating an intrinsic growth.

In connection with the last point, the condition shown in a 6 mm. mongoose embryo (Helly fixation; Delafield's hematoyxlin toto stain) is of the greatest importance. Here a few small clusters occur along the ventral portion of the aorta. They are generally located lateral

420 H. E. JORDAN

to the ventral mid-line, just dorsad of the mouths of the mesonephric branches; several are located just ventrad of the lateral mid-line. Transition stages can here be traced between a 'cluster' of a single cell, with hemoblast characteristics, although still attached as a spheroidal cell to the endotheUal wall, and clusters of approximately a dozen cells. Certain small clusters appear as if the endothelium had buckled into the aorta. Slightly larger clusters consist of a core of endothelial cells passing into intimately associated peripheral hemoblasts. The latter condition is easily comprehensible as a derivation from the evaginated endothelium through proliferation and peripheral differentiation. In a 16 day Loggerhead turtle embryo hemoblasts can be seen differentiating from endothelimn even slightly dorsad of the lateral mid-line.

There seems to be no escape from the interpretation of such clusters of hemoblasts in the pig and other embryos as derivatives from the ventral endothelium of the aorta.

The further question then arises: why is the hemogenic capacity of endothehum of the aorta limited to the ventral wall in the region of the mesonephroi? The evidence from the study of the yolk-sac and the capillaries and vessels surrounding the brain and cord, indicates that young endothelium has a general hemogenic capacity in the pig and certain other vertebrate embryos. The ventral portion of the aorta, from where numerous branches are sprouting at these early embryonic stages, probably contains a younger and less highly differentiated type of endothelium, with greater proliferative capacity, which may explain its hemogenic r61e. Emmel no doubt has the same idea in mind when he suggests a correlation between these clusters and the development and caudal shifting of the ventral aortic branches.

RICHARD HENRY WHITEHEAD

Professor Richard Henry Whitehead, Dean of the Medical School and Professor of Anatomy at the University of Virginia, died at his home in Charlottesville on the morning of February sixth after a brief illness vdth. pneumonia.

Doctor Whitehead was bom in Salisbury, N. C, on July twentyseventh, 1865. He received his academic training at Wake Forest College where he was graduated wdth the bachelor of arts degree in 1886. The same autumn he entered the Medical School of the University of Virginia, and completed the regular two years' course then required for the M.D. degree in one year. He was appointed a demonstrator in Anatomy the following session, in which capacity he served until 1889. The winter of 1889-90 was spent in further study in various hospitals in New York. In the fall of 1890 he was called to become Professor of Anatomy and Dean in the Medical School of the University of North Carolina. From 1896 to 1905 he held also the chair of Pathology. It may be truly said that the Medical School of the University of North Carolina is chiefly the product of his inspiration and effort. The summer vacations from 1900 to 1905 were spent in research work in the Pathological Laboratory of the Johns Hopkins University and in the Hull Anatomical Laboratory of Chicago University.

In 1905 Doctor Whitehead was called to the chair of Anatomy and the Deanship of the Medical School of the University of Virginia. During the ensuing eleven years he gave unstintingly of his strength and sound judgment to the organization and maintenance of a Medical School of the first rank at the University of Virginia. In 1909 the University of North Carolina conferred upon him the degree of Doctor of Laws.

Since 1900 Doctor Whitehead had been active in research. The bulk of his work centers about the interstitial cells of Ley 421

THE ANATOMICAL RECOBO. VOL. 10, NO. 6 APRIL, 1916

422 H. E. JORDAN

dig. His eight papers touching this subject have substantially advanced our knowledge regarding the development, structure and function of these elements. His text-book of the Anatomy of the Brain remains a model of the brief and simple presentation of the fundamentals of a difficult subject. During the past few years various administrative duties of the Dean's office encroached more and more upon his time and strength and necessarily limited his research activity.

Doctor Whitehead was a frequent attendant at the meetings of the American Association of Anatomists. The quiet and unassuming manner of the man, and the clear and concise way in which he presented an occasional contribution made a decided impression upon his colleagues. He was a man who combined in a singular manner the virtues of gentleness and courage; he was a beloved and inspiring teacher; as an anatomist he was animated by the highest scientific ideals, and he displayed a vigor in the prosecution of an investigation and a caution in the formulation of deductions that were an inspiration and a guide both to his students and his colleagues.

In the very prime of his life death summoned the man who has filled a large place in a superbly efficient way; and whom it seems impossible to spare. Anatomical science has lost a loyal devotee, medical education a wise and farsighted counsellor, the Medical School of the University of Virginia a central pillar, and his many friends a perennial source of sympathy and helpfulness. H. E. Jordan.

RICHARD HKNRY WHITEHEAD

424 H. E. JORDAN

A CHRONOLOGICAL LIST OF PROFESSOR WHITEHEAD'S SCIENTIFIC PUBLICATIONS

1900 A contribution to the study of malignant tumors arising in congenital

moles. The Johns Hopkins Hospital Bulletin, No. 114, pp. 221-224.

1901 The anatomy of the brain (a text-book for medical students). F. A.

Davis Co., Philadelphia.

1903 The histogenesis of the adrenal in the pig. Am. Jour. Anat., vol. 2, pp.

349-360.

1904 The embryonic development of the interstitial cells of Leydig. Am. Jour.

Anat., vol. 3, pp. 167-182.

1905 Studies on the interstitial cells of Leydig, No. 2. Their postembryonic

development in the pig. Am. Jour. Anat., vol. 4, pp. 193-197.

A malignant teratoma of the perineum. Jour. Exp. Med., vol. 6, pp.

401-406.

1908 Studies on the interstitial cells of Leydig, No. 3. Histology. Am. Jour.

Anat., vol. 7, pp. 213-2*27.

A peculiar case of cryptorchism, and its bearing upon the problem of the function of the interstitial cells of the testis. Anat. Rec, vol. 2, pp. 177-181.

1909 A note on the absorption of fat. Am. Jour. Physiol., vol. 24, pp. 294-296.

A description of a human thoracophagus, with a consideration of its

formal genesis. Anat. Rec, vol. 3, pp. 447-457.

A case of Cyclopia, Anat. Rec, vol. 3, pp. 286-290. (Proc. Am.

Assoc. Anats.)

The interstitial cells of the testis of an hermaphrodite horse. Anat.

Rec, vol. 3, p. 264.

1911 The early development of the mammalian sternum (with Dr. J. A. Wad dell). Am. Jour. Anat., vol. 12, pp. 89-106.

1912 On the chemical nature of certain granules in the interstitial cells of the

testis. Am. Jour. Anat., vol. 14, pp. 63-70.

1913 The structure of a testis from a case of human hermaphroditism. Anat.

Rec, vol. 7, pp. 83-90.

1914 Vital staining of the interstitial cells of the testis. Anat. Rec, vol. 8, p.

104. (Proc. Am. Assoc. Anats.)

1915 A study of reversal of the circulation in the lower extremity. (With Dr.

J. Shelton Horsley of Richmond, Va.) Jour. Am. Med. Assoc, vol. 64, pp. 873-877.

ON THE DEVELOPMENT OF THE PULMONARY CIRCULATION IN THE CHICK

THEODORE L. SQUIER

From the Zoological Laboratory of the University of Michigan

THREE TEXT FIGURES AND TWO PLATES

INTRODUCTION

Although the chick long ago achieved the position of a classic in embryological studies, certain crucial details in the establishment of its pulmonary circulation have been left in the greatest obscurity. The textbooks for many years have been giving accounts, adequate enough, of the origin of the pulmonary artery, but the development of the corresponding vein has for the most part been mentioned only incidentally in connection with hatching (LilHe). Even with respect to the arterial course in the lung proper, particularly its relation to the capillary system, there remains doubt, for the exact time and mode of completion of this portion of the circuit has not been directly determined in the chick, nor for the most part as closely inferred from investigations on other forms as it might have been. It was for these reasons that I began an investigation of this subject at the suggestion and under the direction of Professor Otto C. Glaser.

METHODS AND RESULTS

The first stage of which I made injections was the 96 hour embryo. If the pulmonary circuit is complete at this time it should be possible to inject the vessels from the vitelline artery or vein. Accordingly, while the embryonic heart was still beating, india ink was injected through the vitelline artery by Knower's bulb method (Knower, '08). Care was used, of

425

426 THEODORE L. SQUIER

course, not to injure the vitelline vessels at any time, and by keeping the embryo covered as much as possible with warm saUne solution the heart action was prolonged and better injections secured. The injected chicks were fixed in Bouin^s fluid, stained with borax-carmine, and after imbedding in paraffin cross sections were cut 10 /x thick.

A cursory examination of such sections prepared from a 96 hour embryo (the length determined from the number of sections was 9.64 mm.) at once showed that the ink had entered all the smaller vessels, and about the lung rudiments a large number of injected capillary tubules were found. From this it is conclusively shown that a very considerable portion of the pulmonary circuit is complete at this time, but whether the entire course is complete and what its exact relations to the rest of the circulatory system are, could only be determined by means of reconstructions.

Because of the minuteness of the structures under consideration, reconstructions in wax could not be undertaken. Instead, the graphic method was resorted to with entirely satisfactory results. I reflected the sections at 300 diameters magnification and outhned the lungs and the surrounding vessels. Then by projecting each of these separate drawings the final reconstruction reproduced in plate 1 was completed. The drawing represents the left lung rudiment and its surrounding plexus of vessels viewed from the medial surface. The right rudiment corresponds so closely to the left with respect to the number and development of pouches as not to warrant a separate description. Throughout I will designate that side of the lung rudiment which faces the middle line of the embryo as the medial surface and the side directed away from the middle line will be spoken of as the lateral surface.

The left lung rudiment at 96 hours consists of three well marked pouches budding from the dorsal side of the bronchus. The first, or most anterior of these, plate 1, g', is the largest. The second and third, h and i, decrease rapidly in size. Posterior to the last pouch, i, a broad swelUng dorsal in position denotes the beginning of a fourth pouch.

PULMONARY CIRCULATION IN THE CHICK 427

The rudiment in question extends through 143 sections, that of the right lung through 135 sections (10 m thick) counting from the bifurcation of the bronchus. Anteriorly the pulmonaryartery forms an extensive plexus, p, the principle portion of which lies dorsal to the bronchus. One arterial branch, s, courses in a ventral direction from the plexus around the median surface of the bronchus and closely approaches a dorsally directed vessel, r, itself a tributary of the median cranial vessel, t, which joins the left pulmonary vein just before this unites with the corresponding vein on the right. Such an arrangement is certainly very suggestive of a previous anastomosis between artery and vein. From the cranial plexus, p, the lung artery, a, proceeds along the external surface of the bronchus and is unbranched until the expanded portion of the lung proper is reached. Immediately before reaching the lung the artery is quite small. It continues along the dorso-lateral surface of the lung rudiment in nearly a straight line and gives off two branches the first of which, plate 2, j, provides for the most anterior lung pouch, while the second branch, k, provides for both the second and third pouches. Distally the artery anastomoses with the vein in the region indicated by x in plate 1, and also it gives off twa lateral branches shown at I and m in plate 2 which end blindly.

On the ventro-medial surface of the lung rudiment three main venous lines of drainage (w, v, w, plate 1) are established. The anterior vessel, u, is the largest, as would be expected, since it drains the oldest and largest of the bronchial pouches. The other two veins show a decrease in size corresponding approximately to the decrease in size of the vesicles which they drain. The left tributary, c/, of the pulmonary vein, formed by the union of the three lateral vessels, turns ventro-medially from the bronchus at an abrupt angle to unite with the corresponding venous tributary from the right lung. The common stem thus formed follows a nearly straight course ventralward and empties into the left auricle of the heart as described by Federow in the case of a duck of 4 days, 13 hours: **Die Einmiindung der Vene durchbohrt den Vorkammerschiedewandgrund schrag

428 THEODORE L. SQUIER

und oflfnet sich an der linken Seite des Septum. Dasselbe ist mit dem Vorkammerboden verschmolzen.'^

No caudal connection exists between the splanchnic plexus and the vessels of the lungs, and in this stage no trace of such connection is indicated.

In order to verify the essential correctness of the reconstruction, I cut sagittal sections 200 m thick of a 96 hour injected embryo. The result was very satisfactory indeed, since the sections were thick enough to show the course of the circulatory vessels even more clearly than would actual dissection of the organs concerned. The thickness of the sections precluded the possibihty of counting the secondary lung vesicles or pouches. The general course of the vessels is the same as found in the reconstruction. Several features of the cranial arterial plexus are shown here (fig. 1) however, that are omitted in the reconstruction owing to the fact that the latter does not include the most anterior part of the plexus. The artery, a, sends a single dorsally directed branch, e, to the gut. Near this dorsal branch two ventral branches are given off and the anterior of these, jy branches immediately into two stems which eventually anastomose with a plexus that empties into the median cranial branch of the pulmonary vein. Cranially an intricate dorsal plexus, d, is given off by the artery and its anastomosing branches extend cephalad possibly as far as the level of the first gill arch (compare with p, plate 1). The plexus, /, surrounding the lung proper is very similar to that found in the reconstruction. The artery, a, follows the dorsal surface of the bronchus (in the figure faintly indicated by a sUghtly darker area) as described in the preceding embryo, and between the last cephalic branch, s, and the level of the lung proper it is unbranched and of uniform diameter. In the section next adjoining that figured a small caudally directed branch arises from the common stem of the pulmonary vein near the union of the right and left main tributaries and anastomoses with the plexus of blood vessels surrounding the gut posterior to the lungs. The same section also shows the median-cranial tributary of the vein and its arterial anastomosis already described in the preceding embryo.

PULMONARY CIRCULATION IN THE CHICK 429

In the 96 hour chick then, the pulmonary circulatory system is complete. The vein stem which empties into the right auricle proceeds dorsally for a short distance and then branches into right and left veins which drain the corresponding lung rudiments.


Fig. 1 Photograph of sagittal section 200 /* thick of injected 96 hour chick showing general course of right artery and vein together with plexus surrounding the right lung rudiment ; X 97. a, pulmonary artery; d, cephalic plexus surrounding gut; j, arterial stems that anastomose with the median cranial vein tributary (not shown); 6, common stem of pulmonary vein; r, left pulmonary vein; /, plexus surrounding the right lung rudiment. Region of bronchus and lung rudiment is indicated by the dark area in the neighborhood of the plexus, /, and the pulmonary artery.

430 THEODORE L. SQUIER

The left vein, near its union with the right, receives a tributanwhich runs in a cranial direction midway between the lung rudiments and which forms anastomoses with ventrally directed branches from the lung artery. These anastomoses may or may not persist at 96 hours.

Sections 10 m thick were prepared from a 72 hour injected embryo measuring 6.56 mm. (the length was determined from the number of sections). The lung anlage consists of a right and left primary rudiment. No secondary lateral vesicles are present. The distal ends of the primary bronchi are slightly swollen into pouches. The common stem of the pulmonarjvein empties into the left side of the sinus venosus at 6, figure 2. From this point the vein stem runs dorsalward for a short distance and then receives its right and left tributaries, c and d, which drain the ventral sides of the right and left lung vesicles respectively, where, as clearly shown by the injections, they form capillary baskets distinguished from those of the 96 hour stage by greater simplicity.

Just before the confluence of right and left veins, a comparatively large vessel empties into the dorsal side of the common pulmonary stem. This last named tributary runs to the ventral side of the gut where it branches and the two veins (e, fig. 2), after forming anastomoses with branches from the pulmonary arteries, parallel each other in a caudal direction until the level of the distal ends of the lung rudiments has been reached. Here their identity becomes lost in a general plexus surrounding the gut.

Frontal sections 200 m thick of this stage were also cut. The lung anlagen are in about the same stage of development as in the 72 hour embryo just described. The right and left primary lung vesicles as shown in figure 3, Z, are without secondarypouches, and the distal end of each is slightly swollen. The common stem of the pulmonary vein receives the right and left vessels, d and c, at a point midway between the two primary vesicles, and nearly midway between the proximal and distal ends of the bronchi. In addition to the main vessels draining the right and left lung rudiments, two cephaUc branches arise from the common stem. The branch, g, which lies to the left,

PULMONARY CIRCULATION IN THE CHICK


431


leaves the stem vein without dividing, while the other, h^ which is considerably larger, bifurcates almost immediately. All of these vessels run cranialward along a median line between the bronchi. Near the proximal end of the left bronchus a lateral branch, j, from the left pulmonary arterj^ crosses the dorsal surface of the bronchus and anastomoses \nth the cranial end



_ u


Fig. 2 Cross section of 72 hour chick through region of the sinus venosus and mouth of the pulmonary vein; X 120. w, auricle; v, ventricle; 6, common stem of pulmonary vein; s, sinus venosus; c, d, left and right pulmonary veins respectively; a, left pulmonary artery; c, median vein tributaries; i, bronchus; I, gut.

A G

M

C

F

L


H


.D

.E N


.^ L


432

PULMONARY CIRCULATION IN THE CHICK 433

of one of the median vein branches. A caudal branch, e, is also present, which connects the pulmonary vein with a well developed plexus, p, surrounding the posterior portion of the gut.

The vessel, e, connecting the gut plexus, p, with the right pulmonary vein is verj'- minute and in the preparation was but slightly injected. The exact relations of the corresponding vessel, /, on the left have not been determined. Whatever they may be, the evidence from the size of the^e veins indicates that at 72 hours the blood current flowing between this splanchnic plexus and the pulmonary veins is feeble. The correctness of this supposition is further indicated by the fact that at 96 hours the connections e and / are either still more weakly indicated or entirely absent. The pulmonary circulation in the 72 hour chick then, is also complete. It differs however from that of the 96 hour stage in two respects : the capillary baskets around the lung rudiments proper are simpler, but the accessory and secondary connections such as those with the splanchnic plexus are more numerous. Furthermore the median cranial tributaries of the pulmonary veins in comparison with the main right and left vein trunks in the 96 hour chick are much larger and as in this latter stage anastomose with the lung arteries.

DISCUSSION

That the pulmonary circuit is complete, though not necessarily in its definitive form in the 72 hour chick is not open to doubt. Granted only that it undergoes gradual correlative metamorphosis during the remainder of the period of incubation, there is left no reason for expecting a circulatory crisis at hatching. The success of my injections shows conclusively that

Fig. 3 Frontal section 200/* thick of injected 72 hour chick; X 120. m, mouth of common pulmonary vein; d, c, right and left pulmonary veins; g, h, median cranial tributaries to common vein; j, lateral branch of artery forming anastomosis with median cranial vein; a, pulmonary artery; n, small portion of plexus of vessels surrounding right lung rudiment; I, lung rudiment areas indicated by deeper shading; e, caudal tributary to pulmonary vein;/, probably a tributary to pulmonary vein with a connection similar to that of e; p, plexus surrounding gut.

434 THEODORE L. SQIHER

blood must flow through the puhnonary system at a very early stage, although owing to relative resistances in this and the general systemic circulation the amount passing through the lungs is undoubtedly small. With the general enlargement of the pulmonary system, and finally upon collapse of the last aortic arch, a circuit adequate for respiratory purposes is not only at hand, but is automatically put in use.

With respect to the early course of the vein and its anastomoses with the artery, several conditions found correspond in general with those of Federow. The close approach of a branch from the median cranial vein to an arterial branch in the 96 hour chick (plate 1, r) has been mentioned above as suggestive of a previous anastomosis which has but recently disappeared. In a duck of 114 hours Federow found a similar condition and says: "Der distale (dem Kopfe nachste) Teil des kranialen Venenastes, der die Anastomosen mit den ventralen Lungenarterien bildet, hat die Verbindung mit dem proximalen Abschnitte des kranialen Astes verloren.

With respect to the anastomosis between the caudal branch from the vein and the plexus surrounding the gut he says:

leh mochte hier noch erwahnen, dass der kaudaie Venenast bei den Vogeln und Saugetieren (bei den ersteren durch die Gefasse der Speisrohre) eine Zeitiang mit der hinteren Hohlvene in Verbindung steht. Wenn auch diese Verbindung zwischen den Gefassen von ganz verschiedenen Systemen in der topographischen Nahe und in der schwachen Differenzierung der Lunge und des Damikanals an dem betreffenden Stadium eine gewisse Erklarung findet, so stelit sie sich (loch sehr bemerkenswert dar. Etwas Ahnliches beobachtet man bei der Entwickelung der hinteren Hohlvene, namlich die Verbindung zwischen der V. omphalo-mesenterica und den Vv. cardinales poster iores.

Federow has dealt at length with the origin of the pulmonary vein and concludes that it is produced at a very early stage of embryonic life as a proliferation of endotheUum from the dorsal sinus wall which projects into the dorsal mesocardium. Into this proliferation the sinus cavity tunnels, thus producing a short vessel which is the anlage of the vein. This vein, verj^ near its point of origin from the sinus, branches into two vessels

PULMONARY CIRCULATION IN THE CHICK 435

whose ultimate divisions terminate in capillary trees that anastomose with capillary outgrowths from the lung arteries. The vein mouth, which at first empties into the sinus, is finally absorbed into its definitive position as a result of unequal growth of the heart wall.

Brown, on the basis of his investigations on the pulmonary system of the domestic cat, disagrees with Federow's view and holds that the pulmonary veins are differentiated out of an indifferent plexus originally present around the primitive gut and from the beginning connected with the sinus. The vessels of this plexus form numerous anastomoses with the surrounding veins and in addition the plexus '^exhibits two well defined connections with the venous portion of the heart, (1) the cephaUc or pulmonary, and (2) the caudal or postcaval. These are constant both in occurrence and position." According to Brown's view, therefore, the pulmonary vein is simply a specially developed part of an indifferent plexus present from the beginning in the region from which the lungs grow.

My own observations on the development of the pulmonary circulation do not furnish the basis necessary to decide between these opposed views. Concerning the presence of an extracardiac indifferent plexus, as claimed by Brown there is no doubt. The primitive gut is certainly surrounded by a network of capillaries, and jt seems probable that as the lung anlage pushes out from the gut a portion of the surrounding capillary plexus would be carried with it. If this capillary system is derived from angioblast originally continuous with that from which the heart itself is formed, it is easy to see how the pulmonary vein might be nothing more than the survival of a pathway rendered permanent in accordance with the mechanical principle that those vessels in which the blood current is strong become permanent whereas those in which it is weak atrophy and eventually disappear (Mall '10). The numerous early connections between pulmonary arteries and veins which for the most part are lost as early as 96 hours seem to harmonize with this interpretation. In the 72 hour chick, in addition to the pulmonary veins, which persist, there are from one to three

436 THEODORE L. SQUIER

or more branches which run toward the head to form the median cephalic connection with the arteries and caudally one or more vessels connect the vein stem with the plexus of the gut. This latter connection shows signs of atrophy even at 72 hours and in the 96 hour chick from w^hich the reconstruction figured in plate 1 was made it has completely disappeared. The median cranial connections are more persistent and in plate 1 the condition of the vessels indicates that the connection with the left artery has been but recently lost. Accordingly then, the pulmonary vein in the chick could be regarded as complete from the beginning and as representing the sole survivor of numerous primitive vessels that drain the region surrounding the lung anlage.

On the other hand Federow's idea that a median dorsal outgrowth from the sinus connects secondarily with the lung capillaries is not to be set aside lightly. In the first place I have found the outgrowth clearly indicated in embryos earlier than those injected; in the second place its position is identical with that assigned to the earliest indications of the pulmonary vein as given by Brown. It seems quite possible therefore that further investigation might harmonize the two views which so far at least do not necessarily appear mutually exclusive.

In conclusion I wish to acknowledge my indebtedness to Professor Glaser whose suggestions and assistance both in the laboratory work as well as in the preparation of my manuscript have been of the greatest value.

BIBLIOGRAPHY

Bremer, J. L. 1902 On the origin of the pulmonary artery in mammals. Amcr.

Jour. Anat., vol. 1, no. 2. Brown, A. J. 1910 The pulmonary vein in the domestic cat. Anat. Rec,

vol. 7, no. 9. F'ederow, V. 1910 Uber die Entwickelung der Lungenvene. Anat. Hefte.

1 Abt. 122 Heft (40 Bd., H. 3). Flint, J. M. 1907 The development of the lungs. Amer. Jour. Anat., vol. 6,

no. 1. Knower, H. E. 1908 A new and sensitive method of injecting the vessels

of small embryos, etc., under the microscope. Anat. Rec., vol. 2,

no. 5. LiLLiE, F. R. 1908 The development of the chick. Mall, F. P. 1906 A study of the structural unit of the liver. Amer. Jour.

.\nat., vol. 5, no. 1.

PULMONARY CIRCULATION IN THE CHICK PLATE 1

THEODORE L. SQUIER


PLATE I

EXPLANATION OF FIGURE

Reconstruction of the left lung rudiment and accompanying blood vessels of a 96 hour chick viewed from the medial surface; X 100.

a, left pulmonary artery df left pulmonary vein

g, hy i\ first, second and third lung pouches respectively

i, blind ventrally directed arteriole

0, blind distal arterial termination

p, dorsal plexus of artery

r, branch from the median cranial tributary of the pulmonary vein

s, ventrally directed arterial branch which probably previously anastomosed with the dorsally directed vessel, r

tf median cranial tributary of the pulmonary vein

M, V, w, veins draining first, second and third lung pouches respectively

j:, distal anastomosis between artery and vein


THE ANATOMICAL RECORD, VOL. 10, NO. 6


437

PULMONARY CIRCULATION IN THE CHICK

THEODORE L. 8QUIER


PLATE 2


PLATE 2


EXPLANATION OF FIGURK


Reconstruction of the left lung rudiment and accompanying blood vessels viewed from the lateral surface; X 100.


a, left pulmonary artery

/, left bronchus

g, h, ij first, second and third lung

pouches respectively J, arterial branch supplying the first

lung pouch


A*, arterial branch supplying second

and third lung pouches /,m, lateral blind arterial branches n, blind distal termination of vein o, blind distal termination of artery


438

THE VITAL STAINING OF MITOCHONDRIA IN

TRYPANOSOMA LEWISI WITH JANUS

GREEN

p. G. SHIPLEY

From the Anatomical Laboratory, Johns Hopkins University

ONE FIGURE

Franga reported in 1907 that there were granulations in the cytoplasm of certain Trj^panosomes (T. costatorum and T. rotatorum) rendered visible by praeagonal staining with neutral red, safranin, methylene blue and pyronin. The last he found to be a true vital stain, since organisms which had been brought into contact with it remained alive and motile for three or four days after they had been stained. He was unable to obtain the same results in trypanosomes parasitic in mammals (T. equiperdum); but Policard ('10) who used a somewhat different technique was able to vitally stain granules in Trypanosoma Brucei, gambiense and equiperdum. He spread a drop of blood into a thin layer between a slide and cover slip, at the edge of which, he placed a drop of concentrated solution of neutral red : the diffusion of the dye into the plasma from the point of contact of the drop with the blood film resulted in a positive stain.

He divided the trypanosomes studied into 3 classes, according to the distribution of granules in the cytoplasm. 1) Those with a few very small granulations. 2) Those with anterior granules and 3) Trypanosomes with anterior and posterior granules. Policard made no attempt to ascertain the chemical composition of the granules which he studied, but he believed that they were not products of the degeneration of the cytoplasm of the organism. He says: ^^Nous savons seulement que leur reaction n'est pas acide et que ce ne sont pas des produits de deg6n6r6scence, puis qu'on les rencontre chez des trypanosomes au debut

439

440 p. G. SHIPLEY

de rinfection. Leur role physiologique n'est pas meme encore soupconne/'

Miehaelis ('99) has recently introduced a vital dye, janus green, which Bensley ('11) and others have shown to have the power of selectively staining mitochondria in Hving cells; and it was with the hope that this dye could throw some light on the nature and significance of the granules described by Policard and Franca, that it was used in the vital staining of trypanosomes.

Cover glasses were prepared by covering them with a thin film of an alcoholic solution of the dye (95 per cent alcohol, 20.00 cc; janus green, 00.02 gram), after the technique used by Cesaris Demel ('07) and others to demonstrate the 'sostanza granula-filimentosa' in red blood cells. The film, which is spread on the cover slip with a glass rod, should be even and so thin as to be almost invisible. A drop of blood from an animal (Mus norvegicus albinus) infected artificially with T. Lewisi was allowed to spread between the dyed surface of a cover glass and a clean slide, and was examined at once. The thin crust of dye substance left on the cover slip by the evaporation of the alcoholic vehicle dissolves in the plasma, and forms a solution which at once stains the trypanosomes swimming about in it. Fresh blood was also studied after being mixed on a clean slide with equal parts of 1 to 10,000 solution of janus green in physiologic saline solution. Control preparations were made of infected blood stained with neutral red, pyronin, and di-ethyl-safranin, a dye which has been used by Cowdry ('14) as a vital stain for mitochondria in the human leucocyte. Fixed smear preparations of blood were made and stained after the methods of Bensley, Meves, Altmann and Benda. The smears were exposed to osniic acid vapor for a few minutes before immersion in the fixing fluids in order to preven' any distortion of the granules from drying.

In fresh preparations the trypanosomes move about so rapidly that, in order to study them carefully, it is necessary to add gelatine to the plasma; or to make smear preparations of the blood as soon as the granules are fully stained, which happens in from two to five minutes.

STAINING OF MITOCHONDRIA WITH JANUS GREEN 441

The first structure to stain is the kineto-nucleus which takes up the dye with remarkable avidity even when very dilute solutions are used, and appears as a very bright bluish green rodlike structure. Other stained granules make their appearance in the cytoplasm almost at once. In T. Lewisi the distribution of these granules in the cytoplasm does not adhere strictly to any of the three types suggested by Policard, and trypanosomes of type one, which contains a few very small granules, are extremely rare. They are scattered through the cytoplasm and are most numerous at the anterior end of the organism (figs. 1, 2 and 4). Often they are clumped in a mass just anterior to the nucleus (fig. 1), or there may be a clump at either or both ends of the nucleus (figs. 2, 3, and 5), in which case the granules anterior to the nucleus are most numerous. The tendency to mass formation is very marked. Occasionally organisms are found having a few very fine granules posterior to the kineto-nucleus (figs. 1, 4 and 5).

The granulations vary from the tiniest granules to large swollen masses, which Policard noted as being frequent in trypanosomes from the blood of animals in extremis when involution forms are most frequent (figs. 5, 6, and 8).

Vacuole like bodies sometimes appear in the cytoplasm, are often as large as the nucleus and they are coloured with the vital dye (figs. 7 and 8). One side of the vacuole is always more deeply stained than the other and some vacuoles have a seal ring appearance in optical section (fig. 8 c). Some of these vacuole like bodies contain small granules in their interior which also stain vitally with janus green (fig. 8 b).

The motility of the organisms is apparently in no way affected by the number of these cytoplasmic granules or by their size, except that the swelling of the granules which occurs in dying organisms goes hand in hand with the slowing of movement which characterizes the beginning of their dissolution.

Both the large granules and the vacuole like bodies probably result from swelling, since all transition forms may be found from the fine granules to the fully distended vacuole. They are probably an index of a degenerative change of some sort, because

442 P. G. SHIPLEY


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STAINING OF MITOCHONDRIA WITH JANUS GREEN 443

they are more numerous in trypanosomes which are losing their motility; and often dead swollen organisms are seen in which the vacuoles entirely fill the cytoplasm. Policard noted vacuole like bodies which he differentiated from food vacuoles, etc., by their brick red staining with neutral red. The death of the trypanosomes is followed by the gradual fading of the stain. The nucleus remains uncoloured throughout. In these preparations, as in the fixed smears, the mitochondria in the white blood cells may be seen stained in the same way as the granules in the cytoplasm of the trypanosomes, and in preparations stained with janus green the leucocytic nuclei react as do those of the trypanosomes, remaining uncoloured by the dye for a long time after the mitochondria have taken their characteristic blue green tint.

It is impossible to say definitely that the granules which Franga and others have seen after supervital staining with neutral red are identical with those which are stained by solution of janus green. The mitochondria of healthy cells do not react in neutral red. Injury to the cell, however, in presence of a basic dye is at once followed by intense coloration of the mitochondria. The trypanosomes swimming about in these dye solutions are cells slowly undergoing toxic death, and are in a condition in which one expects to find mitochondrial staining. It is, therefore, probable that a part at least of Franga's granules are of mitochondrial nature, and therefore, are identical with the granules which stain selectively with janus green. In organisms treated with di-ethyl-safranin the granules are stained but the staining is neither so sharp nor so constant as when the janus green is used.

In permanent preparations fixed and stained by the methods of Benda, Bensley, Meves and Altmann the same granulations give characteristic mitochondrial staining reactions. The kinetonucleus in particular, stains very brilliantly with the mitochondrial dyes and is sharply contrasted with the surrounding cytoplasm. These granulations show also the solubility in acetic acid which characterizes mitochondria in other situations. After fixation in a solution of osmic acid containing five drops of acetic acid (glacial) to 20 cc. of the solution it is not possible to find

444 p. G. SHIPLEY

definite isolated granules. Instead a diffuse staining is seen in the region where they normally occur, and if the percentage of acetic acid in the fixing fluid is increased above this amount even the diffuse staining is not found.

These granules show the characteristic reactions of mitochondria toward fixing and staining fluids, and their reaction to vital stains is Ukewise typical. They exhibit the same sensitiveness to increased acetic acid content of fluids used to fix them, and finally they may be stained in the same way as mitochondria and may be observed side by side with mitochondria in cells known to contain them (blood leucotytes). The conclusion is therefore justified that they are mitochondria.

Extremely interesting in this connection is the reaction of the kineto-nucleus toward mitochondrial stains, both in fixed and vitally stained preparations, indicating as it does a certain similarity between it and the mitochondria of the organism. Whether or no there may be a functional similarity as well, is a question, the answer to which offers the opportunity for a most fascinating study. A close relation of mitochondria to the motor portion of the cell has been asserted, and Benda ('13) has attributed to these granules a function in relation to cytoplasmic contractility because 1) of their microchemical resemblance to the dark bands of striated muscle, 2) their disposition about the axial filament of the spermatozoid and their arrangement and abundance about the roots of the cilia in ciliated cells, expecially those of the amphibian kidney. His view, however, has been opposed by Regaud ('08) who finds that the mitochondria in the ciliated cells of the urinary tubule of cold blooded vertebrates are relatively few in number and have apparently no constant orderly arrangement; and by Faur6-Fr6miet ('09) who finds that in Vorticella the mitochondria which are grouped about the axial filament have no relation to its contraction. The mobility of T. Lewisi is certainly unaffected by the amount of mitochondrial substance in the cell cytoplasm. At any rate a further study of the mitochondria of the Trypanosomata during cell division, especially in the so called abrephaloplastic forms, is necessary before one can include the mitochondria with the kineto-nucleus of these organisms in speaking of differentiated kinoplasm.

STAINING OF MITOCHONDRIA WITH JANUS GREEN 445

BIBLIOGRAPHY

Benda 1913 Cited by Regaud C. R. des de le Soc. Biol.

Bensley, R. R. 1911 Studies on the pancreas of the guinea pig. Amer. Jour.

Anat., vol. 12, pp. 297-388. Cesaris, Demel A. 1907 Studien uber die roten Blutkorperchen mit den

Methoden der Farbung in frischen Zustande. Folia Haematologica

Bd. 18, Supp. 41, S. 1 to 32. CowDEY, E. V. 1914 The vital staining of mitochondria in human blood cells.

Internat. Monatsschr. Bd. 31, pp. 267-284. Fauk6-Fr£miet 1909 Discussion of the work of Regaud and Mawas **Sur

le structure du protoplasma, etc. "Communications Association des

Anatomistes. Nancy. Franca, C. 1907 Coloration vital des Trypanosomes. Bull, de la soc. Portu gais des Sciences Nat., pp. 8-11. Michaelis, L. 1899 Die vitale Farbung, eine Darstellungs methode der Zell granula. Arch. f. mikr. Anat., Bd. 55, S. 558-575. PoLicARD, A. 1910 Sur la coloration vitale des Trypanosomes. C. R. de la

Soc. Biol. 19 Mars. N. 11, pp. 505-507.

Sur les mitochondries des cellules ciliees du tube urinaire. Regaud Cl. 1908 C. R. des Seances de la Soc. Biol., 25 Juillet, T. 65, p. 206. A SUGGESTION AS TO THE PROCESS OF OVULATION AND OVARIAN CYST FORMATION^

A PRELIMINARY REPORT

S. S. SCHOCHET

Departments of Anatomy of the Tulane U niversity of Louisiana ^ and of the Dartmovth

Medical School

It is generally conceded, that pressure atrophy of the ovarian stroma is the chief means by which the extrusion of the ovum becomes possible. During the growth and maturation of the ovum, the cells of the Graafian folUcle, after increasing greatly in number, begin to disintegrate and liquefy. From then onward, due it is thought to the different chemical composition of the liquor, thus forming in the folUcle, or of the general content of the folUcle, an endosmosis seems to be induced by which the liquor folliculi increases to a far greater amount than is thought possible to result from the Uquefaction of the follicular cells. The folUcle so distends that, foUowing the direction of least resistance, one side of it approaches the free surface of the ovary, producing a bulging in this surface, dispersing the ovarian stroma, thinning its tunica albuginea and the overlying epithelium, and results in a compression of the blood capillaries intervening between it and the surface of the ovary. Clark has shown that the capillaries in the summit of the bulging are practically obliterated by the pressure. It is supposed that nourishment thus cut off from the ovarian stroma under compression, the stroma atrophies till its resistance is less than the pressure exerted by the distending folUcle and the content of the foUicle bursts into the body cavity. The liquefying of the follicular cells having continued till the ovum is free within the follicle, the ovum is extruded into the body cavity with the discharge of the liquor folliculi.

Proc. Amer. Asso. Anatomists, New Haven, December 30, 1915.

447

448 S. S. SCHOCHET

The question was suggested by Dr. Irving Hardesty as to whether the liquor folliculi does not exert some special digestive action upon the resisting tissues thus aiding in the process by which the ovum is extruded. The idea carried with it the action of the Uquor folliculi in the phenomena of menstruation.

Accordingly, a series of experiments were made, the result of which may be of interest. The work was undertaken with the view to determine:

1) Whether the liquor foUiculi has a digestive action and if so does it possess a specific enzyme that can be demonstrated by dialysis or other tests?

2) If it possesses such action, under what conditions is it altered? Is it decreased in pathological conditions?

3) If possessed, will a quantitative estimate of its strength indicate the amount of it present.

A review of the literature has failed to suggest any special chemical action of the liquor folliculi, and nothing of its chemical composition except that it is a para-albumen.

MATERIALS AND METHODS

Obviously only small quantities of the liquor can be obtained at best. Human material is not available sufficiently fresh and in sufficient abundance. Therefore, it was necessary to obtain it from ovaries of animals available in large numbers and freshly killed.

Ovaries of the sexually mature hog (sus Scrofa) were used, as these could be very readily obtained in the slaughter houses of New Orleans. All histological observations indicate, that the process of the production of the liquor folliculi in the Graafian folUcle and the process of extrusion of the ovum are the same in the human as in the hog; and it is logical to assume that the liquor plays the same r6Ie as in the human ovulation and that any results indicating its physiological action in the hog must be similar to the human. In the experiments, anmiotic fluid and fluid from ovarian cysts of the hog were used for comparison with liquor folliculi, and also fluid from human ovarian cysts.

PROCESS OF OVULATION AND CYST FORMATION 449

The Graafian follicles may be readily distinguished in the surface of the ovary from the corpora lutea and ovarian cysts by their reddish color and manifest turgidity. The corpora lutea are yellowish spherical protrusions of firm consistency, and the cysts are translucent and usually larger. The liquor folliculi was obtained by inserting into the large follicles a fine needle of small, dry, thoroughly sterilized, all glass syringe. All material was collected at the slaughter house under rigid aseptic precautions from ovaries of freshly killed animals. The hog being one of the animals, which give birth to litters of young, ovaries may be obtained containing a number of mature follicles. Obviously but a few drops of the liquor could be obtained from a single follicle. All ovaries were taken warm just as the viscera were removed. It was noticed that if allowed to cool only a very small quantity or none could be obtained, due probably to coagulation.

The experimental technique used in this work is based on the principles of Abderhalden's dialyzation reaction with its modification by Grtitzner. It may be briefly summarized: 1) in preparation of material to be tested; 2) process of obtaining the liquor folliculi; 3) diffusion tubes; 4) the test; 5) dialyzation; and 6) the controls.

In preparation of the material for the digestive experiments, blood was obtained, defibrinated, and the fibrin thoroughly washed in cold water until all blood was removed from it. The pieces of fibrin containing the faintest tinge of pink were discarded to eliminate whatever reaction any blood or plasma themselves might give. Pieces of fibrous connective tissue, muscle, and ovary were also used. These were boiled in distilled water for five minutes and the filtrate tested for substances reacting with ninhydrin and with the biuret reaction. This was repeated until the filtrate failed to give a reaction with one cubic centimeter of ninhydrin on boiling one minute. The fibrin in Griitzner^s test was prepared according to the method devised by him, which must be omitted as time and space will not permit.

Schlercher and Schull, No. 579 dialyzing tubes were used. These were first carefully tested with albumen to insure imper 450


S. S. SCHOCHET


meability to it. They were then boiled for five minutes just before each test.

In the test, small quantities of liquor folliculi were introduced into the diffusion tubes together with a small piece of muscle, fibrous connective tissue, and ovarian tissue, prepared as above; separate tubes being used for each. Care was taken that all tissues were below the surface of the liquor folliculi to prevent any possible error from decomposition of pieces. The diffusion tubes were next placed in larger tubes of sterile distilled water. A layer of xylene was placed upon the fluids within and without the dialyzer to prevent the growth of bacteria and to prevent evaporation.

Controls were made with exactly the same technique using amniotic fluid, normal saline, and cystic fluid, instead of liquor folliculi. Cultures were made from the liquor folliculi and cystic fluid used; to rule out any possibility of digested protein due to bacterial action.

The period of incubation was twenty-four hours, as some difficulty was experienced in tests of shorter periods of incubation due to weak reactions. Abderhalden also employed and later advised this modification as to time. The temperature of incubation was 38°C. The filtrate or the fluid in the tube surrounding the dialyzers were tested with ninhydrin and the biuret test.

The results obtained may be tabulated as follows:

/. Abderhalden' s diaUjzation reaction OVABIAN TISSUE


MUSCLE


MQAMENT


FIBRIX


Liquor folliculi


+ + +


4


-f-f-f


Cystic fluid (small cysts)


4

Amniotic fluid



Normal saline


2. Grutzner's fibrin test


Liquor folliculi. Amniotic fluid.. Normal saline. .


PROCESS OF OVULATION AND CYST FORMATION 451

Sixteen tests were made with the dialyzation method which was repeated three times, making a total of forty-eight tests to which may be added the tests of Griitzner's Method.

As indicated in the table, the Uquor folliculi gave a positive reaction with fibrin, fibrous connective tissue (lig. nuchae), muscle and especially a strong reaction with ovarian tissue. Slight positive reactions were obtained with cystic fluid from small cysts, and negative results with larger ones. The controls of amniotic fluid and normal saline were found to be uniformly negative with the exception of slight reaction with cystic fluid, (from small cysts) which to my mind is the strongest point in favor of proper technique.

Before drawing any conclusions from the above it might be well to briefly summarize the theories of ovulation.

By ovulation is meant the discharge of a mature ovum from its Graafian follicle. The study of the process involves a consideration of the development of the folUcle and its rupture.

In ovulation the earlier stages have received more attention than the late stages, though it is the latter in reality that must explain the rupture of the Graafian follicle. It is admitted by all that the most mature follicle, very probably aided by the condition of premenstrual congestion, rapidly swells to the size of a large pea, due to the accumulation of the Uquor folliculi, and produces a hemispherical protrusion of the surface of the ovary.

In considering the forces involved in the formation and increased production of liquor folliculi, the changes occurring in the circulation must be considered.

If the great increase of the liquor folUculi were considered the result of a mere transudation into the follicle, it must be realized that the ordinary blood pressure in the capillaries cannot be the only important factor. It is obvious that the pressure within the enlarged follicle is greater than the blood pressure within the capillaries since the capillaries are compressed by the follicle and even constricted in the summit of its protrusion of the surface of the ovary. The question arises whether this increase of tension of the tissues may increase or decrease the

452 S. S. SCHOCHET

flow of blood through the tissues and thereby possibly increase or decrease transudation. Compression of veins would result in congestion of the veins behind the region of compression with increased transudation; compression of an area of capillaries must result in congestion of the contributing arterioles and capillaries not affected by the pressure with increased transudation. It is reasonable to assume, however, that an equiUbrium would soon be estabUshed in which the blood pressure in the veins concerned on the one hand, or the arteries on the other would be no greater than the common blood pressure. The result would be a decreased in the blood supply, in the immediate vicinity of the follicle, beginning in the earUer stages of accumulation of the Uquor folliculi. However, the approach of ovulation, or the later stages of the enlargement of the follicle, is accompanied by a marked congestion of the general ovarian and uterine vascular system, and this congestion must result in a greater transudation from the vessels into the tissue spaces. Therefore, it is very probable that the distension of the folUcle by the accumulation of its liquor folliculi must be due to causes other ^ than increased blood pressure and resultant transudation from the vessels into the follicle.

It is suggested then that the liquor follicuU must accumulate by a process similar to that of secretion, that the chemical nature of the cells of the Graafian follicle or the product of their liquefaction, or both, may induce an endosmosis into the antrum of the follicle, producing its distension to the pressure greater than that of the tissue about it or of the blood in the ovarian vessels. It is quite conceivable that the chemical composition of the liquor folliculi may vary at different periods, in the process under the agency of the increased number and liquefaction of the follicular cells, as well as the amount of the liquor. Also the layer of cells in the stratum granulosum may act as a diffusion membrane whose control of osmosis may vary at different periods, increasing the endosmotic current in the later stages of enlargement. The congestion of the ovarian vessels is probably induced by the pressure irritation of the enlarging folUcle. The chemical character of the follicle mav induce it.

PROCESS OF OVULATION AND CYST FORMATION 453

This congestion results in a greater transudation from the vessels into the tissues, and the increased fluid in the tissue spaces; but this means a greater amount of fluid for the forces within the follicle to act upon, resulting in the greater enlargement of the follicle and this again irritating to a greater congestion, and so on till the follicle may burst and the cause removed. It would be logical to expect that, during the period of congestion, characteristic of the premenstrual period, a more vigorous elaboration of the products of the cells of the follicle (ferments or enzymes) would be made possible by the increased transudation or nutrition in the tissue spaces.

It is, of course possible, that other forces may contribute to the process of ovulation. We have no evidence that the nervous system has any direct influence. The sympathetic is of course, concerned in the altering of a caliber of the ovarian vessels (vaso-dilators), but this is no doubt a reflex, the impulses, which are aroused by the condition of the ovary itself, affecting the terminations of the nerve fibers in it.

From the comparative results with amniotic fluid, cystic fluid, and normal saline, it is suggested that the digestive action of the liquor follicuU is no mere matter of chance, but is a definite factor depending in some way at present unknown upon the particular nature of the liquor. It is a matter of common knowledge that different enzymes are specific in their action, that is to say, each enzyme acts only on one class of material and acts on it in a determined maimer, producing certain specific substances as a result of that action, and that extra-cellular as well as intra-cellular enzymes act only when completely freed from the cell body. As to the enzymic action of the liquor folliculi, the general law which applies elsewhere, may be applied here. The rate at which digestion goes on varies with temperature, with the reaction, the concentration of the digesting substance, and the condition of the material digested. The forces involved may be considered accelerated or aided by the increased temperature in the region of the ovary during the congested stage, more probably accelerated as enzymic action is greater at a temperature sUghtly above body temperature. The chemical

THE ANATOMICAL RRCORD, VOL. 10, NO. 6

454 S. S. SCHOCHET

reaction of the liquor folliculi was found to be alkaline to litmus and phenolsulphopthalein. Practically all the digestive agents of the body act best in the presence of an alkaline reaction. In the experiments here, the liquor folliculi was used in its normal concentration and, as noted above, at a temperature 38*^C. or slightly above the normal body temperature.

THEORIES OF RUPTURE OF FOLLICLE

The purely mechanical theory of Hensen who ascribed the rupture of the Graafian follicle to be due solely to pressure atrophy, due to the increase of the liquor folliculi can no longer be held tenable, as Naegel has shown that an increased pressure causes a thickening of the tunica interna of the theca folliculi. The thickening of the theca folliculi about the enlarging follicle is very evident in all sections, and may be explained as due to two causes:

1. The mere fact that the liquor folliculi does increase in amount, and the follicle increases greatly in size must result in a packing off, or compression of the ovarian stroma about the follicle and thus in a gradual increase in the thickness of its theca.

2. It is well known that in certain other conditions in the body any gradually increasing pressure leads to a proliferative fibrosis, and thus the thickening of the elements of the theca interna (an increase in size and in number of the connective tissue corpuscles with a subsequent increase in the fibrous tissue of the theca interna) and, therefore, may be in part but an expression of one of our simple laws of physiology, namely, that all irritation causes proliferation.

According to the earlier views of de Graaf, von Baer, His, and Waldeyer, that the ovum may be extruded through rupture of the Graafian follicle resulting from the formation of a local area of necrosis or macula pellicuda due to a distinctive preformed non vascular area has been shown to be incorrect by the excellent monograph of C^lark dealing with the blood supply of the ovary and its changes during ovulation.

In the words of Dr. Clark: 'There is undoubtedly a deeper lying cause than the mere growth and pushing forward of the

PROCESS OF OVULATION AND CYST FORMATION 455

follicle until, by erosion of the tunica albuginea of the ovaryit empties itself/' In his excellent monograph, Dr. Clark summarizes his views of the final epoch in the evolution of the follicle to be due to the arrangement of the vessels and the phenomena of congestion of the internal genital organs. He states, A follicle may reach maturity through the influence of the normal circulation, but it requires the increase of arterial pressure due to the menstrual wave to induce rupture and extrusion of the ovum."

It is evident that we must go still farther in elucidating the problem of the rupture of the Graafian follicle and determine, if possible, the correct basis on which to build the argument relating to the causative factors for its rupture.

In interpreting the menstrual wave, it has been assumed by many to be due to the congestion of the internal genital organs. From this point of view then, it would seem that any congestion brought about would induce rupture of the follicle. As a matter of fact the stage of preparation, premenstrual congestion begins for some days before ovulation or actual bursting of the folUcle (stage of destruction) and capillary pressure gives us an inadequate explanation.

It is suggested that it may be the period at which time the liquor foUiculi reaches its maximum amount and the enzyme is liberated in the liquor.

SUMMARY

The rupture of the Graafian follicle is due in part to the digestion of the theca folliculi by a proteolytic ferment or enzyme in the liquor folUculi.

The author wishes to acknowledge his indebtedness to Prof. Irving Hardesty for his constant interest in the progress of this \york, and for many helpful criticisms. To Drs. C. W. Duval and H. N. Kingsford for material made available, and to Dr. F. P. Lord for assistance in the preparation.

456 8. S. SCHOCHET

BIBLIOGRAPHY

AiME, P. 1907 Recherches sur lea cellules interstitielles de I'ovaire chez quel ques mammiferes. These. Nancy. Allen, B. M. 1904 The embryonic development of the ovary and testis of

the Mammalia, Am. Jour. Anat., vol. 3, pp. 89-146. Ancel, p., and Bouin, P. 1909 Sur les homologies et la signification des

glandes k s^cr^tion interne de I'ovaire. Compt. Rend. Soc. Biol.,

November 13, T. 61, pp. 497-98. Arnold, L. 1912 Adult human ovaries with follicles containing several oocytes.

Anat. Rec, Phil., vol. 6, pp. 413-422. Benthin, W. 1911 ITber FoUikelatresie in Saugetierovarien. Arch. f. Gynae kol., Bd. 94, pp. 599-636. BiEDL, A. 1910 Innere Sekretion: ihre physiologischen Gnindlagen und ihre

Bedeutung fur die Pathologic. Urban and Schwarzenberg. Bouin, P. 1902 Les deux glandes ^ s6cr6tion interne de I'ovaire: le glande

interstitielle et le corps jaune. Rev. Med. de L'Est., Nancy, July 15. Bouin, P., and Ancel, P. 1909 Sur les homologies et la signification des glandes

k s<5cr6tion interne de I'ovaire. Compt. Rend. Soc. Biol., T. 65, vol.

61, pp. 464-466. Cent, C. 1912 II cerebello e la funzione ovarica. Riv. sper. di frem., Reggio Emilia 38, pp. 219-290. Champy, C., and Gley, E. 1911 Action des extraits d' ovaires sur la pression

art6rielle. Compt. rend. Soc. de biol. Paris, 71, pp. 409-413. Clark, J. G. 1901 The origin, development and degeneration of the blood

vessels of the human ovary. Johns Hopkins Hosp., vol. 9, pp. 593-676. CoHN, F. 1912 Die klinische Bedeutung der Follikelsprungstellen in Ovarium.

Deutsche, med. Wchnschr., Leipz. u. Berl., 38, pp. 780. CusHiNO, Harvey. 1912 The pituitary body and its disorders, Clinical states,

etc., J. B. Lippincott Company, Philadelphia. CoHNHEiM, O. 1912 Enzymes, T. John Weyler and Sons, New York. Fellner, O. 1909 Zur Histologic des Ovariums in des Schwangerschaft. Arch.

f . mikr. Anat., Bd. 73, pp. 288-305. Frank, R. F. 1911 The function of the ovary. Tr. Amer. Gynec. Soc, vol. 35,

pp. 269-302. Ganfini, C. 1907 Sul probabile significato fisiologico dell'atresia follicolare

nell'ovain di alcuni mammiferi. Arch. Ttal. di. Anat. e di embriol.,

T. 6, pp. 346-357. Harz, W. 1883 Beitrage zur Histologic des Ovariums der Saugethiere. Arch.

f. mikr. Anat., Bd. 22, pp. 374-407. His, W. 1865 Beobachtungen uber den Bau des Saugetirereierstocks. Arch.

f. mikr. Anat., Bd. 1, pp. 151-202. Hoelzi, H. 1893 Uber die Metamorphosen der Graafischen Follikel. Arch. f.

pathol. Anat., Bd. 134, pp. 438-473. Iscovesco, H. 1912 The lipoids of the ovary. Univ. m. Rec. Lond. pp.

395-402. Keller, R. 1912 Blutgerinnungszeit und Ovarialfunktion. Arch. f. Gynec.

Berl. 97, pp. 540-582.

PROCESS OF OVULATION AND CYST FORMATION 457

Kingsbury, B. F. 1913 The morphogenesis of the mammalian ovary (Felis

domestica), Am. Jour. Anat., vol. 15, pp. 345-379. Kingsbury, B. F. 1914 The interstitial cells of the mammalian ovary: Felis

domestica. Am. Jour. Anat., vol. 16, pp. 59-91. Lane-Claypon, Janet E. 1905 On the postnatal formation of primordial ova.

Proc. Physiol. Soc. Journ. Physiol., vol. 32, pp. xli-xlii, March 18. Lob, W., and Gutman, S. 1912 Zur Kenntnis der Enzyme der Ovarien. Bio chem. Ztschr. Berl., xli, 445-460. LoisEL, G. 1904-1905 Les ph^nomen^s de s6cr6tion dans les glandes g<^nitales.

Revue. g<!^n6rale et faits nouveaux. Journ. de I'Anat. et de la Physiol.,

T. 40, pp. 536, 562, and T. 41, pp. 58-93. MacLeod, J. 1880 Contributions k V6tude de la structure de L'ovaire des

mammifdres. Arch, de Biol., T. 1. PflCger , \V . 1863 Uber di e Eierstocke der Sauger und des Menschen . Leipzig . Proca, G. 1912 Sur une action particuliere de I'ovalbumine. Compt. rend.

Soc. biol.. Par. T. 72, p. 843. Regaud, C., and Lacassagne, a. 1913 Sur les processus de la degenerescence

des foUicules dans les ovaires rontgenis^s de la lapine. Compt. rend.

Soc. de biol. Paris, T. 74, pp. 869-871. ScHUUN, K. 1881 Zur Morphologic des Ovariums. Arch. f. mikr. Anat.,

Bd. 19, pp. 442-512. Seitz, L. 1906 Die FoUikelatresie wahrend der Schwangerschaft, insbeson dere die Hypertrophic und Hyperplasia der Thecainterna-Zellen und

ihre Beziehungen zur Corpus luteumbildung. Arch. f. Gynaek., Bd.

77, pp. 203-356. Van der Stricht, O. 1901 L'atr6sia ovulaire et L'atresia foUiculaire du

follicule de de Graaf, dans Tovaire du chauve-souris. Verh. d. Anat.

Gesell. zu Bonn., pp. 108. Vincent, Swale 1912 Internal secretion and the Ductless glands. Lond. Waldeyer, W. 1870 Eierstock und Ei. Leipzig. Young, J. S. 1911 The life history of the ovary. Jour. Obsrt. and Gynec. Brit.

Emp., Lond., vol. 20, pp. 285-290. A PORCUPINE SKULL WITH A PAIR OF SUPERNUM ERARY WELL DEVELOPED INCISORS

IN THE UPPER JAW

MARCUS WARD LYON, JR. George Washington University, Washingtonj D, C.

ONE FIGURE

The specimen here recorded and illustrated is unique so far as my observations on rodents extend and I have been unable to find mention of a similar case in the Uterature on mammals. The anomaly occurs in a young (deciduous premolars and first molars only teeth in place aside from the incisors) male Old World porcupine, Acanthion longicaudum, nimiber 153973, United States National Museum, collected along the Kendawangan River, southwestern Borneo, July 10, 1908, by Dr. W. L. Abbott. This specimen was briefly referred to by me in a faunal paper in 1911.^ The condition evidently being rare seems worthy of a special note.

The order Rodentia (Gidley^ has recently shown that the Lagomorphs constitute an order apart from the rodents) is principally characterized among other things by the possession of a single pair of heavy cutting teeth in both upper and lower jaws. It is only in those rodents possessing more than three cheek teeth, in general the so-called Sciuromorphs and Hystricomorphs, that deciduous teeth occur. The one, or sometimes two, premolars usually found in these groups are preceded by milk molars. The first cheek tooth in the specimen here illustrated is a milk molar and was destined to be succeeded by a permanent premolar.

The incisors of rodents, usually considered to be the second

1 Proc. U. S. Nat. Mus., vol. 40, p. 114, April 25, 1911. « Science, n. s. vol. 36, pp. 285-286, August 30, 1912.

459

460 MARCUS WARD LYON, JR.

pair of incisors of the generalized mammalian dentition,' are to be regarded as members of the permanent dentition rather than permanently retained milk incisors. In mammals when only one set of teeth is functional, it is probably the permanent set as is exemplified in the early shedding of the milk teeth in seals and in bats. If the small additional incisor here illustrated is correctly interpreted, it is further evidence that the cutting teeth of rodents are members of the permanent set of teeth. A consideration of the use of the rodent incisor makes it at once apparent that a rodent must possess permanently functioning incisors as it could ill afford to dispense with such necessary teeth during the period of casting off the milk incisors and the incoming of the permanent ones.

In the porcupine skull under consideration a pair of well developed supernumerary upper incisors is present. The incisors .in the; lower jaws are normal in every respect. These extra teeth are fairly large. Their shape, size, and relation to the upper incisors are illustrated, natural size, right and left sides, in figure 1 and need no detailed description. The entire incisor socket has been exposed on the right side of the skull and it is there seen that the supernumerary tooth extended posteriorly more than half the length of the normal tooth. During the process of cleaning or handling the skull, a few millimeters of the small right incisor were broken off at the free end. The root of the tooth was situated further back than is shown in the illustration ; that is the small incisor on the right side should be placed further back in the socket, and the apparent free edge of the tooth on the right side is not what was its cutting edge. The free distal edge of the small left incisor is worn by contact with the opposing under incisor. The free edge of small now broken off right incisor was without doubt similarly worn. Although showing evidences of wear these teeth could have been of no service to the animal. The left incisor is worn in the reverse direction as compared with the normal rodent incisor, that is, the anterior surface of the tooth is worn away more than the

« Weber, Die Saugetiere, p. 480, 1904.

SUPERNUMERARY INCISORS IN UPPER JAW 461

posterior, well shown in the lower view, figure 1. This condition of wear was evidently brought about by the protection afforded the posterior surface as it lay in contact with the hard anterior surface of the normal tooth. The supernumerary inci


Fig. 1 Natural size view of skull of Old World porcupine, Acanthion longicaudum, number 153973, United States National Museum, from southwestern Borneo, collected by Dr. W. L. Abbott. It shows a small pair of extra incisors lying in front of the normal incisors.

462 MARCUS WARD LYON, JR.

sors seem to have an ill-defined layer of enamel covering them all aromid. In the center of the free extremities of these incisors is seen the remains of the dental canal just as it is also seen in the free extremities of the normal incisors. The posterior end of the small extracted tooth, the right one, shows typical incisor structure, a large pulp canal with walls thin at the base and progressively increasing in thickness anteriorly. Both small incisors show indistinct longitudinal striations and the left one has a well marked groove on the anterior face of the lower half of the exposed portion. There is no indication of a bony septum in the alveolus separating the small from the large incisor. There is nothing about the supernumerary teeth to indicate that they would not have persisted until or throughout adult life.

Rudiments of incisors have been described in the house mouse by Woodward^ and in the Sciuridae by Adloff,^ both authors giving reviews of the subject and many bibhographic references. The position of these vestigial teeth, occurring only in early embryonic life, as illustrated by these authors is essentially the same as with the porcupine, that is anterior to the permanent tooth.

To accoimt for these anomalous teeth in the porcupine three explanations may be considered. First, that the occurrence is simply pathologic without any other significance. Second, that the small incisor corresponds to one of the incisors lost from the normal manomalian dentition in the evolution of the rodents. In that case it ought to he in a separate alveolus. Third, that it is a hypertrophied and persistent milk incisor, the view that I consider the most rational. The occurrence of persistent or least partially persistent milk incisors is not infrequent in man, the mammal in which abnormal conditions most frequently come to notice.

In conclusion, I wish to express my thanks to the authorities of the United States National Museum, particularly to Mr. Gerrit S. Miller, Jr., Curator, Division of Mammals, for the privilege of studying and recording this specimen.

Anat. Anz., vol. 9, pp. 619-631, 1894. •Zool. Anz., vol. 20, pp. 324-329, 1897; and Jena Zeitschr. Naturwiss., vol. 32, pp. 347-410, 1898.

THE CARRYING ANGLE

MORRIS RUSH

Ernst Philip Boas

From the Mt. Sinai Hospital^ Neiv York City

ONE FIGURE

The late Dr. Morris Kush had begun a study of the carrying angle, which was cut short by his untimely death. It had appeared to him that the obliquity of the forearm in extension was greater in women than in men, in other words that the carrying angle was smaller in women; and he had planned to ascertain its size in adults and in children of both sexes, and to discover if possible the causes of the differences which he believed he would find. Among his papers were found some two hundred measurements of adult males and females. He did not have the opportimity to work these out thoroughly, but he was able to determine that the right carrying angle is smaller than the left one and that the angle in females is smaller than in males. From this point I have taken up the work along the lines which he had hoped to develop. I did not obtain measurements of children because I soon discovered that to endeavor to solve all of the questions suggested by an exhaustive study of the problem would be too formidable an undertaking at the present time.

The instrument with which Dr. Kush measured the carrying angle was of his own invention. It is depicted in the accompanying illustration. The instrument is placed on the anterior aspect of the extended and supinated arm with the bar (A), which is fixed to the protractor (C) at a right angle to its base, lying on the upper arm. The two side pieces (B) are then appUed to the two epicondyles of the humerus. This brings the bar (A) into a line with the long axis of the humerus.

463

464


MORRIS KUSH


The lower bar (D) is then adjusted to the long axis of the forearm and the resulting angle is read off on the protractor. This angle, which is the one which appears in the tables, is the supplement of the carrying angle. The instrument admits of error because the upper bar is not apphed to a fixed point on the upper arm. I have remeasured a number of the individuals whom



Dr. Kush had measured, and have found considerable variation between his figures and mine. But Dr. Kush had acquired a constant technique for himself, and so the relative values of the different measurements are significant, even if their absolute values may be questioned.

The bisacromial and intercristal diameters were measured with a pair of calipers.

In table 1 are recorded 105 measurements of adult males, and in table 2, 100 measurements of adult females. Theindi THE CARRYING ANGLE 465

viduals measured were patients at the Mount Sinai Hospital, New York City. The greater number of them are Russian Jews from the lower east side of New York City.

For purposes of ready comparison, all of the averages with their mean square errors are tabulated in table 3. The true carrying angles, i.e., the supplements of those in tables 1 and 2 are given.

A study of these figures justifies the conclusion that the carrying angle is smaller in females than in males. In 1895 Potter (1) made some measurements of the carrying angle, using "a narrow three foot rule, hinged at its center on the flat, having a disc, divided into degrees, attached firmly to one arm of the rule. The average size of the angle in 90 females was 167.35 degrees and in 95 males 173.17 degrees. He did not differentiate between the right and left arms. If in the present series we average the values of the right and left carrying angles we get 165 degrees for females and 167.7 degrees for males. The averages of the left carrying angles alone, in females is 166.6 degrees and in males 169.27 degrees.

A number of other observers report measurements of the carrying angle. Bertaux' (2) figures are 168 degrees for females and 169 degrees for males. Brauneand Kyrklund's (3) are 166.6 degrees for a series of eleven males. The average size of the carrying angle, without regard to sex, found by Malgaigne (4) was 165 degrees. F6re and Papin (5) record observations on a series of 194 cases. When we calculate the averages, we find the average for the right side to be 160 degrees, for the left side 161.2 degrees. Mall (6) reported measurements of the angle between the axis of flexion of the elbow joint and the long axes of the humerus and of the ulna respectively. The sum of these angles is the carrying angle, which may therefore be calculated from his tables. His averages for negro laborers are:

Males right arm 170.0 degrees. 23 cases

Males left arm 169.0 degrees. 26 cases

Females right arm 166.5 degrees. 10 cases

Females left arm 167.9 degrees. 11 cases

460


MORRIS KUSH


TABLE 1 Adult Juaie — 105 cases


AGE


RIGHT

KLPPLEMEXTARY

CARRYING

ANGLE


LEFT

SUPPLEMENTARY

CARRYING

ANGLE


BIBACROMIAL DIAMETER


1NTERCRI»»TAL DIAMETER



d- gre -« 


degrees


inches


inckei


47


19


15


38


29


m


17


7


39


30


38


14


8"


36


27


29


20


12


34


28


50


12


3


39


27


21


18


16


26


25


30


12


10


37


27


25


12


9


34


26


50


10


13


39


31


28


18


14


39


28


13


4


4


28


25


33


18


15


36


29


50


10


10


37


29


26


13


13


37


27


47


14


4


38


27


46


9


6


36


27


48


16


13


36


25


48


17


9


39


30


35


23


16


39


30


28


15


8


35


27


29


15


13


37


28


22


16


11


39


27


40


13


14


35


28


39


6


4


38


25


23


15


14


38


27


64


10


6


34


27


32


8


7


37


29


34


18


20


35


27


26


11


9


38


28


64


14


12


39


30


78


15


12


38


29


19


25


20


34


25


68


15


5


37


32


50


12


16


37


31


60


12


8


39


30


23


9


6


37


26


15


20


14


32


24


57


11


7


37


30


53


7


12


35


29


49


18


16


35


29

THE CARRYING ANGLE


467


TABLE 1— Continued


AOE


RIGHT

HUPPLBUENTART

CARRYING

ANGLE


LBIT

8UPPI BMBNTART

CARRYING

ANGLE


BISACROMIAL DIAMEtER


INTERCRISTAL DIAMBTBR



(Ugreea


degrees


inches


incl.es


37


15


6


38


30


36


13


13


37


27


35


11


7


38


28


33


12


10


38


31


m


5


5


40


30


25


16


14


38


28


20


11 38


28


18


18


16


35


27


13


12


7


39


28


19


17


14


37


28


31


26


12


36


27


58


15


12


38


29


45


10


13


35


26


18


21


16


32


27


30


11


12


36


28


58


7


5


35


25


20


14


11


35


27


39


16


13


39


29


43


14


14


37


29


29


12


10


36


27


52


13


10


37


27


45


15


13


35


27


29


6


3


36


28


42


16


13


39


29


30


16


14


35


27


30


16


14


39


28


22


16


16


38


27


51


13


12


35


25


58


10


8


40


28


28


18


12


40


29


45


12


14


38


30


54


19


19


38


31


25


11


14


32


28


54


11


4


35


29


16


10


3


38


29


62


13


10


36


30


46


13


10


40


31


41


13


10


35


28


18


7


5


40


29


28


10


9


40


28


36


17


12


36


28

468


MORRIS KUSH


TABLE l—Continued


AQB


RIGHT

SXTPPLBMENTART

CABRYINO

A>raLB


LEFT

8UPPLBMENTARY

CARRYING

ANGLE


BI8ACROMIAL DIAMETER


INTERCRIBTAL DIAMETER



degrees


degrees


inchcji


incheg


48


10


5


37


28


39


8


7


40


29


40


12


8


35


29


30


9


4


29


29


31


10


13


40


30


41


12


8


39


28


45


21


17


38


32


56


8


5


40


29


28


12


12


37


27


32


15


12


38


28


51


20


14


35


26


43


15


12


35


29


67


13


13


34


27


52


10


7


35


28


46


16


15


35


30


26


9


13


38


29


58


18


12


36


28


30


13


12


38


29


21


17


14


43


30


22


17


18


33


26


39


17


8


33


27


44


16


13


34


30


24


12


9


37


27


22


16


13


40


28


Average


13.66


10.83


36.5


28.07


Mean square deviation. . .


16.68


15.18


7.46


3.24


E


±4.08


±3.89


±2.73


±1.8


In whites the angle is about 169 degrees. The cases reported are too few to allow a comparison of males and females and of right and left. Nagel (7), using an instrument similar to Dr. Rush's, made an exhaustive study of the angle of the elbow. From a study of thirty males and thirty females he found that the carrying angle was 170 degrees in the former and 168 degrees in the latter. There was no difference between the right and left sides. Thus all of the authors agree that the carrying angle is smaller in females than in males, although

THE CARRYING ANGLE


469


TABLE 2 Adult female — 100 cases


A OB


RIOHT

SUPPLEMENTARY

CARRYING

ANQLB


LMT

8UPPLEMBNTARY

CARRYING

ANGLB


BISACROUIAL DIAMETER


INTERCRIBTAL DIAMETER degree*


degree*


inch€9


inchee



20


15


9


35


30



34


12


12


30


32



31


15


19


35


30



22


18


11


28


29



25


18


11


37


28



50


13


15


33


28



17


14


12


34


29



50


13


8


32


29



22


13


16


35


30



58


20


17


30


27



38


16


9


35


30



25


11


10


32


27



22


20


14


29


25



54


25


20


33


28



25


20


16


33


24



21


13


10


32


25



26


15


12


32


27



21


17


15


35


29



26


9


8


30


26



38


15


13


33


30



32


15


13


29


26



53


15


15


32


29



27


20


18


32


27



25


11


14


35


27



49


19


15


32


27



43


15


11


33


30



40


15


9


33


29



38


20


19


32


30



28


15


8


34


29



22


17


15


33


27



20


14


10


35


29



15


20


16


32


27



41


18


15


32


28



59


18


15


33


29



38


14


11


35


30



40


16


13


35


31



35


8


5


33


25



32


20


16


34


29



23


18


15


31


27



36


15


13


33


27


THE ANATOMICAL RECORD, VOL. 10, NO. 6

470


MORRIS RUSH


TABLE 2— Continued


AGE


RIGHT

SUPPLEMENTARY

CARRYING

ANGLE


LEFT

SUPPLEMENTARY

CARRYING

ANGLE


BISACROMIAL DIAMETER


INTERCRI8TAL DIAMETER



degreea


degrees


inches


ineke<(


27


18


17


35


28


48


14


11


32


28


53


14


12


36


29


26


18


17


33


28


54


15


18


37


32


37


22


16


31


28


25


17


12


32


28


42


19


12


34


27


48


15


14


30


28


36


22


20


32


28


20


20


18


33


28


30


20


16


31


29


19


19


15


33


25


28


18


15


29


26


48


15


10


30


30


24


17


8


34


28


27


8


6


34


31


31


12


4


29


28


54


19


17


30


30


48


21


14


30


27


29


14


13


34


29


30


12


7


30


27


38


14


13


31


29


44


14


16


30


27


46


14


10


32


25


47


26


13


32


28


50


18


19


33


30


55


17


25


32


29


59


19


13


31


29


24


23


20


32


26


35


20


12


30


28


23


15


13


33


29


42


12


12


30


27


35


14


11


34


27


52


16


13


33


30


34


13


8


35


30


29


20


15


31


28


40


16


10


32


27


36


18


14


35


30



19


13


32


26


22


10


7


31


26

THE CARRYING ANGLE


471


TABLE 2— Ck)ntinued


AOE


RIGHT

SUPPLEMENTARY

CARRYING

ANGLE


i-EFT

SCPPLKMENTARY

CARRUNO

ANGLE


DI8ACROMIAL DIAMETER


INTERCRLSTAL DIAMETER



degre a


degreeg


inclet


irule»


25


24


23


33


29


26


20


18


33


26


28


15


16


34


30


29


18


16


32


26


28


18


15


33


27


39


20


18


33


31


24


13


11


33


30


41


15


8


32


29


28


16


11


30


25


32


19


18


32


29


42


16


11


30


26


14


19


18


30


26


42


12


12


34


30


25


16


14


35


31


55


16


15


36


33


48


16


12


30


26


32


20


11


32


27


17


20


14


34


27


33


12


9


31


28


Average


16.47


13.42


32.46


28.15


Mean square deviation. . .


12.12


14.11


3.63


3.13


E


±3.48


=*=3.76


±1.9


±1.75


TABLE 3


8 EX


RIGHT CARRYING ANGLE


LEFT CARRYING ANGLE


BISACROMIAL DIAMETER


INTERCRIBTAL DIAMEIER


Male

Female


degrees

166.3±0.39 163.5±0.35


degre « 

169.2±0.38 166.6±0.38


\nc\e9 36.5±0.27 32.5±0.19


inclet 28.1±0.17 28.1±0.17


there is disagreement as to the degree of difference, and as to the absolute size of the carrying angle.

It was at first thought that this difference might be due to the relatively greater intercristal diameter in women which might cause a compensatory increase of the obliquity of the forearm in extension, in other words a decrease in the carrying angle. The coefficient of correlation between the right carrying angle

472 MORRIS RUSH

and the intercristal diameter was therefore calculated. It is —0.01 in males and —0.131 in females, too small to have any significance. The coefficient of correlation between the right carrying angle and the bisacromial diameter is also very small — 0.054 in males and —0.091 in females. The figures show that there is no correlation between the carrying angle and the difference between the bisacromial and intercristal diameters. The smaller carrying angle in women, therefore, is not due to their narrower shoulders and relatively broader pelvis.

No anatomical feature appears to account for the difference in size of the carrying angle in the two sexes. The cause of this difference, whatever it may be, acts equally on the right and the left arms, for the coefficient of correlation between the right and left angles is high, +0.639 in males, and +0.67 in females. A glance at the averages in table 3 suggests that the dissimilarity between the two sexes may have a physiological, rather than an anatomical basis. In males as well as in females the right carrying angle is distinctly smaller than the left one. This is probably due to physiological factors.

The likelihood of this interpretation is enhanced by measurements of the carrying angle in left handed individuals. In table 4 are recorded the carrying angles of twelve left handed persons.

In this series the right carrying angle is on the average only 0.33 degree smaller than the left one, while in right handed individuals it is about 3 degrees smaller. Left handed persons are usually, to a certain degree, ambidextrous, while right handed persons favor the right hand more exclusively; therefore, if the size of the angle depends on physiological moments, the difference between the two sides will not be so marked in left handed individuals. Moreover, among these few observations there are two which are widely divergent from the others, 154 degrees, right and 167 degrees left, and 163 degrees right and 173 degrees left. These may be incorrect, especially since Dr. Kush had placed a question mark next to the first of these observations. If that were so, the average left angle would be smaller than the average right one.

THE CARRYING ANGLE


473


TABLE 4 Left handed individuals


SEX


RIGHT CARRY I NO ANOLB


LETT CARRTINQ ANGLE



degrees


dtgreet 167


164


Female ■



169 154


166 167 165


164 170


167 163


173 162


160


Male :



167 170


166



167 168


166 171


167 168


171


Sum


1994


1998

Thus we find the carrying angle smallest in the arm which is most used. By analogy it is possible that the difference in the angle in the two sexes is dependent on physiological factors. It would be interesting to compare the carrying angles of series of individuals having widely different occupations, e.g., railway porters and clerks.

On the basis of different observations, Nagel arrived at the same conclusion. From a study of the relationship of the carrying angle to the angle between the axis of flexion of the elbow and of the long axes of the humerus and ulna respectively, to the olecranon angle, to the angle of torsion of the humerus, and to various other anatomical landmarks, he became convinced that the size of the carrying angle was determined by no anatomical factor. He found that in many individual cases intensive use of the arm, as shown by a strongly developed musculature, caused greater obliquity of the forearm. For instance, in athletes the carrying angle was 6 degrees smaller than in selected weak armed individuals. Vice versa children showed only a slight obliquity of the forearm. Braune and Kyrklund made the same observation in children. Although NageFs averages showed no difference in size between the right and left angles.

474 MORRIS RUSH

persons who were markedly left handed were found to have smaller left than right carrying angles. In one case the difference was 8 degrees. From all of these facts, Nagel concluded that the size of the carrying angle was determined by physiological factors.

Although irrelevant to the body of this paper, it may be mentioned that the coefficient of correlation between the intercristal and bisacromial diameters is -1-0.389 in males and -1-0.45 in females. There is therefore a definite relation between the two.

SLMMARY

The carrying angle in females is about 3 degrees smaller than in males.

The right carrying angle in males as well as in females is about 3 degrees smaller than the left one.

In left handed individuals the right and left carrying angles are about equal. (This conclusion should be verified by a larger number of cases.)

There is no relation between the carrying angle and the bisacromial or intercristal diameters.

The difference in the size of the carrying angle in the two sexes, and in the right and left sides is not dependent on anatomical, but on physiological factors.

There is a close correlation between the right and left carrying angles, and also between the intercristal and bisacromial diameters.

LITERATURE

(1) Potter, H. P. 1895 The obliquity of the arm of the female in extension.

The relation of the forearm with the upper arm in flexion. Jour. Anat. and Physiol., 29.

(2) Bertaux 1891 L'humerus et le Femur, etc. Th^se pour le Doct. en Med.

Lille. Quoted from Nagel, I.e.

(3) Braune, W. and Kyrklund, K. 1879 Archiv f. Anatomie u. Physiologic,

Anat. Abt. p. 321.

(4) Malgaigne, J. F. Traits d'anatomie Chirurgicale et de Chirurgie experi mental, 2d ed. 2, 634.

(5) FfeRE, C. et Papin, E. 1902 Notes sur quelques variet^s de la direction

du membre superieux. Jour, de I'Anat. et de la Physiol., 38, 105.

(6) Mall, F. P. 1905 On the angle of the elbow. Am. Jour. Anat., 4, 391.

(7) Nagel, K. 1907 Untersuchungen ueber den Armwinkel des Menschen.

Zeitschrift f. Morphologic u. Anthropologic, 10, 317.

THE FORMATION OF THE CRANIAL SUBARACHNOID

SPACES

LEWIS H. WEED Prom the Anatomical Laboratory of Johns Hopkins University

One of the interesting changes in viewpoint in anatomy in the last half century has been the development of a truer conception of the fluid-containing cavities of the body. From the belief that these spaces were largely alike (i.e., serous), realization has come that there exist at least two types of these peculiar fluid-sacs, excluding from the consideration synovial cavities and bmrsae. For the first of the two great classes, the term 'serous cavities' has been retained and is now more or less generally limited to such spaces as are derived from the coelom. None of these are to be regarded as being intimate portions of the lymphatic system; rather is their connection with this closed vascular network probably more of a f imctional than anatomical type.

Distinguished from this first group is the second type, comprising two fluid-systems, possessing characteristic organs of elaboration and specialized mechanisms for the absorption of their individual fluids. These cavities, the cerebro-spinal spaces and the aqueous chamber of the eye, are to be considered on these bases of a specialized mode of fluid-production and drainage, as apart from the serous cavities as they are now known. The two systems, that of the central nervous system with its cerebro-spinal fluid, and that of the eye with its aqueous humor, possess an extraordinary number of analogous processes, as pointed out by Henderson ('12) and amplified by Wegefarth and Weed ('14).

Following an investigation of the physiological anatomy of the cerebro-spinal (particularly the meningeal) spaces in the ordinary laboratory mammals (Weed, '14), attention was directed to the problems of these perimeduUary cavities in the embryo.

475


THE ANATOMICAL RSCOBD, VOl. 10, NO. 7 MAT. 1916

476 LEWIS H. WEED

The results of a portion of this work were reported before the Christmas meeting of the American Association of Anatomists (Weed, '16). At that time a method of replacing the existent embryonic cerebro-spinal fluid with a foreign substance was presented, permitting the substitution to be made without increasing the normal intramedullary pressure. The procedure was employed on living pig embryos, which were subsequently kept alive for varying lengths of time in a 38"^ incubator. Using this method and introducing an isotonic solution of potassium ferrocyanide and iron-ammonimn citrate (later precipitated as Prussian blue), evidence was presented which indicated that in pig embryos the first extraventricular spread of the fluid occxured at a stage of about 14 mm. and that a total filling of the perimedullary space took place when the embryos attained a length of 26 mm.

The passage of fluid from the cerebral ventricles into the perimedullary spaces occurred only from two localized areas of ependymal differentiation in the two halves of the roof of the fourth ventricle. The superior of these areas was found to fimction actively during only a short period of growth; it was finally occluded by the changing relations of the developing rhombic structures. The more inferior area remains as a functional membrane during early fetal life and possibly throughout the adult existence.

The question naturally arose as to the character of the tissues into which the replaced foreign solution was carried by the production of more of the normal cerebro-spinal fluid. For the course of this foreign solution, under the experimental conditions, must be assumed to indicate exactly the course and perimedullary distribution of the true cerebro-spinal fluid. Not only is the question answered by the results of these replacements, but also the histological changes are such as to give insight into the processes concerned in the formation of the subarachnoid spaces.

Surrounding the central nervous system in the stages before the outpouring of cerebro-spinal fluid from the cerebral ventricles, is the undifferentiated perimedullary mesenchyme. This

CRANIAL SUBARACHNOID SPACES 477

tissue is of a loose character, forming a syncytial network of rather small mesh, but fragile. The nuclei of these cells are oval; the cytoplasm is largely devoted to the long processes which connect with adjacent cells. Adhering to these cytoplasmic strands are tiny albuminous coagula, of such amoimt as to be hardly noticeable; also in the meshes of the mesenchyme very small quantities of this protein-material may be identified. These albuminous coagula represent undoubtedly the albumen of the tissue-fluids in these undifferentiated stages.

This rather fine-meshed mesenchyme is the tissue into which the replaced foreign solution and the embryonic cerebro-spinal fluid passes when the elaboration of the fluid exceeds the intraventricular capacity. Shortly after the initial transit of the fluid into the extraventricular spaces (at 14 mm. in the pig), the process of differentiation of the tissue to fonn the final subarachnoid spaces occurs. Already the cranial blastemal condensation has become evident in the basilar regions, delimiting the loose meshes of the perimeduUary mesenchyme. This undifferentiated mesenchyme, then, with the outpouring of the ventricular fluid, undergoes a metamorphosis into a far looser tissue; the process is apparently one of disruption of certain of the mesenchymal strands to form the heavier and stronger trabeculae of the subarachnoid spaces.

The first evidences of this enlargement of the mesh in the periaxial mesenchyme is met with in the region around the medulla oblongata. It becomes first noticeable in stages of from 15 to 18 mm. thus following shortly the extraventricular spread of the cerebro-spinal fluid. The first change does not concern a real disruption of the syncytial strands but resembles more the spreading apart of the cell-bodies by the introduction of more fluid into the so-called tissue-spaces. Thus in a given area of this mesenchyme under consideration, the number of nuclei wiU diminish as the process of differentiation proceeds.

After the length of 18 mm. is passed, at which stage a great augmentation in the extraventricular spread of the cerebrospinal fluid occurs, the phenomenon of the disruption of the mesenchymal strands in the peribulbar tissues may be made out.

478 LEWIS H. WEED

Many of these strands may be observed broken off, sacrificed to a few larger persisting trabeculae. The cells which give rise to these disrupted strands appear to recede until one of the larger surviving elements is reached, when they adhere and apparently aid in the future production of a permanent arachnoidal trabecula. But associated with this breaking down of the mesenchymal mesh and this formation of a smaller number of persisting strands, is another phenomenon of great importance. The larger meshes formed by the process appear filled with a fluid much richer in protein than were the original much smaller interstices. This is shown by the great quantity of the albuminous coagulmn foimd, on histological examination, in all of the enlarged spaces in the tissue. The occurrence of this large amount of albuminous coagulmn is apparently related directly to the distribution throughout this tissue of the embryonic cerebro-spinal fluid, for this embryonic fluid is very rich in proteinmaterial as can be seen by the partial filling of the embryonic cerebral ventricles with the clotted albmnen. In this respect, the embryonic fluid differs markedly from the adult, .where the protein-content is surprisingly low.

This general plan of the formation of the subarachnoid channels attains its maximinn in the transformation of the primitive mesenchymal meshes into the larger cistemae of the adult cerebro-spinal fluid. The process is best illustrated in the posterior cerebello-bulbar angle where the cistema cerebello-medullaris is formed. The initial process of dilatation of the mesenchymal interstices is observed in embryo pigs of 18 to 20 mm. ; at a stage of 23 mm., the mesenchymal strands are already broken down in part and the exposed surfaces of these are covered by an extensive albuminous coagulmn. Likewise, at this length, the larger spaces are replete with similar coagula, indicating the presence of a protein-rich fluid. During the next 10 millimeters' growth, extensive chai\ges in the future cisternae occur; the whole region is now an almost unbroken space, filled with a dense coagulmn and interrupted by a few remaining mesenchymal strands. The outer portion of the cisterna is formed by a

CRANIAL SUBARACHNOID SPACES 479

continuous membrane, which, after somewhat further differentiation, will become the outer continuous layer of the arachnoidea.

In addition to this formation of the subarachnoid spaces in the adult through the enlargement of the embryonic mesenchymal spaces, the perimedullary mesenchyme imdergoes in these same locaUties condensations which result ultimately in the formation of the arachnoid membrane and the trabeculae dividing up the cavum subarachnoideale. Mention has already been made of the adhesion of the cell-bodies of the disrupted mesenchymal elements to the persisting strands — ^the initial step apparently in the ultimate differentiation of the mesotheUal cells which line these spaces. Gradually with the increasing growth of the embryo these cells seemingly become arranged in definite columns covering the persisting arachnoidal trabeculae. At the same time a differentiation of these primitive mesenchymal elements occurs, the cells ultimately being transformed into the typical cuboidal mesotheUum of the subarachnoid spaces. This differentiation begins first in the basilar portions of the cranium and spreads upward, in a way similar to the course of development of the cranium and of the enlargement of the pericerebral spaces.

While such a general process as outlined accoimts for the formation of the arachnoidal trabeculae and the subarachnoid spaces, it has but Uttle bearing on the development of the outer intact membrane of the arachnoidea. This portion of the arachnoidea (which might be termed the arachnoid membrane as distinguished from the arachnoid trabeculae) first appears as a distinct Une of mesenchymal condensation separating the mesenchyme into the primitive arachnoid and dura mater. This rather thin zone of cellular density in reality represents not only the outer surface of the arachnoidea but also the inner surface of the dura mater. At first these develop in close fusion but as the length of 50 mm. is attained in fetal pigs, a separation of the two membranes over the cerebral hemispheres is possible. At this stage also a mesothelial polygonal cell-pattern may be made out on the inner surface of the dura by silver nitrate reductions. With this cleavage of the two surfaces, the arachnoid

480 LEWIS H. WEED

membrane rapidly differentiates, forming an intact layer over the subarachnoid spaces. The cells covering the surface membrane seem to change gradually into the cuboidal type, similar to those covering the arachnoidal trabeculae.

The general process, then, of formation of subarachnoid spaces in the cranium of the pig, concerns a dilatation of the early mesenchymal spaces, a disruption and breaking down of certain of the syncytial strands, and a survival of certain selected strands to form the permanent subarachnoid channels. In addition to this rarefaction of the mesenchyme, there is a process of condensation in this tissue, giving rise ultimately to the arachnoid membrane and reinforcing the trabeculae. Associated with the breaking apart of the syncytiiun of the perimedullary mesenchyme, there are' invariably foimd large coagula of albuminous material, apparently an index of the circulation through the spaces of the embryonic cerebro-spinal fluid.

Such a view of the formation of the arachnoidea from the middle germ layer is in accord with the investigations of His C65), of KoUiker (79), of Farrar (^06) and others. His has described the formation in the spinal meninges of dorsal and ventral spaces, derived from the perispinal mesenchyme. Likewise, Farrar differentiates, in the spinal meninges of the chick, three laminae, the middle one of which alone becomes '* the loose irregular reticulum of the pia-arachnoid." The results of Sterzi's ('01 and '02) researches on the comparative anatomy of the meninges afford an interesting comparison to the plan of development of the subarachnoid spaces outlined here. Streeter's ('16) description of the mode of formation of the perilymphatic spaces indicates that in the internal ear a similar process holds.

This report presents one of the phases of a study of the formation of the cerebro-spinal spaces. The complete communication, with many illustrations, will be published, under the title of The Development of the Cerebro-Spinal Spaces in Pig and in Man as Volxune 5, Number 14, of the Contributions to Embryology, Carnegie Institution of Washington, PubUcation No. 225.

CRANIAL SUBARACHNOID SPACES 481

LITERATURE CITED

Farrar, C. B. 1906 The embryonic pia. Am. Jour. Insanity, 63, 295.

Henderson, T. 1912 Glaucoma. London.

His, W. 1865 Die Haute und Hohlen des Korpers; ein academisches Programm. Basel.

KdLLiKER, A. 1879 Entwickelungsgeschichte des Menschen und der hoheren Thiere. Leipzig.

Sterzi, G. 1901 Ricerche intomo all'anatomia comparata ed aH'ontogenesi delle meningi, e considerazioni sulla filogenesi. Atti del R. Instituto Veneto di scienze, lettere ed Arti. 60, Parte II, 1101. 1902 Recherches sur Panatomie compar^e et sur I'ontogendse des meninges. Arch. Ital. de Biol. 37, 257.

Strebter, G. L. 1916 Development of the Scala Vestibuli and Scala Tympani and their connections in the human embryo. Anat. Rec. 10, 250.

Weed, L. H. 1914 Studies on cerebro-spinal fluid. No. II. The theories of drainage of cerebro-spinal fluid with an analysis of the methods of investigation. Jour. Med. Research, 31 (N. S. 26), 21. No. III. The pathways of escape from the subarachnoid spaces with particular reference to the arachnoid villi. Ibid., 31 (N. S. 26), 51. No. IV. The dual source of cerebro-spinal fluid. Ibid. 31 (N. S. 26), 83.

1916 The establishment of the circulation of cerebro-spinal fluid. Anat. Rec, 10, 256.

Wbqefarth, p. and Weed, L. H. 1914 The analogous processes of the cerebral and ocular fluids. Jour. Med. Research, 31 (N. S. 26), 167.

BOOKS RECEIVED

The receipt of publicationB that may be sent to any of the five biological journab published by The Wiftar Institute will be acknowledged under this heading. Short reviews of books that are of special interest to a large number of biologists will be published in this journal from time to time.

DIRECTORY OF FOREIGN STUDENTS IN UNITED STATES AND CANADA, 1915-1916. . Published by the Committee on Friendly Relations among Foreign Students, Charles D. Hurrey, General Secretary, 124 East 28th Street, New York City. 69 pages.

STUDIES IN SURGICAL PATHOLOGICAL PHYSIOLOGY FROM THE LABORATORY OF SURGICAL RESEARCH, New York University, 1915.

Foreword. There is a growing tendency among groups of men studying different aspects of the same subject to collect and republish in one volume the results of their investigations. This practice is most helpful and it is one which will be greatly appreciated by other investigators.

IJie present volume of papers by associates in the Laboratory of Experimental Surgery of New York Univeirsity, bearing as it does on vital subjects in scientific medicine, is a happy example of such efficient codperative effort and one which may well be emulated by others. The contents should be familiar to every surgeon since its subject matter is representative not only of the surgery of today but of tomorrow.

William J. Mato.

Introduction. The contents of this volume have been arranged in aocordanoe with the newer viewpoint that surgery should deal first with the greater problems of function and diagnosis, therapeutic progress being obviously dependent upon these; and second, with the details of method and technie. This viewpoint is presented in some detail in the article on " Medicine and the World War." in which the authors discuss the growing importance of laboratories of surgical research, not alone for teaching purposes but for the furtherance of the new science of preventive surgery.

A group of papers detailing certain studies upon the alimentary and neural canals and vertebral column follows and it will be seen that biology and particularly its branches, physiological chemistry and heredity, are the underlying basis of this work.

The next group of papers upon the Abderhalden reaction chronicle a series of efforts to apply chemistry directly to surgical diagnosis; and, whatever the final deduction, these studies are in harmony with the biological era into which surgery has passed.

Following these are the details of a series of studies of the ureteral neuro-musculature, of the function of the ureterovesical valve and of an operative procedure logically based upon these functional findings. Perhaps better than any other, this series illustrates the present tendency in surgery to substitute functional for anatomical facts as a basis for new operative therapeusis; to correlate function and morphology and to place reason above technie. One might properly speak of this trend as pragmatic surgery.

The intricate subject of shook has been studied carefully and theprovisional results tend to disprove some of the most widely circulated hypotheses as to its nature and origin.

Finally, technical and miscellaneous papers are grouped together.

For the information of laymen who may examine these papers, it is stated that all the experimental work herein detailed was done upon animals after complete anesthesia and that morphine has been freely used to prevent post operative pain. Physicians and surgeons, of course, understand that no operative work can be done on animals satisfactorily except under full anesthesia.

John William Draper, Editor, 482

THE WANDERING CELLS IN THE LOOSE CONNECTIVE TISSUE OF THE BIRD AND THEIR ORIGIN

VERA DANCHAKOFF

Rockefeller Institute, New York City

The problem whether the different blood cells in the embryonic and adult organism are the products of development of various differentiated anlages, or whether there is one common anlage, the differentiation of which is controlled by definite environmental conditions was the object of many spirited investigations. This problem is closely connected with a further question of deserving cojisideration, namely, as to whether the adult organism preserves a stock of imdifferentiated mesenchynaal cells, endowed with a faculty of polyvalent differentiation.

The purpose of this note is to simmiarize morphologic and experimental data recently acquired, thought to aid in the elucidation of problems announced, concerning the wandering cells in the loose connective tissue of the hen.

1. MORPHOLOGICAL DATA

As may be gathered from the title of this conmiimication, the loose connective tissue contains permanently a certain amoimt of wandering cells. This is especially true for the embryonic connective tissue. Just as the circulating blood contains many kinds of differentiated blood cells, so the loose connective tissue of an adult hen includes, beside the fibroblasts, many kinds of amoeboid cells. Among these are included small lymphocytes, wandering histiotopic cells, granular wandering cells. Mast cells and occasionally plasma cells and true polymorphonuclear special leucocytes. All of these cells are foimd in the adult organism, but the majority of them appear in the embryo. They are nimierous in the connective tissue of the adult hen. The free amoeboid cells are sometimes arranged in the form of character 483

484 VERA DANCHAKOFF

istic cords and groups. Thus in some regions the free amoeboid cells form complete layers wjiich appear in sections as uninterrupted Unes. They are densely arranged in some of the thin connective septa between small fat accumulations. The morphological structure of different kinds of cells encountered in the connective tissue of the adult hen were described by Dr. Solucha in the year 1908.

In 1908 I pubUshed the result of my studies on the embryogenesis of these elements in the chick embryo. Later this problem was worked out for the reptiles and the account of this work became a part of the work about the general haematopoiesis of the reptiles. The cells mentioned above as well as the tissue of other haematopoietic organs, all develop in .the last instance from a conmion, undifferentiated mesenchymal anlage.

The mode of development of the mesenchymal cells into amoeboid cells is easily deducible. The long irregular processes so characteristic of the mesenchymal cells are withdrawn and the cell becomes free, potentially spherical, but very mobile. Sometimes innumerable short and irregular pseudopods cover the whole surface of the cell. Such a cell may remain as a wandering cell of the connective tissue, undoubtedly capable of fiu'ther differentiation. In other cases it may immediately undergo further differentiation and assiune the morphological structure of the large lymphocyte. This latter must be a very active cell, for its appearance is usually followed by a further differentiation, this depending on the environmental conditions.

The environmental conditions for the differentiation of an erythro-granulopoietic organ are more or less definite; a large amount of nutritive material, a region isolated from mechanical disturbance favor an intensive development of haemocytoblasts. The localization within or outside the vessels detennines the ftu-ther differentiation of the haematocytoblasts. A distension of the capillary net and a subsequent retarding of the blood current, offer again favorable conditions for multipUcation and differentiation of the haematocytoblasts.

It is much more difficult to define the specific environmental conditions, which direct the differentiation of the various cell

WANDERING CELLS IN TISSUE OF THE BIRD 485

groups found scattered in the connective tissue. However, some indications show the existence of intimate relations between definite environmental conditions and differentiation. A striking example is presented by the differentiation of the Mast cells along the vessels of the omentum; these follow the vessels in whole ranges and envelop them by their long processes, which, as well as the whole cytoplasm, are filled up by metachromatic granules. An analogous intensive development of the Mast cells is seen under the epitheUum of the intestine. Another instance of the interdependence of cell differentiation on the environmental conditions is offered by the development of the granulocytoblasts in the spleen of the reptiles and birds, this being chiefly confined to the pulpa in the normal embryos, while the small lymphocytes are principally developed in the external layers of the organ and in the splenic follicles.

The embryogenesis of the diffuse granulo-lymphatic tissue is easily estabUshed. The question of its reproduction in the adult organism is however obscure. Are the numerous granular and lymphatic cells in the connective tissue the direct offspring of analogous cells split off from the mesenchyme during embryonic life, securing their existence through their own reproduction? Or do these cells represent a renewal by cells brought by the blood current, or finally is the mode of their reproduction analogous to the one, observed in the embryonic development and may they not differentiate in the adult organism as well as in the embryo from the same source, namely, from young mesenchymal cells? A definite answer to these questions would greatly faciUtate the solution of the problems stated at the beginning of this paper.

The pecuUar structure of the granular cells of the connective tissue, which are not found in the circulating blood, speaks with great certitude for their local differentiation. The presence of mitosis in the non-granular amoeboid cells confirms the possibility of an independent reproduction of these cells in the connective tissue.

In studying the granulo-lymphatic cells in the connective tissue of the adult hen, I observed numerous small agglomerations of true lymphadenoid tissue. The center of such an ag 486 VERA DANCHAKOPP

glomeration is usually occupied by a mesenchymal syncytixun, as described by Mall and Sabin in the case of the lymph glands of mammals. This sjrncytium must be considered as a yoimg, undifferentiated tissue. Mall derived the connective reticulum from this tissue. Another line of differentiation is characteristic of the syncytium in the lymphatic nodes scattered in the loose connective tissue of the hen. This seems to consist in the development of free amoeboid cells from the mesenchymal syncjrtium cells. The syncytimn of the small agglomerations of the lymphadenoid tissue consists of ramified cells, connected with their neighbors by rather short processes. The whole aspect of this syncytium strongly reminds one of such regions of the embryo body where the mesenchyme multiplies intensely and therefore becomes denser. Both in the embryo and in the adult organism the mesenchymal syncytimn appears to be the source of the free amoeboid cells. Only the adult organism fails to show intense differentiation of free cells at the expense of the mesenchymal syncytiimi in the lymphnodes, as is so easily observed in the loose mesenchyme in the embryo. The multiplication of the granular and lymph cells in the connective tissue proceeds in the adult chiefly in a more homoplastic way, at the expense of intermediate stages of differentiation.

It is known that most of the birds and hens particularly do not possess special lymph glands and it is permissible to assimie a homology 'between the numerous small agglomerations of lymphatic tissue and the granular and lymphatic cells, diffusely scattered in the connective tissue of birds on the one hand and the precisely locaUzed lymph glands of the mammals on the other hand.

The conception of the haematopoietic organ, as established in recent years, requires not only the presence in it of proliferation and differentiation of various blood cells, but also the presence of ancestral mother cells. At the expense of such young cells in the lymphnodes the regeneration of various amoeboid cells takes place normally in the connective tissue of birds, and most intensely after loss of blood elements. Accordingly the loose connective tissue should be considered as a haematopoietic organ both in the adult organism and in the embryo.

WANDERING CELLS IN TISStlE OP THE BIRD 487

The data referred to above are obtained as a result of the study of fixed material by the use of technical methods giving minute morphological details.

The existence of numerous granular and lymph cells diffusely scattered in the connective tissue of the hen, as well as of numerous localized centers of mesenchymal syncytium, must be admitted as uncontested facts. However, the whole line of development and differentiation is merely the result of interpretation, which may give a true understanding of the development, supposing no links in the series of observation to be omitted. But the omitting of certain data may give rise to the wildest speculations. One may recall only the possibiUty of admitting the small lymphocytes found in accumulations in the bone-marrow of birds, as being the common stem-cells for granular leucocytes and erythrocytes (Venzlaflf). The vaUdity of the interpretation may be accepted if they are confirmed by experimental tests.

2. EXPERIMENTAL DATA

Observations have shown the presence in the loose connective tissue of the adult hen of swarms of amoeboid cells. Small agglomerations of mesenchymal syncytium are also irregularly scattered about in the connective tissue. Around these agglomerations the amoeboid cells appear in greater numbers and the peripheral layers of the mesenchymal syncytium contain numerous free cells in the form of large lymphocytes, which apparently differentiate at the expense of the cells of the syncyliium and partly reproduce themselves by mitosis.

The following questions, if possible, should be tested experimentally, a) Are the different amoeboid cells of the connective tissue the product of the development and differentiation of the mesenchymal nodes? If so, the mesenchymal nodes would appear to be stocks of undifferentiated cells, preserved in the adult organism from the time of the embryonic development and retaining their faculty of further differentiation. If by some means it were possible to destroy the differentiated cells without destroying the stem cells and if such destruction were followed by a new regeneration of differentiated cells, it would be easy to define the

488 VEieA DANCHAKOFF

source and the mode of regeneration in the adult organism. Such a destruction of lymphoid tissue was obtained by the use of the X-rays, which given in definite doses destroy chiefly the lymphoid elements, leaving the younger stem cells uninjured. An intense regeneration follows Jthe loss of the lymphoid elements and chiefly proceeds at the expense of the cellular syncytium in the lymphnodes, b) The second question to be tested experimentally is: What are the relations to other haemopoietic organs of the lymphoid elements found in the connective tissue? In other words, is the anlage for the amoeboid cells of the connective tissue and for the blood cells identical? This latter question may be answered affirmatively on the basis of experiment only in case, for example, we succeed in changing the actual line of differentiation of the loose mesenchyme into another one — if by means of certain interv^ention we may force the loose mesenchyme of the embryonic body which normally in certain regions gives rise only to lymphatic cells, to differentiate, let us say, into granular leucocytes. Such a metaplasis of the mesenchyme in the embryo body is obtainable, as will be shortly related.

Whether the adult organism preserves a stock of young mesenchymal cells is difficult to say at present. However, this assumption is ti-ue for the embryo and may be based not only on morphological observations but also on experiment.

In studying the factors of resistance to heteroplastic tissuegrafting, Dr. Murphy of the Rockefeller Institute made a verj^ interesting observation. The tumor grafts give intense growth on the allantois of a chick embryo. The spleen graft of an adult chick made simultaneously with a cancer graft inhibits the cancer growth. At the same time the spleen of the embryo becomes much larger.

As already mentioned in my other papers, the spleen and other agglomerations of lymphatic tissue may be considered as localized hypertrophic centers of diffuse analogous processes, present in all parts of the loose connective tissue, which is especially true for the embryo.

If so, we should certainly expect, after grafting, the same changes throughout the whole mesenchyme, as are noticed in the

WANDERING CELLS IN TISSUE OF THE BIRD 489

Spleen. The changes in the spleen were not studied, but they obviously existed, for the organ became many times larger. In order to obtain uncompHcated results I made in my experiments single spleen grafts to 7-10 day embryos. If after grafting, a hypertrophy of amoeboid cells, or whatever changes they may be, were confined both to the spleen and to other mesenchymal haematopoietic anlages and if these changes were identical in all regions, we could deduce herefrom that the mesenchyme of the whole embryo body and the mesenchyme of the spleen is equivalent.

The single grafts of adult spleen on the allantois of a chick embryo were also followed by enlargement of the host spleen. On the other hand, the study of the loose mesenchyme of the embryo body and of other haematopoietic anlages showed intense and widespread changes. These changes consisted in the spUtting oflf from the mesenchyme of haematocytoblasts in the form of the morphological unity of a large lymphocyte. It would be too long to mention all the regions of the embryo body where such an intense spUtting takes place. I will merely state that no place exists in the embryo body where the mesenchyme does not show this process. Inmunerable haemocytoblasts lie along the vessels in the form of thick cords, they are diffusely scattered in places where the mesenchyme is looser, the haemocytoblasts are crowded in groups in every skin papilla, strands of haemocytoblasts separate the smaller and larger muscle bundles and muscle fibers; even the mesenchyme in the centers of developing feathers shows an analogous splitting.

The splitting off of haemocytoblasts may be normally observed in the mesenchyme. The grafts may generalize, accelerate and intensify the same development through all the regions, where mesenchyme is found in the embryo body. The extension of identical reactions to the whole loose mesenchyme and the mesenchymal haematopoietic organs amply confirm the value of the equivalence of the mesenchyme in the spleen, the thymus and in other parts of the embryo body, where an identical reaction takes place.

The intense spHtting off of the haemocytoblasts in the loose mesenchyme of the embryo body, of the spleen and of the

490 VERA DANCHAKOPF

thymus is accompanied by differentiation of these cells, analogous in all these regions. The study of this differentiation gives decisive data about the equivalence of the anlage of the lymphoid elements in the connective tissue and the anlages of other haematopoietic organs. For the differentiation of the mesenchyme now consists in a development of granulocytoblasts and granular leucocylies, even between muscle fibers.

I cannot here analyze the causes which lead to this wonderful metaplasis of the mesenchyme. I would merely like to point out the value of the results obtained for the clear understanding of the r61e of the embryonic loose mesenchyme as of an undifferentiated haematopoietic anlage. Under normal conditions the mesenchyme gives rise to a certain number of amoeboid cells, the direct offsprings of which remain as a part of the wandering cells of the loose connective tissue in the adult organism. This process undergoes intense stimulation which here evidently depends upon resorption and circulation of certain enzymes, furnished by the grafted spleen cells. At the same time a differentiation takes place in the lymphoid cells, which in many places is normally absent.

Not only the diffuse mesenchyme of the body, but the spleen and the thymus become at certain periods after grafting centers of a most intensive true granulo-leukopoiesis (giving rise to special leucocytes).

What under these circumstances becomes of the specificity of

the lympho- and leukoblasts? The same cells, which under ordi • nary conditions give lymphatic non-granular cells as products of

their differentiation, now become the source of the most intensive

leukopoiesis.

ThuSj the embryonic mesenchyme appears to be a diffuse atilage for both lymphopoiesis and granuloleukopoiesis. The faculty of polyvalent development for the embryonic mesenchjone is proved by the study of the changes in the mesenchyme, resulting after the grafting of spleen from an adult chick on the allantois of the embryo.

Other important results are obtained by a closer study of the changes in the spleen occurring after grafting. These results he WANDERING CELLS IN TISSUE OF THE BIRD 491

come an additional proof of other polyvalent differentiation possibilities of the mesenchyme. They demonstrate onceanore, that only environmental conditions direct the differentiation of the haematocytoblasts derived from the spleen mesenchyme, for,' numerous haemocjrtoblasts may under definite conditions differentiate in the spleen into erythroblasts.

The spleen of the chick embryo is normally a lympho- and granuloleuko-poietic organ. In the earUest stages of development it appears in the form of a dense, scarcely vascularized mesenchymal anlage. The spUtting off of lymphoid haemocytoblasts is gradual; the latter remain outside the vessels and many of them differentiate into granulocytoblasts. The developing vessels contain mostly differentiated erythrocytes. The development in a spleen after grafts proceeds very intensely. The splitting off of lymphoid haemocytoblasts is most active. At the time when the spleen anlage becomes vascuolarized, nimierous Ijonphoid haemocytoblasts pass into the limien of the vessels. The blood current in the spleen being at this time very slow, many of them remain here and differentiate inside the vessels into erythroblasts and erythrocytes.

• This differentiation of the haemocytoblasts (that is, of the large lymphocytes), into erythroblasts inside the vessels with a very slow current again shows that the haemocytoblasts are polyvalent cells, of which the differentiation is directed by environmental conditions.

The results of the experiments confirm fully the deductions made with regard to the observations of the embryogenesis of the different blood cells.

The study of the changes in the embryo, obtained by grafting have shown that the loose mesenchyme of the embryo at certain stages is equivalent in all the regions of its localization. The mesenchyme is moreover polyvalent in its potencies of development. It may give typical connective tissue cells as well as all the different kinds of blood cells. The differentiation of the mesenchymal cells is directed by the force of external conditions, whether these conditions consist in the presence or absence of different food material, or in the special physico-chemical milieu of the vessels. Their proUferation seems to depend upon specific stimuli, which may consist in enzyme-Hke material.

These experimental results give strong support to the monophyletic views regarding the origin of different blood cells, and seem to stand in contradiction to the polyphyletic origin of the various blood cells.

The completely different cells (the red and the different kind of white blood cells) are so completely different only because they present the final steps of various directions of differentiation. These cells, as such, are certainly not capable of interchanging, but, as the above described experiments have shown, the various fines of differentiation of their conunon stem cells may be interchanged.

THE MICROSCOPIC STRUCTURE OF THE LEG MUSCLE OF THE SEA-SPIDER, ANOPLODACTYLUS LENTUS

H. E. JORDAN

Department of Anatomy, University of Virginia

SEVEN FIGURES

This contribution fulfills the second part of a projected comparative microscopic study of volimtary muscle. The present aim is to devote attention to hitherto undescribed specimens. Leg and wing muscle of various insects has become classic for the demonstration of sarcoplasmic cross striation, and has furnished the chief basis for our conceptions of the detailed structure and the functional phases of voluntary striped muscle. Judging from the published illustrations of insect muscle, however, the leg muscle of the sea-spider offers by comparison a material of exceptional clearness; and for this reason alone it would seem to merit a detailed description, more especially since it furnishes in short longitudinal series a complete set of structural transition stages correlating with functional phases.

The first study in this series pertained to Limulus muscle (Anat. Rec, vol. 10, no. 3, 1916; page 2100- A point of peculiar interest in this connection concerns the fact that both the Xiphosura and the Pycnogonids (Pantopoda) are usually placed in 'incertae sedis' in the current text-books of zoology. A quite general concensus of opinion considers them more or less closely related, and ranks them both between Crustaceans and Arachnoids. Limulus muscle, however, is in appearance very much more like vertebrate than like insect muscle; while the

' The complete paper with illustrations will appear in a forthcoming volume from the Department of Marine Biology of the Carnegie Institution of Washington.

493

494 H. E. JORDAN

muscle of the marine arthropod, Anoplodactylus, is of the typical insect type.

The material of Anoplodactylus lentus Wilson (formerly known as Phoxichilidium maxillare) was collected from the piles of the U. S. Fish Commission Wharves at Woods Hole, Mass. Various fixing methods were employed. The material here to be described was preserved in Flemming^s fluid and stained with the iron-hematoxylin picric-acid-fuchsin combination.

The leg muscle fibers are long cylinders embedded in groups in a finely granular, otherwise homogenous, 'connective tissue' magma, the sparsely scattered vesicular nuclei of which are very minute (figs. 1 and 2). (The extreme minuteness of the nuclei is characteristic of all of the sea-spider tissues ; the chromatin is imiformly collected in one or several spheroidal granules.)

The muscle fibers in cross section have a circular or elliptical outline. They vary greatly in diameter (figs. 1 and 2); a certain amount of this variation is undoubtedly due to differences in degree of contraction and development. The fiber is invested by a meager, very delicate, layer of homogenous sarcoplasm, limited peripherally by a faint membrane, perhaps a sarcolemma. The nuclei are disposed peripherally and lie just beneath the 'sarcolemma.' Certain fibers are surrounded by a considerable space (fig. 1), a contraction artifact; when such is present the outer boimdary is sharply marked by the 'sarcolemma;' the nucleus when present lies within the space. With picric-acidfuchsin the connective tissue stains a very faint yellowish pink; the structure which I am inclinded to regard as the homologue of the sarcolemma of vertebrate muscle remains unstained.

The fiber consists of a solid column of finely granular sarcoplasm throughout which are scattered the relatively coarse myofibrils. These are collected in the form of radially disposed lamellae (figs. 1 and 2) which appear to be undergoing a peripheral radial longitudinal splitting and a central tangential division. In analogy with growing Limulus fibers, and those of the newlyhatched rain-bow trout, these appearances are most probably to be interpreted in terms of growth through myofibril increase by longitudinal fission.

STRUCTURE OF SEA-SPIDER MUSCLE 495

In longitudinal section the prevailing fiber consists of from ten to twelve coarse myofibrils. All of the striations, with the exception of the Z line (telophragma), are clearly limited to the fibrils. Longitudinal sections reveal also the entire series of phases involved in the contraction process. These will be next described.

Figure 3 represents a resting, non-contracted, or relaxed fiber. Two complete sarcomeres are included. The conspicuous striping results from the Q discs, which consist of horizontally aligned deep-staining bacillary segments of the myofibrils. Between two successive Q discs appears a secondary striping in what corresponds to the J disc of vertebrate muscle. The J disc at this phase has about twice the length of the Q disc. The conspicuous stripe of the J disc is a deep-staining granular * line' which bisects the disc, the ground membrane of Krause (Z stripe; telophragma). The best proof of the membrane nature of this 'stripe' which accrued from the study of the Limulus muscle, namely its attachment peripherally to the sarcolemma and centrally to the nuclear wall, is lacking in the case of the sea-spider muscle. What I have described above as a possible sarcolemma never shows the characteristic festooning of Limulus and vertebrate muscle. I am unable to determine any continuation of the Z stripe beyond the most peripheral myofibrils. But two observations point to its true membrane nature: 1) it has a deUcate zig-zag outline, the non-granular portion passing between adjacent fibrils; 2) in cases where single peripheral fibrils are separating, the point of attachment of the separating fibril to the fiber is almost invariably at the level of the Z stripe. However, that the Z stripe is more than simply a membrane is indicated by the fact that its 'granules' correspond to the line of the fibrils — the myofibril apparently swells at the point of attachment to the ground membrane — and by the further fact that its character (whether granular or of sharp contour), its grade (whether fine or relatively robust) and the depth of its staining reaction vary somewhat with the phase of contraction.

Approximately midway between the Z line and the next succeeding Q disc appears a shadow of some width, the iV line

496 H. E. JORDAN I 2


!|. WO


.1


L


m

3


ffff Vn&Q


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'fi-,

STRUCTURE OF SEA-SPIDER MUSCLE 497

(nebenscheibe), thus dividing this half of J into a central definitive J disc and a terminal portion of the original J substance, the E disc (endscheibe). N is clearly due to the presence of some

EXPLANATION OF FIGURES

Leg muscle of the Pantopod, Anoplodactylus lentus. The tissue was fixed in Flemming's strong solution; the sections were cut at 5 microns, and stained with iron-hematoxylin and picric-acid-fuchsin. The drawings were made by aid of a Bausch and Lomb camera lucida. Figures 1 to 6 were made with a B. & L. 1.9 mm. oil immersion objective and a number 10 ocular; figure 7 with a ^ Leitz oil immersion lens. Figures 1 to 6 are magnified 1600 diameters; figure 7, 2000 diameters. The illustrations were reduced one-fifth in reproduction.

Fig. 1 Cross sections of two adjacent muscle fibers, enveloped by a finely granular homogeneous * connective tissue' magma. The space about the larger fiber is a fixation artifact, m.n., muscle nucleus; ct.n.j connective tissue nuclei. The myofibrils are arranged in radial lamellae.

Fig. 2 Cross section of a group of four adjacent muscle fibers, showing the variation in diameter and the disposition of the muscle and connective tissue nuclei.

Figs. 3, 4 and 5 Longitudinal sections of three fibers showing the structure in the relaxed condition and in two successive early stages of contraction. The fibers are so placed as to put the middle Z line on the same level. The arrows connecting the next series of Z lines above show how the sacromeres decrease in length during contraction. A comparison of the three figures shows also how the Q disc lengthens and finally divides in the midline to produce the // disc, how each half of the original Q disc unites with an N disc in the formation of a contraction band, and how the original / disc disappears in the process of contraction.

Fig. 6 Longitudinal section of a complete fiber showing successive stages in contraction in longitudinal series from the relaxed condition to the tensely contracted condition. The succession of contraction phases is indicated on the left by the numerals 1 to 6. The numerals S to 6 designate the later steps in the formation of the contraction band, c.h. Both at the extreme upper end and in the mid portion (2 to 2') the fiber has suffered a stretching. In the stretched middle portion (representing a midphase of contraction), the M membrane is conspicuous, having apparently the same structure and staining reaction as the Z membrane. The brackets on the left connect the constituent elements of a future contraction hand (disc). At the lower end the contracted widened fiber is connected with a chitinous midrib of the leg by tendinous tissue (T), the fibers of which are continuous with the myofibrils and which towards their termination show the characteristic staining reaction of collagenous fibers with acid fuchsin.

Fig. 7 Longitudinal section of a fiber showing the complete set of cross striations. Insect fibers of similar structure are generally interpreted as in the relaxed ('extended') condition. Figure 6, however, shows that a fiber of this structure represents a midphase of contraction upon which is superimposed a stretching.

498 H. E. JORDAN

substance in the myofibrils, determining a deeper staining reaction of this portion as compared with the enveloping J substance. It has no interfibrillar representative and can therefore not be due to the presence of sarcoplasmic granules (V granules') as suggested by Heidenhain (2). Such a fiber gives not the slightest indication of the presence of an H disc nor of an M membrane (mesophragma) commonly described for the resting insect muscle fiber. The nature and significance of these stripes will be discussed below; where will appear also the proof that the fiber in question is actually in the relaxed condition.

It should perhaps be definitely stated at this point that this study proceeds upon what would seem to be a perfectly legitimate assumption, namely, that a difference in staining capacity mirrors a structural difference — if indeed perhaps only a difference in the relative amount or even concentration of physically identical material — and that a variation in structure, as revealed by staining reaction, is correlated with a phase of function. These structural differences and physical alterations are, at least in part, probably fundamentally molecular and so associated with chemical changes. The advantage that living muscle, as compared with fixed and stained material, is sometimes assumed to possess is illusory. More can apparently be learned regarding the actual morphologic changes underlying contraction with well preserved stained material than with living material. Enough can be seen from Uving material to give sufficient assurance that the stripes of preserved material are not fixation artifacts. Staining renders more conspicuous the actual structural and chemical alteration.

In the light of the above it would seem to be clear that the J disc can not be regarded as simply an optical effect, due to a difference in refractive index between Z and Q as suggested by Meigs (3) in the case of the wing muscle of the fly. It is much too wide to satisfy the requirements of such a hypothesis. Moreover, the difference in staining capacity between Q and J, and the presence of N render such interpretation imtenable. The same argument applies in greater part also to the H disc as related to the M 'membrane' which appears in certain phases of contraction.

STRUCTURE OF SEA-SPIDER MUSCLE 499

Figure 4 represents an early stage of contraction, figure 5 a still later intermediate stage. Figures 3, 4 and 5 are so placed as to put one Z line, connected by arrows, on the same level; the next Z line above is also connected by successive arrows. It is apparent that there occurs a gradual shortening of sarcomeres corresponding with the degree of contraction. In the phase illustrated in figure 4 the shortening is considerable. Coincident with this shortening of the sarcomeres occurs also an alteration in the Q disc ; the other stripes remain apparently unaltered to any appreciable extent, and need not again be specially mentioned.

The change in the Q disc pertains to a terminal movement of Q substance, as indicated by the staining quality between midportion and termini, causing the ends to appear swollen and to become deep-staining, and the intervening portion to remain slender and to stain progressively less deeply towards the midline. It can be seen also that the Q disc, taken as a whole, has a slightly greater length, and the J disc a much shorter length, as compared with the relaxed fiber of figure 3. There is as yet no clear indication of the H disc and the M line.

An almost identical condition is illustrated also for the muscle of the antenna of the lobster by Dahlgren and Kepner (4), and a very similar condition for the muscle of the ichneumon fly larva by Prenant, Bouin et Maillard (5), for a fiber of Dytiscus and the wing muscle of the wasp by Schafer (6), a muscle fiber of Cassida equestris by RoUet (7), and for the wing muscle of the water beetle Hydrophilus piceus by Ranvier (Schafer (6) ).

Figure 5 represents a later step in the same process begun in figure 4. The sarcomeres have suffered a further slight shortening; so have also the J discs; and Q has increased appreciably in length. The chief change pertains to the Q disc; the deeper staining constituents of this disc have moved still further apart leaving a mid-portion of considerable width imstained, the H disc. Moreover, the deep staining ends of the Q segments are no longer of spherical granular shape as in figure 4, but now have a bacillary form.

It seems clear that there has been a movement of substance from the midline of Q towards its terminal borders, and that the

500 H. E. JORDAN

resulting H disc is a phenomenon of this intermediate phase of contraction. Moreover, at a certain point in the movement the terminal portions of Q fuse with the N lines, thus obliterating the intervening portion of J. At a later stage in this particular phase this combined disc (Q + N) may stain deeply as a whole; this then gives the appearance frequently illustrated as the typical uncontracted ('most extended^ phase (e.g., Schafer, Quain's Anatomy, vol. 2, pt. I, p. 182, fig. 282).

As will be described in connection with figure 6 this movement oi Q + N continues towards the Z line until E also dif appears, and Q + N fuses with the Z line to form the contraction band. The latter is of course bisected by the Z Une which has now become masked by the presence of the Q substance of the contraction band. At this final stage the fiber consists of a series of deep staining contraction bands separated by alternating interband unstainable segments.

In proof of the correct seriation of the three fibers just described, we may now consider a single fiber which shows the several main stages in contraction in longitudinal series.

Figure 6 illustrates a fiber contracted at its lower end; at this end it is attached to a chitinous midrib of the leg by a tendon {T)] the opposite end, several sarcomeres beyond the last one shown, is similarly attached to the lateral chitinous wall. In passing from the attached lower end upward the contraction bands become in general less narrow and compact until at point 4 the band is clearly double. At this point also, and just above it, it may be seen that the contraction band consists of deep staining segments of the fibrils, without interfibrillar representatives except for the Z membrane. Conspicuous also in passing from points J^io 5 to6 and on towards the tendinous attachment is the gradual widening of the fiber as a whole, which is correlated with a lessening interval between successive contraction bands and a thickening also of the constituent myofibrils. Contraction clearly means, in part at least, a thickening of the fiber due to a thickening of 'the constituent fibrils; correlated with a movement and condensation of the Q substance, and a contraction in length and a rearrangement of the unstainable (J)

STRUCTURE OF SEA-SPIDER MUSCLE 501

substance of the fibrils. The length of the sarcomeres at 1 is to that of the second from the end of the contracted portion as 3 to 1; the width as i to ;? +.

The most instructive picture with respect to contraction appears at the point 5, where the upper dark staining line of granules (similar to the condition illustrated in fig. 4) is closely apposed to a second (lower) dark staining line (similar to the condition shown in figure 5, but a somewhat later stage); and the two are about to come still closer together (as at point 4) to form a contraction band (as at point 6). The several steps in the process of contraction are shown at points 1, 2\ 3, 4> ^ and 6. Points ly 2 (and 2') and 3+4 show clearly conditions similar to those illustrated in the separate fibers, figures 3, 4 and 5.

Point 1 of figure 6 represents an intermediate stage between figures 3 and 4. Q is here characterized by a terminal row of sharply defined spherical granules, indicating the initial step in the movement of the condensing Q substance towards the Z membrane. At points 1 and 2' the migration has progressed to a stage somewhere between figures 4 and 5. At points 3, 4 and 5 are shown the final steps in the same process.

What requires further consideration in the case of this fiber is the fimctional interpretation of the upper end, and also of the several sarcomeres between 2 and 2\ This interpretation will be attempted after consideration of the fiber illustrated in figure 7. It should be stated here that the fibers of figures 3, 4 and 5 are closely associated in the same leg; figure 6 is from a second specimen ; and figure 7 from a third. These five fibers (from three different specimens) represent all of the chief structural characteristics of the very many fibers studied in many specimens.

Figure 7 represents perhaps the rarest type of fiber. It contains all the striations, including even the M line. The Z line is conspicuous and granular; both Q and J are of considerable length; and the M line, while very pale and delicate, is nevertheless immistakably present. Such structural condition is generally interpreted as that of the relaxed fiber. In the Ught of what we have learned from figures 3 to 6 this fiber would seem to demand interpretation in terms of a midphase of contraction. But it

502 H. E. JORDAN

corresponds strictly neither with figure 4 nor with figure 5; where, among other differences, no indication of an M membrane can be discerned. To anticipate what I intend to prove from figure 6, the fiber of figure 7 represents, I beUeve, a fiber at about the stage of contraction shown in figure 4 which has become stretched considerably under the tension of an outlying tensely contracted portion of the same fiber. An early phase of contraction could alone account, I beUeve, for the conspicuous presence of H, and a stretching for the length of the Q discs and the faint presence of an M membrane.

To return to figure 6: The lower end is clearly tensely contracted, the upper end (point 1) just passing from a relaxed condition to an early phase of contraction. Points 2 and 2' represent sUghtly later steps in the contraction process than point 1. In contraction the fiber clearly thickens (e.g., point 6 and below); the fiber is stouter from points 1 to 2 and from points 2' io 6 than in the intervening portion; but points 2 and 2' are at the same stage of contraction, and they represent a transition step between the phase shown at point 1 and that shown at points 3 and 4. The intervening portion then it seems must be interpreted in terms of an alteration of a structure represented at points 2 and 2'. Stretching alone offers the simplest explanation of the actual conditions. The fiber here is somewhat thinner, and the Q disc much longer, than in other portions of the same fiber. Also the J disc is here longer than in the portions just above and below. The greater length of the Q disc and the shortness of the J disc at point 1 may be explained on the basis of a fusion of Q with N in the terminal movement of Q substance at this phase of contraction. The two uppermost sarcomeres may be explaimed on the same assumption of a stretching at this point. This portion of the fiber may be conceived to have been in a relaxed condition, like that illustrated in figure 3; the stretching of the fiber due to the pull of the tensely contracted opposite end brought the adjacent fibrils so closely together that when stained the Q disc appears almost as a single mass, in which separate fibrils are with difficulty distinguishable.

It appears then that the fiber of figure 7 is similar to that portion of the fiber 6 between points 2 and 2'. A difference inheres

STRUCTURE OF SEA-SPIDER MUSCLE 503

in the relatively greater length of J, and the granular more deeply staining character of the Z line. Both may possibly be explained on the basis of a greater degree of stretching in fiber figure 7, as compared with the similar portion of the fiber figure 6. Granting that the Z line is a true membrane, to which the myofibrils are attached, spanning the interfibrillar space (as was clearly shown in Limulus muscle), then in a contracting fiber — where the Z membrane tends to become more deUcate and stain less intensely, due to its being stretched in a horizontal plane on account of the swelling of the fiber — when stretched longitudinally the Z membrane becomes less taut, thus increasing its bulk and consequently its discernible staining capacity.

Similarly may be explained the visibiUty of an M membrane both in fiber figure 7 and in the similar portion of fiber figure 6. Neither in fiber figure 5, where the H disc is wide and conspicuous, nor in the contracted portion of figure 6, can an M membrane be seen. In both cases a longitudinal shortening is associated with an increase in diameter; this must of necessity produce lateral tension upon a membrane which may be present spanning the diameter of the fiber. If we suppose such a membrane to be actually present, but ordinarily invisible due to the limitations of microscopic vision, then relaxation of the tension upon such a membrane, due to longitudinal stretching, might bring the membrane within the lower limits of visibility. This, I believe, may be the explanation of the appearance of an M line in figures 6 and 7.

It must be admitted that this explanation, however, is perhaps weakened somewhat by the delicacy and light staining reaction of also the Z niembrane in fiber figure 6 between points 2 and 2' , Aside from this objection, however, which may be met on grounds of variability in the staining reaction of Z under various conditions (due to varying amounts of Q substance left, following a contraction band), the above explanation for the conspicuous character of Af in fibers figures 6 and 7 would seem to offer support to Heidenhain^s (2) contention that an M membrane (mesophragma) is invariably present in striped muscle, but below or at the limit of microscopic vision (about 0.2 ^ in width). That the inferred M membrane, however, differs structurally from

504 H. E. JORDAN

the Z membrane seems clear from the fact that it does not produce a peripheral festooning of the sarcolemma in vertebrate fibers, nor is it attached centrally to processes of the nucleai wall as was shown in the case of the Z membrane of Limulus muscle, both skeletal and cardiac.

However, this latter argument for a difference in structure between the Z and M membranes may be met by the supposition — for which the appearances respecting the M membrane described in figures 6 and 7, and their most probable interpretation, offer a tolerably sound basis — that the M membrane is of such extreme delicacy or relatively lesser extensibility as to be unable to withstand the tensions demanded and met on the part of the relatively more robust and extensible Z membrane in the formation of festoons in the sarcolenmia. Regarding the actual occurrence of an M membrane generally in striped muscle, as maintained by Heidenhain (2), concerning which there is much discussion and scepticism, I now incline to beUeve that its observation and non-observation on the part of different investigators depends upon slight individual differences in acuity of microscopic vision. My observations on the sea-spider muscle yield no evidence in support of Schneider's (8) suggestion, that the M stripe is a transient structure representing the initial stage in the formation of the Q disc following a contraction band (disc).

Before taking up the question of the interpretative value with respect to function, of the microscopic structural details above outlined, we may give attention to the manner of attachment of the muscle fiber to the chitin. We have here a clear case of muscle-tendon continuity, a condition which O. Schultze (9) maintains prevails in general even in vertebrate muscle. The evidence for the myofibril-tendofibril continuity in this muscle is twofold: 1) direct observational — the myofibril can be readily seen to continue as the tendon fibril; 2) indirect, inferential — the ^ connective tissue' in which the muscle fibers are embedded is non-fibrillar, whereas the tendon connection consists of coarse fibrils, like the myofibrils, among which are many small nuclei similar to the muscle nuclei. Moreover, there is here nothing in the nature of a sufficiently coarse sarcolenmia to permit of the

STRUCTURE OF SEA-SPIDER MUSCLE 505

attachment of the robust tendon fibrils, in the manner described by Baldwin (10) for certain vertebrate muscle; and the tendon stains increasingly more deeply with fuchsin as the point of attachment to the chitin is approached.

The morphologic picture contributes the following information regarding striped muscle activity in contraction: in contraction the fiber as a whole shortens and thickens; the increase in diameter of the fiber is associated also with a thickening of the fibrils, as first maintained by RoUett (1885, (7) ). There is no evidence to suggest that the interfibrillar sarcoplasm has appreciably either augmented or decreased. The thickening of the fibril in contraction involves a rearrangement mainly of the stainable Q substance. This Q substance moves terminally, causing a bisection of the original Q disc ; this movement involves the A^disc and proceeds as far as the Z membrane, where, together with a similar contribution from the next succeeding Q disc, is formed a contraction band. Muscular motion appears thus to be of the nature of a streaming of a portion of the myofibril protoplasm. The transfer of the Q substance is limited distally in contraction by the Z membrane, and proximally in relaxation by the M membrane. In contraction, halves of successive Q discs fuse at the Z line; in relaxation, halves of successive contraction bands fuse at the M lines. Contraction and relaxation are thus apparently functions of the internal arrangement of adjacent sarcostyles (myofibrils), coordinate action at any level being attained by the attachment of the fibrils to the Z and M membranes. The morphologic evidence then seems to support Englemann's theory (11) that muscular contraction results from an interchange of fluids (gels) between different parts of a sarcostyle (sarcomere).

The minute structure of the ^contraction bands' (discs), as illustrated in figure 6, and their relationship to the Z membrane is essentially identical with the simpler types of intercalated discs of vertebrate heart muscle, a circumstance which would seem to furnish strong support for my interpretation (1) of intercalated discs as irreversible contraction bands.' Contraction in cardiac and skeletal muscle is an essentially similar process; similar contraction bands occur in both instances. Moreover the sim 506 H. E. JORDAN

plest types of intercalated discs are very similar to contraction bands; and both are similarly related to the Z membrane. Assuming an irreversibiUty of certain contraction bands, due perhaps to unusual strains, the simpler types of intercalated discs are readily conceived to result. From the simpler, the more complex types may be derived through the operation of the simpler mechanical stresses incident to rythmic contraction.

The foregoing discussion assumed the identity of the deeply stainable substance and the Q substance. The Q disc is generally described as composed of anisotropous substances, the J disc of isotropous substance. But the segregation of these two physically different granules is by no means complete; the fibril as a whole contains anisotropic materials; these are relatively more abundant in the Q disc. The question then arises as to whether the streaming more deeply staining substance in contraction is an aggregation of anisotropic substance. This would be the a priori inference but the observations of Schaf er (with gold chlorid staining) and Englemann (with the polariscope) contradict such a conclusion. Englemann and Schaf er maintain that the anisotropic material remains segregated in the clear disc between the terminal contraction bands. However, apart from a refinement of Englemann^s theory of muscular contraction, which views the movement of the intrafibrillar material in terms of an absorption of the isotropic substance by the anisotropic, this apparent contradiction between the microscopic and polariscopic evidence need offer no special difficulty.

, The proximate factors in muscular contraction are: 1) nerve impulse; 2) shortening and thickening of segments of the myofibrils between successive Z membranes ,coincident with a streaming of stainable substance from M on either side to Z. The functional unit is thus the sarcomere. The fundamental closely related phenomena are streaming and shortening.

A consideration of the various theories of muscular action — chemical, chemico-dynamical, physical, physico-chemical, electrical, etc. — or the formulation of a new theory is outside the scope of this investigation, which seeks to limit itself to a description of structural details. However, it may be stated that the rapidity of the reversible action of muscle contraction seems

STRUCTURE OF SEA-SPIDER MUSCLE 507

an obstacle to a confident acceptance of any theory thus far proposed. If one confines himself simply to the obvious facts, namely, a movement of material from midline of sarcomere to the terminal Z membranes and back again to either side of the M membrane coincident with contraction (shortening) and relaxation (lengthening), the most extreme rapidity offers no difficulty. This 'streaming' may perhaps represent an imbibition of more fluid colloids by relatively more solid proteid gels within the sarcomeric element of the myofibril, as has been suggested; the point of absorption being marked by the advancing more deeply staining Q line. The shortening and the thickening of the myofibril may thus be the result of a swelling and consequent roimding up of more soUd presumably oval colloidal particles. But underlying this there must of course reside a functional (energy) metabolism. This involves chemical phenomena. These are known to include oxidation of carbohydrates with an evolution of heat, and the Uberation of lactic acid and carbon dioxide. The morphologic evidence would seem to indicate that attempts at interpretation of muscle function must limit attention to internal changes of the myofibrils. This evidence, namely, an apparent streaming of a tinctorially, therefore structually and perhaps chemically, distinct substance, may be brought into accord with the now more generally prevailing theory of muscular contraction expressed in ternas of alterations of surface tensions between adjacent coUodial particles in the myofibrils, by regarding the line of the 'streaming' stainable substance in its passage toward the Z membrane as the level of progressive chemical changes upon which the surface tension phenomena depend.

A final point concerns the significance of the complex cross striation as exhibited by the sea-spider muscle and insect wing and leg muscle in general, in terms of power and rapidity of contraction. Insect muscle is commonly regarded as highly differentiated and perfected for rapid function. A high degree of differentiation no doubt obtains, as is indicated, irrespective of the clear and complex striation, by the peripheral location of the nuclei; but that such condition is specifically closely correlated with extreme rapidity of function as exhibited by wing muscles

THE ANATOmCAL RECORD, VOL. 10, NO. 7

508 H. E. JORDAN

and certain leg muscles of insects, is clearly not the case; for the sea-spider moves its legs relatively slowly. As regards the force of the muscular movement it is more difficult to arrive at a conclusion since factors of relative weight and distance of movement enter here; but there appears no striking evidence that the leg of the sea-spider is capable of very powerful muscular contraction, as in the case of certain leaping insects. We appear to be on firmer ground when we speak of an arthropod type of striped muscle and a vertebrate type of striped muscle. Both are highly differentiated, with a general correspondence, but with differences in detail. Rapidity and force of contraction do not seem to be necessarily correlated with a visibly complex striation. As concerns the sea-spider and the Limulus muscle, the former is a typical arthropod muscle, the latter corresponds more closely to the vertebrate type. The chief points of difference concern the visible presence of the N disc and the M line and the wider and more conspicuous character of the Q and J discs in arthropod as compared with vertebrate voluntary striped muscle.

BIBLIOGRAPHY

(1) Jordan, H. E. 1916 A comparative microscopic study of cardiac and

skeletal muscle of Limulus. Anat. Rec. vol. 10, no. 3, p. 210.

(2) Heidenhain, M. 1911 Plasma und Zelle. Fischer, Jena.

(3) Meigs, Edward B. 1908 The structure of the element of cross-striated

muscle and the changes of form which it undergoes during contraction. Zeitschrift fur Allgemeine Physiologie, 8, 1.

(4) Dahlgren and Kepner. 1903 Principles of Animal Histology (Mac millan), p. 85, fig. 83.

(5) Prenant, Bouin et Maillard Traits d'Histologie. Masson et Cie. ,

Paris.

(6) Schafer, E. a. 1912 Quain's Anatomy (Longmans) vol. 2, part 1, pp.

182 and 184.

(7) RoLLET, A. 1885 Untersuchungen iiber den Bau der quergestreiften

Muskelfasern. Denkschr. der Math. Nat. Kl. der kaiserl. Akad. der Wissenschaften zu Wien, Bd. 49.

(8) Schneider, K. C. 1908 Histologisches Praktikum der Tiere.. Fischer,

Jena.

(9) ScHULTZE, O. 1912 Ueber den direkten Zuzammenhang von Muskel fibrillen und Sehnenfibrillen. Arch. f. Mikrosk. Anat., Bd. 79.

(10) Baldwin, W. M. 1912 The relation of muscle fibrillae to tendon fibrillae

in voluntary striped muscle of vertebrates. Morph. Jahrbuch, Bd. 45.

(11) Engelmann, T. W. 1895 Ueber den Ursprung der Muskelkraft. Nature,

March 28.

THE CORRELATION OF CERTAIN CHEMICAL FINDINGS WITH HISTOLOGICAL STRUCTURE^

FREDERICK S. HAMMETT From the Biochemical Laboratories of the Harvard Medical School, Boston, Mass.

It may be stated that in general those substances found in tissues are the products of the cellular activity of the tissues themselves. Undoubtedly many tissue constituents have been brought there as such by the blood; but under average conditions of uniform diet the determinable integral constituents are the products of the metabolic processes of the cells and cell nuclei upon the substances brought to them by various means. The glycogen of the liver and muscles, the phosphatids of the brain and the purines of all tissues are specific enough examples to demonstrate the possibility of tissue constituents being in general the product of the specific tissue activity.

During the work on uric acid in tissues it was noted that certain tissues invariably contained more uric acid than others. This difference was so marked and so constant in occurrence whatever the kind of animal under investigation, that in order to explain these differences, recourse was had to the histological structure of the tissues in question as well as to their function.

It seems plausible therefore to divide tissues into two general classes, basing the classification upon the histological structure and the uric acid content and strengthening the comparison by a consideration of the function.

In the one group it is possible to place all those tissues whose nuclear mass is relatively great in proportion to protoplasmic mass such as liver, spleen, kidney and general glandular tissue. In the other group may be placed those tissues in whose cells the nuclear material is relatively small in proportion to protoplasm, such as muscle tissue.

In the former group we find without exception that uric acid is present in quite considerable quantity. In the latter group the uric acid content is uniformly low in amount.

Histological evidence points to the fact that glandular tissue activity is inherently bound up with nuclear activity. The changes in gland activity are accompanied by well marked changes in nuclear structure. The specific function therefore of these tissues is closely associated

The work herein reported was carried on while the author was working in the Department of Bio-Chemistry of Harvard Medical School and represents a part of the work required for the Degree of Doctor of Philosophy.

509

510


FREDERICK S. HAMMETT


if not entirely bound up with nuclear metabolism. With muscle tissue however we are unable to trace any direct histological connection between tissue activity and marked changes in nuclear structure. The main function of muscle tissue depends upon its protoplasmic metabolism. Increase in muscle activity has yet to be proven to yield an increase in uric acid excretion that can be traced to increased nuclear activity. Whereas both MareS and Smetanka have conclusively proven that increase in glandular activity causes increase in nuclear activity and metabolism, with increase in uric acid excretion.

It is not assimied that the nuclei of muscle cells have no part in the production of uric acid; but it is considered that the nuclear activity of muscle cells is differentiated from that of gland cell nuclei in that in the former the protoplasmic activity is predominant while in the latter nuclear metaboUsm is the main factor in the functioning of the tissues.

From the results of the herein reported analyses the foregoing conclusions seem justified. The proposition is advanced that the comparative analysis of the tissues of an animal as regards their uric acid content gives some index of the relative nuclear activity of the tissues examined.

A survey of the appended tables demonstrates the points under discussion. The figures represent milligrams of uric acid in one hundred grams of tissue.

Distribution of uric acid in cat tissues BLOOD


MT78CLB


LIVEB


KIDNBY


8PLEBN


LUN08



mg.


mg.


mg.


mg.


mg.


mg.


1


0.34


0.47


13.51


6.61


7.76


3.67


2


0.47


0.37


6.60


4.60


4.09


3.15


3


0.41


0.29


4.76



3.37


0.46


7


0.10


0.26


3.72



0.61



8


0.12


0.15


4.10



1.98



9


0.20


Trace


4.73



0.30



10


0.29


0.79


2.83



4.76



11


0.15


0.28


2.27



1.30



13


0.83


1.20


2.54


Rabbit


LIVER


LUN08


niQ.

0.72 0.60


mg.

4.96 5.69


mg.

1.02 0.80


mg. 26.60 10.13


mg.

2.89

1.38


mg. 1.43

CHEMICAL FINDINGS — ^HISTOLOGICAL 8TRUCTUBE


511


Hen BLOOD



KIDNST


BPLBBN


LITBB



W


R



1

2


mg. 4.51 3.68


mg.

1.14

0.68


mg.

1.83

0.74


mg.

21.06

40.59


mg.

10.5 11.19


mg. 27.05 26.59


Man MU8CLB


LIVBR


BPLBBN



mg.


mg. .


mg.


3


1.29


3.40


5.47


4


0.62


2.41


1.55


5


0.92


2.04


2.39


6


0.59


2.48


5.55


Dove


HtTSCLE


KIDNBT


LIVBB


mg. 3.30


mg. 52.33


mg. 15.74


Turtle


Cat


ITDBCLB


LIVBB


mg. 0.42 White


mg. 1.03


mg. 5.71


Inasmuch as the hen and dove excrete their nitrogen as uric acid thus differentiating them from the Mammalia it would not be accurate for the purpose of the argument to include the blood and kidney analyses as examples. With these exceptions the differences as previously discussed are plainly apparent.

CONCLUSIONS

From a comparison of the relative uric acid content of body tissues with their histological structure both at rest and after activity it is possible to correlate the function of the tissues with their nuclear

512 FREDERICK S. HAMMETT

activity and thus draw a line of demarcation between a predominately protoplasmic activity and a predominant nuclear activity.

LITERATURE CITED

Mares, F. 1887 Sur I'origine de I'acide urique chez I'homme. Arch. Slaves

de Biol., 3, p. 207. Smetanka, F. 1911 Zur Herkunft der Harnsaure \mm Menschen, Pflugers

Archiv, 138, p. 217.

COLLATERAL ROOT CANALS AND MULTIPLE FORAMINA IN HUMAN TEETH

J. R. CALLAHAN

Director oj the National Dental Association in the Cincinnati General Hospital

THREE FIGURES

It is essential to the proper understanding and treatment of foci of systemic infection arising from the presence of infected pulp tissue confined within the root canals of devitalized teeth, that the anatomy of the root canals of human teeth be more thoroughly understood.

Anatomical text-books mention and illustrate the 'foramen' which transmits the afferent and efferent blood vessels and terminations of the nervous system of the pulp.

Three or more years ago Fischer discovered what appeared to be multiple foramina. Later Feiler showed, by removing cemental tissue from root ends with sand paper disks, that the multiple foramina of Fischer were canals or holes in cemental tissue produced by the action of 'antiformen' a powerful solvent of organic substance.

The surfaces of root ends so treated show that these minute canals or holes do not connect with the pulp canal, therefore are not true dental foramina.

The accompanying photomicrographs show clearly what I have for three years, in papers on root canal filUngs, referred to as collateral root canals and multiple foramina.

Figures 1 and 2 are ground sections of teeth from which the pulp tissue was removed. The canals were then filled with stained rosin solution. The stained rosin solution entered the tubuU, therefore the dentine as well as the collateral canals show dark. Figure 3 is a ground section of a tooth from which pulp tissue was removed. The tooth was then placed in hematoxyUn stain over night. This specimen shows a rather frequent collateral branching of the root canals in a canine tooth. I am not yet prepared to say in what per cent of teeth these canals occur.


513

514 J. R. CALLAHAN

A SHORT SPINAL CORD IN THE TOAD

W. M. SMALLWOOD

Contributions from the Zoological Laboratory j Syracuse University y C. W. Hargiii, Director

TWO FIGURES

While studying the nervous system of anura in our beginning zoology this fall, I noticed one specimen having most of the neural canal empty save for some nerves. On closer examination, it was seen that the regular ten pairs of nerves were present with their usual distribution. In each intervertebral space a pair of nerves left the neural canal. The specimen was set aside for further study.

When the central nervous system was removed entire, it was readily seen that all of the parts were present and normal except the length of the spinal cord. The letter X in figm-e 1 indicates the position of the sacral vertebra and the beginning of the urostyle into which the fiUum terminale of the spinal cord normally penetrates for a varying distance. This makes the length of the spinal cord less than one-half what it should be and was in all the others examined. During the past ten years I have had opportimity to examine more than 2000 such dissections and this is the first case of such a marked shortening.

Sections of the cord were made by using the Weigert stain and the general results are shown in figure 2. This is a cross section through the region where the seventh, eighth and ninth nerves left the stub of the cord. The following summary shows the more important modifications:

First. The neurocoele is not in the center of the section but pushed ventrally.

Second. The gray substance occupies a greater proportion of the total area in this cross section than in a spinal cord of normal length for anura.

Third. The dorsal gray commissure is greatly enlarged.

Fourth. The dorsal column is nearly double the normal size.

Fifth. There is a small funicular region.

Following Herrick's^ classification of the somatic sensory into exteroceptive (pain, touch, heat) and proprioceptive (muscle sense); there is an interesting structural dififerentiation in the region illustrated by this section. The numerous exteroceptive fibers mostly end

^ Herrick, C. J. Introduction to Neurology, 1915, p. 132.

515

516


W. M. SMALLWOOD


in the dorsal column but in this region three nerves enter close together instead of each pair being separated by a vertebra. In order to ^ able to take care of the large amount of stimuU coming in over these three nerves, the profound changes mentioned above has taken place.



Fig. 1 Short spinal cord in a toad. The letter X indicates the position of the sacral vertebra and the beginning of the urostyle.

Fig. 2 Camera lucida drawing of cross section of spinal cord of toad through stub of cord. The area outlined with dashes is the dorsal gray commissure.

PROPERTIES OF COLLOIDS IN RELATION TO TISSUE STRUCTURE

RAPHAEL ISAACS University of Cincinnati

The study of fresh tissues under the microscope by dissection or compression methods or in hanging drops, is accompanied by several difficulties. The clear-cut pictures of the fixed and stained tissue are absent, as well as the fibrillar structures characteristic of fixed sections. The tissue colloid changes rapidly on removal from the body, swelUng or coagulating. It becomes necessary, therefore, to ascertain what properties of colloids can be determined by optical or micro-physical methods, and what changes colloids of different kinds undergo when subjected to conditions under which fresh tissues are examined. Much of this work has already been done by the colloid chemists, and the experiments and conclusions of this paper are mostly repetitions of those experiments which are necessary to illustrate the essential points, together with such modifications as were found necessary in appl3dng the subject to our special needs. The results are applicable both to the study of single cells and to masses of tissue.

The colloids used were gelatin, egg-albumin, fibrin, gluten, ascitic fluid, and others. Test tube and sUde experiments were made, so that the results could be checked up in each case under the microscope.

RELATION OF CONSISTENCY, SIZE, AND REFRACTIVITY OF TISSUE

COLLOIDS

The first series of experiments were carried out to test the relationship between consistency and refractivity. When fresh tissues remain under the microscope for some time, some of the elements, as the nuclei, and 'fibrillae,' appear much clearer. Their refractiveness increases. A similar series of changes can be studied in strips of watersoaked gelatin of equal size, when dehydrated with solutions of alcohol of varying strengths. A strip taken from a 40 per cent solution is smaller, tougher, and more refractive than the corresponding strip taken from a 30 per cent solution. The behavior may be simuned up by saying that the smaller the size of a shrinking colloid, the greater the toughness (tensile strength) and refraction. This would indicate that the increase in refraction in nuclei and fibrils in freshly mounted tissues, removed from an available source of oxygen and in the presence of accumulated waste products, is due to a gelation or partial dehydration of

517

THE ANATOMICAL RECORD, VOL. 10, NO. 8 JUNE, 1916

518 RAPHAEL ISAACS

the colloids of these structures. The accompanying increase in toughness is well shown when the tissue is teased. The change in size is often measurable, as in the contraction of the clot in tissue cultures.

The 'stiffness' of a colloid solution further depends, to a considerable extent, on the distance between the walls of the container. The viscosity is such, that the narrower the vessel, the stiffer is the colloid. It is thus possible that a considerable proportion of the supporting properties of the intercellular connective tissue substance, which appears in the living as a structureless colloid, may be due to the position it holds with reference to the tissue cells bounding it.

RELATION OF CONCENTRATION TO OPTICAL DIFFERENTIATION

To what extent can morphological appearances, based on differences in states or concentrations of colloids, be differentiated imder the microscope? Studies on gelatin, egg-albumen, globulins, and gluten solutions of varying dilutions, show that there are two modifying factors which must be considered in studying the minimal optical dLifferences between various concentrations. These factors are the thickness of the layer and the state of the colloid, whether sol or gel. In appreciably thick layers one is able to make more delicate distinctions under the microscope, than in extremely thin layers, where the concentrations of a given colloid may vary considerably without optical change. Nevertheless, a very small amount of a colloid can change the optical properties of a medium. The gel state, for a given concentration, is optically distinguishable from the sol state. Differences of concentration in the gel state are more easily seen than corresponding differences of concentration in the sol. Changes from one state to the other are frequently met with in tissues. They are often aided or hastened for histological purposes by the use of reagents. It is probable that the freezing process in making frozen sections of tissues, leaves structures in the tissues which have been produced by a change of sol to gel state. Hardy ('99, p. 187) describes precipitation networks in frozen colloids. Such changes are reversible in gelatin, but irreversible in the case of some of the tissue constituents.

RELATION OF CONCENTRATION TO STRUCTURE ON FIXATION

A. Fisher ('99), Mann ('02), and Hober ('14), among others, have pointed out that a structureless colloid solution, such as egg-albumen or gelatin, exhibits, on fixation, a network. Hardy ('99) has appUed this phenomenon to the interpretation of the structure of 'fixed' protoplasm and Isaacs ('16) has applied it to explain the appearance of connective tissue fibrillae as one of the post-mortem changes in preserved tissue. An additional factor, as Hardy ('99, p. 187) points out, is the apparent structural entity produced by 'shear strains' in colloids. These shear effects are doubly refractive, and correspond to some of the fibrillar

PROPERTIES OF COLLOIDS 519

structures described in connective tissue and other cells. Collagen fibers can be simulated by exerting shear strains on the structureless jelly of freshly coagulated fibrin in clotting blood or plasma.

The pattern of a precipitated colloid is the same for concentrated and dilute solutions. The former, however, is denser and coarser, the latter more deUcate and thin. This gives us an index to the state of the tissues at the time of their death, when one is studying fixed material. The more dilute regions leave, on precipitation, deUcate networks, while the stiffer, more concentrated regions leave coarser and denser coagula. Such changes mark the transition from young to older tissue, and show the variations in water content with variations of physiological state.

FACTORS IN THE FORMATION OF THE DEHYDRATION AND PRECIPITATION PATTERNS

A. Fisher ('99) has described the variation in the size of precipitation granules when various colloid substances are treated with fixing agents. When the action of the fixative is complete, it is usually found that the granules have arranged themselves in the form of a network. The fines formed are often strikingly homogeneous and definite, and resemble fibers and fibrillae. A study of coagulation, drying, and precipitation in gelatin or egg-albimien shows the process of network formation to consist of two steps. First, there is a union of particles of the colloid substance to granules or crystals which may be visible under a magnification of 900 or even less. This is accompanied by the second process, which consists of a winding together of these granules, imbedded in the more liquid parts of the colloid, so that a network, resulting from the diffusion currents, is produced. This would seem to be compatible with Hardy's conception of colloidal solutions as consisting of colloidal particles floating in an extremely dilute solution of the colloid in the solvent (Mann, p. 47). The currents continue until all parts are precipitated. The precipitation process, as also the dehydration process, involves a separation or displacement of liquid from solid. With most fixatives, the liquid set free is enough to destroy their isotonicity, even for various parts of a cell, whether near or far from the surface. Others, as formalin, produce little change in the colloids, when used alone, and there is little change of size or separation of ingredients

From this description, it is to be expected that each colloid will have its own precipitation pattern, since different substances form different sized precipitation granules (A. Fischer), and exhibit different degrees of viscocity. Likewise different fixatives produce different patterns because they precipitate granules of different sizes in a given colloid, and because the rate of action is different.

520 RAPHAEL ISAACS

RELATION OF CLOUDY SWELLING TO FIXATION PATTERN

The action of acids on colloids has been treated by different authors, most recently and thoroughly by Martin H. Fischer ('15). Of special interest in this connection is the postmortem swelling of tissue following an increase in the acid content, and the precipitation, under the same conditions, of the colloids of the casein type, such as the globulins (cloudy swelUng), and probably chromatin. The so-called zynaogen and secretion granules may also fall in this class (Fischer). Fixation of such tissue is equivalent to precipitation of dilute colloids, and if the precipitated albimiinous granules resist the solvent action of acids in the fixative, they become thoroughly woimd up in the deUcate precipitation network in the protoplasm of the cell.

EFFECT OF 'PHYSIOLOGICAL' SALT SOLUTIONS

A series of experiments were carried out to test the effects of 'phy^iologicar salt solutions on the colloids of the cell. The action of salts in maintaining the outUne of the cell may be explained on either an osmotic or a colloid basis. In either case it is evident that the different parts of the cell, — the nucleus and cytoplasm, are in equilibrium at any given moment as far as water content is concerned. As soon as the oxygen supply of the cell is decreased, we have an increase in the acid content, and the cell will swell if free water is present. A solution such as Ringer's or Locke's prevents this, the former by "the antagonistic action of salts and acids, the latter through this plus neutraUzation, since Locke's solution contains sodium bicarbonate. Any simple system which would prevent this increased absorption of water by the cytoplasm must do so also for the nucleus. The nucleus, however, is denser than the cytoplasm.

Experiments were performed to see if one solution could accomplish this. As the nucleus and cytoplasm may be composed of different colloids or of different densities of the same colloid, two systems were prepared. The first contained fibrin and gelatin in water equiUbrium, the reaction being acid; the second, two strengths of gelatin under the same conditions. On treating either system with a salt solution, swelUng could be reduced, but it soon became evident that unless the system was kept exactly in one state of hydration, the relative density, consistency, or refractiveness of one moment were not comparable to those of the next, because the different colloids, as well as the colloids of different strengths, absorb and lose water at different rates and the sizes do not change in proportion. A third possibiUty consists of the fact that the nucleus may be more, or less acid than the cytoplasm, under the same conditions, and therefore may absorb different amounts of water. From the fact that nuclei appear brighter in tissues undergoing the first postmortem changes, it seems possible that the nucleus may actually undergo some dehydration while the cytoplasm is becoming hydrated.

PROPERTIES OF COLLOIDS 521

The application of this to Uving cells in Ringer's solution is evident. Acid is constantly being produced and must be neutraUzed or counteracted. As long as the water content of the nucleus and cytoplasm is kept exactly as in the original condition, we are justified in calUng our observations on these cells normal. As soon as the conditions vary, the nucleus will become too tough or too Uquid for the corresponding variation in the cytoplasm. This no doubt takes place in the body within certain Umits, under varying physiological conditions, and accounts for the fact that cells can grow in tissue cultures, after being washed in salt solutions. Too great a variation, however, is fatal, as a result, no doubt, of solution of the cell constituents on the one hand or their coagulation on the other.

SUMMARY AND CONCLUSIONS

It is thus evident that when tissues are removed from the body for examination in the living condition, any change in refraction indicates a dehydration, a gelation or a solution process, which, beyond narrow limits, tends towards irreversible changes in the state of the cell colloids. Both dehydration and gelation increase the refraction. An optical change in the tissue colloids is noticeable only after considerable change in concentration, but is more readily noticeable if the change is from the sol to the gel state or the reverse. The pattern produced on coagulation or fixation is due to a shrinking and to diffusion currents, which cause the granules which are precipitated to whirl around, finally becoming wound up in the parts which coagulate more slowly. The pattern for a dilute and concentrated colloid is the same, the former yielding a more delicate fixation figure than the latter. The physiological state of a tissue at the time of fixation can thus be worked out. The action of physiological salt solutions in preventing swelling due to acid intoxication is not uniform for all the constituents of the cell. Slight variations in strength of the salt solution may give false impressions as to the relative densities of the cell constituents, because of the different rates of dehydration.

522 RAPHAEL ISAACS

BIBLIOGRAPHY

Fischer, Alfred 1899 Fixirung, Farbung, und Bau des Protoplasma. Jena.

Fischer, Martin H. 1915 Oedema and nephritis. N. Y.

Hardy, W. B. 1899 On the structure of cell protoplasm. Joum. of Physiol., vol. 14, no. 2.

H5BER, Rudolf 1914 Physikalische Chemie der Zelle und der Gewebe. Leipzig und Berlin.

Isaacs, Raphael 1916 An interpretation of connective tissue and neurogliar fibrillae. Anat. Rec, vol. 10, no. 3.

Mann, Gustav 1902 Physiological histology. Oxford.

A SIMPLE PARAFFIN RIBBON WINDER

ROBERT T. HANCE Zoological Laboratory, University of Pennsylvania

TWO FIGURES

Several years ago McCliing designed a cylinder revolving on an axle borne on two standards for the purpose of picking up the paraffin ribbon as it left the knife of the microtome and winding it lightly aroimd the drum. The value of such an apparatus is obvious. Long serial sections may be woimd entire onto the cylinder doing away with the danger of scattering the ribbons by a sudden puff of wind as sometimes happens when they are laid on paper in the usual way. It further does away with the necessity of piecing portions of ribbons together on the slide since a continuous ribbon allows strips of exact length to be cut.

The writer designed a machine for the same purpose (described in the Trans. Amer. Mic. Soc, December, 1913) to be used with the Leitz base sledge microtome which carries the knife much higher than most cutting machines. It is not nearly so simple to make, however, as the one to be described in this paper.

McClung*s model has been adopted by one of. the American microscope manufacturers. Since the device is so exceedingly useful and the price of the machine on the market is beyond the 1 mits of the average student^s resources, the writer recently made a ribbon winder that can be built in half an hour and costs approximately eleven cents. It is so simple that every student of histology or embryology can make one for his or her own use.

DESCRIPTION

The dimensions may vary with the materials at hand and the measurements of the machine pictured will be given merely to present a general idea of the size relations.

Base. 8 inches by 3| inches, | inch thick.

Uprights, A one inch dowel stick was used although a broom handle or even a flat board would serve. A quarter inch hole was drilled through the center of the dowel which was then cut across the diameter of the hole. This gave two sticks with a semicircular groove in one end. Each of these sticks was now cut to an exact len^h (in this case 3J inches) and fitted into holes bored in the base the centers of which were 5f inches apart.

523

524 ROBERT T. HANCE


Figure 1

A SIMPLE PARAFFIN RIBBON WINDER 525

Cylinder, The cylinder was made of 2| inch cardboard mailing tube. I have made them of smaller sizes which work just as well. A twenty inch length of this tubing may be purchased for five cents and then cut into five lengths with a hand scroll saw or coping saw. Thin, flat corks, 2k inches in diameter, are bored to take a glass rod and are then fitted into the ends of the tube. A glass rod eight inches long is thrust through the holes. This forms the axle which fits into the semicircular grooves in the uprights as shown in figure 1. The tube is finally wrapped with paper to give a smooth surface and the apparatus is finished.

Some workers prefer the roller to have flanges on either end. These may be easily made by gluing disks of heavy cardboard against the ends of the drum as shown on the lowest cylinder in figure 2. On the


Figure 2

same dnmi is shown a cork fitted to the axle on one side which the worker can add should the glass rod not give suflScient purchase for easy turning.

Instead of a glass rod for the axle a quarter inch dowel might be used. Dr. McClung has suggested that a wooden mailing tube would serve for the cylinder and might even cost less than the cylinder with cork ends described above. Since these maiUng tubes are turned up on a lathe the ends show the centering marks and make it easier to locate and drill the center for the axle than with the corks.

When in use it is necessary to slide the apparatus side ways so that the ribbon is wound spirally. That the mechanism may move smoothly three so-called domes of silence (such as are used on chairs) may be fastened to the bottom. These, however, are not absolutely essential.

The ribbon may be wound over and over itself without danger of sticking. The ribbon on the top cylinder of figure 2 is between 40 and 50 feet long and is unbroken. It may be allowed to remain on the

526 ROBERT T. HANCE

drum in perfect safety until the worker has time to mount !t. Recently I have allowed a ribbon to remain on the cylinder uncovered and subjected to the usual variations in a room for four weeks and at the end of the time mounted the ribbon without trouble and without loss of a section.

A storage rack for the cylinders. When a worker has several blocks of material to cut it is of convenience to do them all at one time. For this purpose a number of drums are necessary and to take care of the drums a storage rack was made as shown in f.gure 2. The dimensions are the same as for the last base described with the exception of the uprights which are twe've inches high. When one cylinder is filled it is placed on the hooks fastened into the upright as shown in the photograph and a fresh cylinder is placed on the working base.

SOME APPARATUS FOR THE MICROSCOPICAL LABORATORY

SIMON H. GAGE AND BENJAMIN F. KINGSBURY

Laboratory of Histology and Embryology ^ Cornell University ^ Ithacaj N. Y,

PIPTEBN figures'

As new needs in the work of a laboratory constantly arise with advancing knowledge, modifications in old apparatus or entirely new forms are developed to meet those needs. Fortunately also, progress in knowledge is usually all along the line, hence the special needs in any given part are met and overcome by advances in other parts.

The devices here described have grown up to meet our special needs, and have been rendered possible, in large part, by recent advances in applied electricity, and by the production of new forms of glass.

In order to present the matter in the most compact and intelligible form, each piece of apparatus is shown drawn to scale.

1. Movable stand for microscopical slide trays, and for reagent boards (fig. 1). This stand is of the correct dimensions for holding 37 of the standard slide trays (fig. 2) or a less number of reagent boards, or a combination of the two.

It is constructed of four posts, a top and three fixed shelves. The three compartments thus produced are fitted with runs so spaced that two of the slide trays are admitted. Next the fixed shelves, three slide trays or a reagent board can be inserted. For ease in moving the stand, the corner posts are supplied with furniture slides or casters.

The sides are covered with fiber-board; the front is open as shown. If desired, a door may be added to enclose the front. If this is done, the door should be so hinged that it can swing around to one side and be entirely out of the way when inserting or removing the slide trays or reagent boards.

In our hands these stands have been found of special help for the following purposes :

a. For classes in histology, or especially in embryology, each group of students is supplied with a stand in which are the histologic specimens or the embryologic series needed for a given period or study.

6. For advanced students or investigators it has been found convenient to have one of these beside the work table to hold the series being worked on, or to contain needed reagents, or to hold series and ribbons of sections that are being prepared. As a tray of ribbons

' Engravings of all the figures were supplied by the authors.

527

528


SIMON H. GAGE AND B. F. KINGSBURY


should be covered by an empty tray for protection against draughts or mechanical injury, it is necessary to have the runs far enough apart to receive at least two trays.

c. Finally, in an investigation in which it is necessary to have at hand for consultation a large number of series, embryologic or histo


Fig. 1 Movable stand for slide trays and reagent boards. (About l/19th natural size), r, reagent boards for glass bottles and jars; si, slide trays.


r- — 1

\i- ! Fig. 2 Standard slide tray (about l/7th natural size).

logic, the stand may be loaded with the needed series from the collection and placed beside the work table where they are convenient and also safe. In a word, it has the same advantages for specimens that a movable, revolving bookcase has for books.

2. Standard slide tray (fig. 2). The slide tray here shown is designed for 60 slides 25 x 75 mm., 10 in a row; 35 slides 38 x 75 mm., 7 slides in a row; 25 slides 50 x 75 mm., 5 in a row. Each row is separated by a low partition from the others. The partitions are only on one face. The smooth face is for ribbons of sections, etc. One side of the frame is grooved and the other tongued so that the trays may be

APPARATUS FOR MICROSCOPICAL LABORATORY


529


stacked, and remain in place. Besides the employment of the trays for general uses about the laboratory, they have been found most economical and convenient for storing the thousands of slides which go to make up a large embryologic and histologic collection. For the collection the slide trays are kept in lockers in which the runs allow the trays to be arranged in groups of five, more or less.

3. An electric paraffin melter (fig. 3). It is composed of a straight sided porcelain pitcher with a metal cover to exclude dust, placed in a galvanized iron receptacle lined with asbestos. A slot in the side for



Fig. 3 Electric paraffin melter (about 1/lOth natural size). /, Porcelain pitcher with a metal top. This is the paraffin container and, as shown, its handle projects from a slit in the warming jacket. The pitcher rests on cross pieces, f , Base supporting the pitcher and its jacket, and containing the electric sockets. In this one two lamps are shown, c, the electric supply cable. Both the metal cases are lined with asbestos paper.

the handle allows that to project, and the pitcher of melted paraffin can be removed from the heater and returned without distiu-bing the heater.

The heater, as shown in the figure, is composed of two parts, the upper with cross pieces to hold the pitcher and the lower to contain the electric bulbs. We have found two 20 watt lamps sufl&cient. No regulator is used, for when the temperature is great enough to melt the paraffin the radiation is sufficient to hold the temperature between 55 and 60°C. We have found this a very convenient laboratory accessory.

4. Electric spreading plate and infiltrating oven (figs. 4 and 5). For spreading parafl&n sections a level, smooth, warm surface on which to rest the glass slide is a great convenience, and it is almost necessary where the slide must remain for 9, considerable time on the warm plate without melting the paraffin, especially in glycogen staining. This plate is heated by an electric lamp of 10, 15, or 20 watts, depending on the season, and no regulation is necessary. If the lamp is atone end as shown in the figure, the plate is warmer at that end and one can get almost any desired temperature by moving the slide over the smooth surface nearer or farther from the lamp (see also fig. 12).

530


SIMON H. GAGE AND B. F. KINGSBURY


The metal box is lined by asbestos board, the spreading plate on top alone being uncovered. The box is placed in an asbestos lined, metal tray. In this tray may also be placed the infiltrating dishes, and a beaker for keeping paraffin melted.

This piece of apparatus is especially useful for the advanced student or investigator as no one else uses it and he can be confident that the tissues or embryos he is working with will not be disturbed or mixed up. It is perfectly safe too, if properly installed. That is, with a porcelain lamp receptacle and with proper insulation on the cable passing through the metal wall. Figs. 4 and 5. Electric spreading plate and infiltrating oven (about l/8th natural size). A, top, B, bottom; a, 6, c, t, lampsocket, separable plug, cable and porcelain insulating tube; r, porcelain receptacle for the lamp; tc, small windowto show whether the lamp is burning; 1 and 2, 5 and 6, pyrex glass infiltrating dishes; 8 and Jl^, metal infiltrating dishes.



Fig. 6 Leveling plate for spreading paraffin sections, and for imbedding (about 1/lOth natural size). L, small gas lamp; T, thick brass plate with three supporting legs, two with screws for leveling.

5. Gas-heated leveling table for spreading sections (fig 6). If one must use a gas or an alcohol lamp for heating the spreading table it has been found by us that a heavy plate of brass with three legs is best, especially if the two at the end are fitted with a screw thread and a check nut to fix the legs in position when the table is level. The thick metal top can be made very smioth, and by heating one end any desired temperature can be obtained. The heavy metal top helps to make the table steady also. The ordinary thin metal tables cannot be made sufficiently level and the heat is not so uniform and continuous. Even if one has an electric spreader (figs. 4 and 5), such a table as this is very desirable for imbedding in paraffin, for the paraffin in the paper

APPARATUS FOR MICROSCOPICAL LABORATORY


531


boxes can be heated to any desired temperature before cooling the bottom and putting in the specimen.

6. Drying oven (figs. 7 and 8). For diying balsam mounted preparations, especially series of sections, and for drying spread sections on the slide, some form of steady heat, and a space free from dust is needed. The drying oven here shown was designed to receive 16 of the standard slide trays (fig. 2). It may be heated by gas or by the use of one or more electric bulbs.



7 8

Figs. 7 and 8 Warming and drying oven (fig. 7, l/25th; fig. 8, l/22d natural size).

Fig. 7 The oven proper (1), is 52 cm. long, 38.5 cm. wide and 45 cm. high, and has a capacity of 16 slide trays. The support (2), is 52 cm. long, 38.5 cm. wide and 25 cm. high. It is wired for four electric lamps.

Fig. 8 The oven (1) and support (2) in section. The arrows indicate the currents of warm air extending into and out of the oven. A, asbestos lining of the outer shell ; B, one of the nimierous ventilating holes from the air space into the oven; C, flue for the escape of the air at the top; H, runs for the slide trays (S); D, door of the support; PT-L, wiring and lamp.

In general construction it is something like an incubator but is designed so that a current of warm air passes from the heating chamber below, up through the air jacket and through holes in the inner wall between each tray into the general space of the oven whence it circulates upward and escapes through a ventilating flue.

The outer wall is lined with asbestos to avoid radiation. For convenience it is composed of two parts, the lower serving as support and container for the heating device.

In the one here shown there are four lamp sockets. One can get almost any desired heat by using one or more of the lamps and by selecting lamps of greater wattage for the higher temperatures. By using a Uttle care one can have almost any desired temperature without special regulation.

532


SIMON H. GAGE AND B. F. KINGSBURY


7. Devices for using artificial daylight in microscopic work (figs. 9, 10, 11, 12, 13, 14).2 At last the great boon of an artificial daylight has been given to the worker with the microscope by means of the recently invented forms of glass light-filters. These have been prepared for the welsbach mantle Ught and for the incandescent electric light, especially the nitrogen-filled mazda lamp.

From our experience with these glass filters, the daylight ^lass should be ground on one or both sides with a very fine abrasive.



Fig. 9 Artificial daylight lantern for a single worker (about l/15th natural size). Af, microscope; A^, nitrogen-filled mazda lamp of 100 watts; a, aperture for the daylight glass. This is about 5 cm. in diameter. The arched covering at the top is open on one side only, at the right in this figure. Compare figures 10 and 11, and for details of structure, figure 12. It will be noted that the table rail is cut out in front to avoid interference with the legs of the observer, and the table drawer is at the right and can be pulled out without moving. The seat is a revolving, adjustable piano stool.

' For a discussion of the requirements for the production of artificial daylight, and the means so far employed, and the uses of artificial daylight see:

Herbert E. Ives. Artificial Daylight. Journal of the Franklin Institute, vol. 177, May, 1914, pp. 471^99. 19 figures.

Simon H. Gage. Artificial Daylight for the Microscope. Science, N. S. vol. 42, October, 1915, pp. 534-536. One curve.

M. Luckiesh. Artificial Daylight. Science, N. S. vol. 42, November, 1915, pp. 764r-765.

APPARATUS FOR MICROSCOPICAL LABORATORY


533


Some form of microscope lamp may be used, or if one is to use the nitrogen-filled mazda lamp, a cheap and efficient lantern can be cheaplyconstructed by any good tinner by using tin and making a square or round pail-Uke lantern. The lamp fixture is attached to the cover as shown in the diagrams, and opposite the filament of the lamp is an aperture in which is placed the dayUght glass filter. As the lamp gives off much heat, the lantern must have large ventilating holes at the bottom and at the top.

On the inside the tin is coated with aluminum bronze. The reflecting surface increases the Ught passed through the glass filter and,


Fig. 10 Artificial daylight lantern for two workers on opposite sides of the table (about l/9th natural size). M, microscopes on opposite sides of the table; a, opposite the apertures for the daylight glass (see figure 12 for the construction of the lantern). The arched covering over the lantern is open on only one side. Compare figure 11 in which the closed sides are toward the observer, while in this figure the open end faces the reader.



Fig. 11 Lantern for artificial daylight for two workers on the same side of the work table (about l/l5th natural size). M, microscopes placed obliquely to face the two openings for daylight glass in the lantern; a, daylight glass in adjacent sides of the lantern. (For the construction of the lantern see figure 12.)


THE ANATOMICAL RECORD, VOL. 10, NO. 8

534


SIMON H. GAGE AND B. F. KINGSBURY


more important still, it reflects the radiant energy instead of absorbing it as would a black surface. This aids in keeping the heat at a minimiun. The outside of the lantern is painted dead-black to avoid unpleasant reflections. If but a single worker is to use the lantern, then only one aperture with the daylight glass is needed (fig. 9).

If two workers on opposite sides of the table are to use a single lantern, then there should be openings with the daylight glass on opposite sides of the lantern as shown in figures 10 and 12. If the two workers must be on the same side of the table or work-bench, then the two openings can be made as shown in figure 11, and the microscopes placed somewhat obhquely on the table.

Finally every laboratory has need of some means for demonstrating


V


n

Gl

a n


V Fig. 12 Lantern for daylight glass in section (about l/4th natura size). /, top of the lantern supporting the lamp. It sets down on the lower part like the cover of a pail, v, ventilating slits. The tin from these slits is turned up at right angles; sc^ the porcelain socket with key switch, and the asbestos insulated cable for the current; N, the 100 watt nitrogen-filled mazda lamp; w, the flat warming plate on the top of the cover. It is a brass plate about three millimeters thick. The temperature on this plate is about 40 to 45° C. and serves for spreading paraffin sections, etc. 2, the lower part of the lantern containing the daylight glass. It is square in cross section as shown by i4, B.

a, daylight glass in the apertures of the lantern. The pieces of glass are about 5 cm. in diameter. I, lugs to hold the daylight glass in position; A, the partition containing the lamp socket seen from below; v, ventilating slits; n, nuts at the ends of the bolts holding the metal clamp for the lamp socket; B, bottom of the lantern. It is about 15 mm. from the table, thus permitting an intake space for air. Paraffin infiltration can be done here if one is careful. (.4, B, are only about l/9th natural size.)

APPARATUS FOR MICROSCOPICAL LABORATORY 535

the most difficult preparations or those showing a special feature in the most perfect manner. To meet this need a round table was constructed and a single lamp installed in the middle (fig. 13). The supply cable is received in the vertical tube about 2 meters above the floor to keep it entirely out of the way when it must be carried for some distance.


Figs. 13 and 14 Demonstration table for artificial daylight (about l/25th natural size).

Fig. 13 shows the table with lamp and its fixture in position. S, standard screwed into a flange in the table; N, the nitrogen-filled lamp of 200 or 250 watts; T, tube for carrying the supply wires to the lamp. The tube passes through a collar at the end of the standard and may be fixed in any position by means of a set screw. The tube is about 150 cm. long and serves to hold the supply wires about 2 meters above the floor. The standard can be unscrewed if it is necessary to take the table through a narrow door.

Fig. 14 shows the table with the 8 shields containing daylight glass in the apertures (a), and also some microscopes in position. Below the table is one of the shields (sh) with the projecting wing at the right to lap over the next shield and prevent the escape of stray light.

In the demonstration table here shown there is room for 8 microscopes. Shields of wood or metal are set up and overlap at the edges to avoid stray Ught. Each shield contains a disc of the daylight glass, and all are lighted by the single nitrogen-filled mazda lamp of 200 or 250 watts in the center of the table (fig. 13). To avoid the very brilliant light extending upward, a disc of cardboard or tin is put over the lamp, but plenty of space must be left around the edges for ventilation. Some holes through the table top under the lamp should be made for ventilation also. In all of the devices shown the Ught is amply sufficient for all powers up to and including the 1.5 mm. oil inunersion.

As pointed out by Brewster nearly a century ago, and still later by Nelson and Wright, the clearest microscopic images are obtained in the microscope when the source of light is relatively small. As these small

536


SIMON H. GAGE AND B. F. KINGSBURY


pieces of daylight glass are practically the sources of light, this prime requisite for a good light is furnished. It will be found in using the daylight glass, as here indicated, that one can utiUze a considerably larger part of the aperture of the objective without the fogging [and loss


•2 Vio't Blo« 

rw' r


f


Qr»«n Yellow Orang* Rad



M .43 .46 47 A9


.63 .66 .57 .59 M 63 , Wav« Ungth in Mierona ^)


Fig. 15 Curve of sunlight, the light from a nitrogen-filled mazda lamp, and the mazda light filtered through daylight glass (172CD).

The numerals on the bottom show the wave lengths, and those on the left the intensity in the different parts of the visible spectrum.

of detail coming with a large aperture when one uses a large source of light like a window.

In figure 15 is shown a curve of the intensity in the different parts of the visible spectrum for sunlight, for the light of a nitrogen-filled mazda lamp, and for the lamp-light filtered through the daylight glass.

The great excess of red, orange, etc., in the unfiltered mazda lampUght shown in this curve is most strikingly illustrated by looking at such a lamp burning out of doors in full daylight.

THE ANATOMY OF A THREE-LEGGED KITTEN

W. B. KIRKHAM AND H. W. HAGGARD

From the Sheffield Scientific School and the Osborn Zoological Laboratory, Yale

University

THREE FIGURES

The animal which is the source of this paper was an ordinary, tiger-striped, female kitten which came into the possession of the laboratory through the kindness of our colleague. Dr. Henry Laurens. It lived in the laboratory for a number of weeks, and when about six months old was killed and dissected to detennine the exact nature of the anomaly.

The female parent of this kitten had previously given birth to other litters of normal offspring, and this animal which we studied was one of a litter of three, of which the other two were normal. Fxirthermore, all the members of a litter born since our specimen are normal. The male parent of our kitten is unknown.

The kitten, aside from its having only three legs, was a hvely, healthy animal, with sleek fur, and a playful disposition. It balanced itself well, planting the right fore foot under the midline of its body to compensate for the missing left limb (fig. 1). There was no external evidence of any part of the left fore limb except a lump at the shoulder which indicated the presence of at least a part of the shoulder girdle, and the skin was drawn smoothly over the shoulder without any sign of puncture or folding.

As soon as the animal was chloroformed the skin was reflected from the left side of the body, and at once there came to view, beneath a thin layer of fascia, a point of bone (fig. 2). The shoulder muscles (deltoids, clavo-brachial, supra- and infraspinatus, teres major and minor, and subscapular) were all

537

538 W. B. KIRKHAM AND H. W. HAGGARD


Fig. 1 Photograph of the three-legged kitten when about six months old. The right fore limb is swung under the body to compensate for the missing left limb.


Fig. 2 Left scapula, rudimentary humerus, and deep shoulder muscles of the three-legged kitten. 1, scapula; 8, spinodeltoid; 9, acromiodeltoid; 10, distal end of the humerus; //, capsule of the shoulder joint; 12, mass of muscle fibers representing the combined latissimus dorsi and the pectorals; IS, anomalous band of muscle fibers encircling the rudimentary shaft of the humerus. The unnamed stumps of muscles appearing below the joint capsule are those of the supraand infraspinatus.

ANATOMY OF A THREE-LEGGED KITTEN 539

normal on the left side of the body with respect to origin, insertion, and innervation. This is not surprising, since the skeletal parts with which they are connected were only slightly abnormal, as described in detail later on.

The appearance of the left fore limb after the removal of most of the shoulder muscles is shown in figure 2. Two muscle anomalies were clearly evident. First, the fibers of the latissimus dorsi, pectoralis ntiajor and pectoralis minor muscles, all three of which were thinner than usual, but possessed of normal origins, became fused into a conamon sheet which was inserted on the inner surface of the humerus, where normally the latissimus dorsi alone is inserted, the normal place of insertion of the pectorals having failed to develop in this humerus. The second muscle anomaly was a mass of fibers which was wrapped completely around the humerus from the point of insertion of the pectorals and deltoids to within a short distance of the tip. Into this mass of muscle ran the musculospiral, musculocutaneous, median and ulnar nerves, but we feel no justification in offering a theory as to the homology of this anomalous ntiass of fibers. In addition to the above mentioned anomahes should be noted the entire absence of any recognizable triceps, biceps or coracobrachial muscles, a fact to be correlated with the absence of their points of origin from the abnormal left scapula and humerus. Possibly these three muscles are among those represented by the unnamed mass mentioned above.

The right and left scapular and humeral bones of the threelegged kitten are shown, drawn to the same scale, in figure 3. The clavicles, which are not represented, were normal on both sides. The left scapula differs from the right in the following respects: (a) the anterior superior border, owing to the narrow neck, forms a greater angle with the axillary border, (b) the spine of the left scapula, perhaps owing to the imdeveloped state of the infraspinatus muscle, is lower than that of the right, (c) the suprascapular notch, on the anterior superior border, is missing, owing to the absence of the coracoid process, (d) the axillary border of the left scapula shows, in correlation with the abnormally narrow neck and head, a decided thickening at its

540 W. B. KIRKHAM AND H. W. HAGGARD

junction with the neck, (e) the neck of the left scapula is only about half the width of that of the right, (f) there is an entire absence from the left scapula of the coracoid process, including the areas for the attachment of the coracobrachialis and biceps muscles and the supraglenoid fossa, (g) correlated with the complete absence of the coracoid process, the glenoid cavity of the left scapula is imperfectly developed, possessing not more than half its usual area, and it is convex rather than concave. In connection with the above mentioned anomahes it should be noted that according to Jayne ('98) the coracoid process and the Fig. 3 Outer aspect of the right and l^ft scapular and humeral bones of the three-legged kitten, all drawn to the same scale. 1, left scapula; 2, left humerus; 5, epiphysial groove; 4, shaft; 6, right humerus; 6, abnormal depression in neck of left scapula; 7, right scapula; 5, epiphysial groove; 5, shaft.

upper portion of the glenoid cavity of the scapula develop in the cat from distinct ossification centers. Also, the work of Braus ('08) and of Harrison ('15) ha^ shown that in Amphibia the anlagen of the shoulder girdle and of the fore Umb are entirelydistinct, since experimental removal of the latter does not prevent the development of the former.

The left humerus of this specimen is chiefly characterized by its representing so small a part of the normal bone. Like the right himierus the left shows a clearly defined epiphysial groove, but on the left humerus there is an entire absence of the lesser tuberosity, together with about half of the articular surface

ANATOMY OF A THREE-LEGGED KITTEN 541

adjoining that tuberosity. The part of the shaft present on the left humerus corresponds to the lateral surface of the normal bone from the anterior mid-line laterally to the deltoid ridge. It is everywhere covered with a thick layer of compact bone, and possesses no marrow cavity, its whole interior being filled with spongy bone.

What caused the production of this three-legged animal must probably await conclusive settlement until such anomalies are experimentally controlled, but taking into accomit all of the available evidence it would appear that after starting to grow out normally from the body the left fore limb bud encountered some obstacle which pressed it against the body, as well as checked its increase in length; such of it as had already grown then imderwent further development into bone, muscle, connective tissue, etc.

MARCH 1916

542 W. B. KIRKHAM AND H. W. HAGGARD

LITERATURE CITED

Braus, H. 1908 Gliedmassenpfropfung und Grundfragen der Skeletbildung. I. Morp. Jahr., Bd. 39, Nr. 194.

Harrison, R. G. 1915 Experiments on the development of the limbs in Amphibia. Proc. Nat. Acad. Sci., vol. 1.

Jayne, H. 1898 Mammalian anatomy. Philadelphia.

KiRKHAM W. B. AND Haggard, H. W. 1915 A comparative study of the shoulder region of the normal and of a wingless fowl. Anat. Rec, vol. 9, no. 2.

Reighard, J. AND Jennings, H. 8. 1902 Anatomy of the cat. New York.

ScHAEFFER, J. P. AND Nachamoffsky, L. H. 1914 Some observations on the anatomy of the upper extremities of an infant with complete bilateral absence of the radius. Anat. Rec, vol. 8, no. 1.


On The Development Of The Biliary System In Animals Lacking A Gall-Bladder In Postnatal Life

Richard E. Scammon Institute Of Anatomy, University of Minnesota

Ten Figures

It is well known that the gall-bladder is always absent in some species of vertebrates. There seems to be no general rule governing the distribution of this peculiarity. Thus in the cyclostomes a gall-bladder is found in the myxinoids but is absent in the fully developed Petromyzon. It is present as a rule in fishes although absent in a few species of sharks. A gallbladder is said to be present always in amphibians and reptiles; it is wanting in a number of birds including the pigeons. Among mamnaals no gall-bladder is found in a number of rodents and in noany ruminants, pachyderms and some other ungulates. It is absent in the Cetacea. In other forms the gall-bladder may or ntiay not be present. This is the case in several species of birds and in a few mammals. With the latter may be included man where congenital absence of the gall-bladder is an unusual anonaaly. In man the gall-bladder alone ntiay be lacking, or more rarely both the gall-bladder and the larger biliary ducts.^

Only three of the forms consistently lacking a gall-bladder are commonly available for embryological study. These are the

This work was done with the aid of a grant from the Research Fund of the University of Minnesota.

The comparative list given above is by no means a complete one. A much more detailed statement is to be found in the Milne Edwards, Lecons sur la physiologie et I'anatomie comparce, T. 6, p. 454, ct seq. Later papers upon the partial or complete absence of the biliary system in man are: Hochst3tter, Arch. f. Anat., 1886; Latham, Journ. of Anat., 1838; Bubenhofer, Anat. Hefte, 1905; and Beneke, Marburger Universititsprogramm, 1905.

543

544 RICHARD E. SCAMMON

lamprey (Petromyzon), the pigeon and the rat. The development of the biliary system in these species will be taken up in the order given above.

PETROMYZON

I have made no study of Petromyzon material but include here a brief summary of the findings reported in the literature.

The early history of the liver in Petromyzon has been studied by Gotte C90), v. Kupflfer ('93), Brachet ('97) and others. A complete biliary system is developed including a large gallbladder and a common bile-duct of considerable diameter. These structures are lined with a high colunmar epithelium which later becomes somewhat flattened and they are surrounded by definite mesenchymal sheaths. The biliary apparatus takes part in the general rotation of the gut which occurs in this form and it persists during the Ammocoetes or larval period.

The later history of the common bile-duct and gall-bladder has been followed out in some detail by Nestler ('90). He found that in an Ammocoetes 13 cm. in length the bile-duct still opened into the anterior end of the mid-gut and was connected distally with the gall-bladder. Near its posterior or intestinal end there was a short segment in which the lumen was almost completely obliterated by the increased height of the lining epithelium. In a specimen a centimeter longer the common bile-duct was still connected with the gall-bladder but its connection with the intestine was lost and its caudal end obliterated. In this region was found a collection of 'Zellballen' or follicles which were connected with the part of the duct still intact. In still later stages the duct proper was found to have entirely disappeared but the follicles persisted. Nestler apparently regarded these follicles as cystic remains of the common bile-ducts or of outpouchings from it. The gall-bladder first became much reduced in size and the epithelium fining it increased in height. The surrounding sheath of mesenchymal tissue also was thickened. Later the structure was completely obfiterated.

These observations were confirmed in the main by Bujor ('91) who, however, accepted the earfier view of Langerhans ('73)

DEVELOPMENT OF THE BILIARY SYSTEM 545

that the follicles observed near the cranial end of the common bile-duct were pancreatic. This view has been confirmed by the recent careful researches of Picqu6 ('13).^

PIGEON

In the pigeon, as in birds generally, there are two ducts leading from the liver to the intestine. Of these the anterior or cranial one is commonly termed the ductus hepato-entericus. The posterior one is divided into two parts by the point of attachment of the gall-bladder. The portion between the hepatic trabeculae and minor ducts and the gall-bladder is called the ductus hepato-cysticus, and the part between the gall-bladder and intestine the ductus cysticus or ductus cystico-entericus. Since the pigeon, as a rule at least, loses its gall-bladder in the course of development, the terminology above is not always an appropriate one. For simplicity the two main ducts will be designated in the following description as the anterior and posterior hepatic ducts.

Brouha ('98) has pubUshed the only information which we have concerning the later development of the liver in the pigeon. He modelled and described two stages, embryos of 59 and 100 hours incubation respectively, in coimection with his extensive study of the development of the liver in the chick. The liver forms from the floor of the foregut and the anterior intestinal portal, but the anlage, which is hollow in the chick, is solid at first in the pigeon. In Brouha's younger specimen the anlage was already partially divided into anterior and posterior diverticula as in other birds. In the older specimen this division was quite distinct and the anlage of the gall-bladder was represented by a distinct expansion of the left side of the extreme posterior end of the hepatic gutter at the point of origin of the posterior hepatic diverticulum or duct (Brouha, '98, pi. VIII, fig. II, V. B.). Brouha did not follow its later history.

' Although Nestler does not describe a pancreas in Petromyzon, he figures and labels as pancreas a group of tubules in the wall of the mid-gut on the side opposite to the follicles associated with the ductus choledochus.

546 RICHARD E. SCAMMON

The youngest pigeon embryo I have examined is 7 mm. in length, of 72 hours incubation (Minn. E. C. 588). In development it stands between the two stages described by Brouha. The anterior and posterior divisions of the hepatic diverticulum are differentiated into short wide ducts which connect with a considerable network of hepatic trabeculae surroimding, in part, the large right omphalo-mesenteric vein. All three pancreatic anlagen are present. The dorsal one forms a pouch which is


Fig. 1 A reconstruction of a portion of the mid-gut, liver and pancreas of a pigeon embryo 7 mm. long (Minn. E. C. 588). Right lateral view, X 50. A./i., anterior hepatic duct; D.p.j dorsal pancreas; G.b.j gall-bladder; L.v.p.y left ventral pancreas; PJi., posterior hepatic duct; R.v.p., right ventral pancreas.

almost as large in cross section as is the gut at this level. The ventral diverticula are smaller pouches which arise from the gut just above the posterior end of the hepatic gutter. A right lateral view of a reconstruction of this region is shown in figure 1. In the reconstruction the hepatic trabeculae are cut away near their origin from the hepatic diverticula. The posterior hepatic diverticulmn is very short and ends by dividing into two parts. The right division becomes continuous with the hepatic trabeculae and represents the ductus hepato-cysticus of other

DEVELOPMENT OF THE BILIARY SYSTEM 547

birds. The left division is the anlage of the gall-bladder and at this time is simply a short rounded swelling. This swelling is due almost entirely to the thickening of the epithelium, there being no true lumen in the cystic anlage (fig. 3).

The posterior hepatic diverticulum lengthens rapidly, extending forward and to the right. The cystic anlage is carried along


Fig. 2 A reconstruction of a portion of the mid-gut, liver and pancreas of a pigeon embryo 8 mm. long (Minn. E. C. 590). Right lateral view X 50. Abbreviations as for figure 1.

with it and is soon located some distance from the gut in the margin of the hepatic parenchyma. The right branch of the bifurcation which connects with the hepatic trabeculae straightens out so that its axis falls in line with that of the main duct and the two form a single tube. The thickening representing the gall-bladder is strongly evaginated forming a small pouch which

548 RICHARD E. SCAMMON

joins with the posterior hepatic duct by a short broad neck. Its walls become thinner and its lumen is thereby widened, but as a whole it increases very little in size. An embryo (Minn. E. C. 590), 8 mm. in length and distinctly older than the one first described, shows these changes in progress. Figure 2 is a


G.b.

Fig. 3 Transverse section of the liver and the duodenal region of a pigeon embryo 7 mm. long (Minn. E. C. 588). X 400. G.b., gall-bladder; H.t., hepatic trabeculae; P.h., posterior hepatic duct; R.v.p.f right ventral pancreas.

right lateral view of a model of this specimen, and figure 4 a section through the long axis of the gall-bladder.

In later stages the entire mass of the liver is much enlarged. Both the anterior and posterior hepatic ducts increase rapidly in length and in caliber, but the anterior one grows more rapidly

DEVELOPMENT OF THE BILIARY SYSTEM 549

than the posterior. Although the liver and its ducts continue to increase in size, the gall-bladder renotains about constant until the embryo reaches a length of 12 to 15 nam. Thereafter the gall-bladder becomes somewhat smaller both absolutely and relatively than in earher stages. Both the lumen and the walls of the sac tend to dinMnish, and folds or sacculations naay appear in its fundic end. The surroimding mesenchyma begins to take the form of a definite sheath with concentrically placed


Fig» 4 Section through the long axis of the gall-bladder of a pigeon embryo 8 mm. long (Minn. E. C. 590). X 400. C.d., cystic duct; G.6., gall-bladder.

nuclei. The neck or true cystic duct becomes elongated and slender. Figure 5, an oblique section of the gall-bladder of an embryo 15.5 mm. long (Minn. E. C. 586), shows these changes in progress.

With the growth of the liver the gall-bladder changes its position, being carried away from the margin of the Uver parenchyma where it was formerly located. In later stages it comes to Ue more and more in the posterior part of the dorsal ligament.

THE ANATOMICAL RECORD VOL. 10, NO. 8

550 RICHARD E SCAMMON

The cystic duct is reduced in caliber and the lumen becomes very small or is occluded. From the specimens which I have examined it appears that the bladder generally becomes separated from the duct and renoains for a period as an isolated thick


Fig. 5 Section through the long axis of the gall-bladder of a pigeon embryo 15.5 mm. long (Minn. E. C. 586). X 400.


Fig. 6 Section through the gall-bladder and posterior hepatic duct of a pigeon embryo 26.8 mm. long (Minn. E. C. 596). X 400. The gall-bladder is the larger of the two epithelial tubes.

walled cyst lying in the dorsal hepatic mesentery close to the posterior hepatic duct. This condition is shown in figure 6 of a section of the gall-bladder in an embryo 26.8 mm. long (Minn. E. C. 596).

DEVELOPMENT OF THE BILIARY SYSTEM 661

Later the gall-bladder degenerates in most cases. In an embryo 46 mm.* in length there was no definite cyst but in the region formerly occupied by the gall-bladder there was found a snaall cluster of deeply staining and degenerating cells which probably represented its ren^iains. There was no trace of this


Fig. 7 Section through the posterior hepatic duct and gall-bladder of^ajpigebn embryo 62 mm. long. X 400.

structure in an embryo 55 mm. in length. A specimen 62 mm. in length, however, showed a small persistent gall-bladder. It was of about the same diameter as the duct to which it was attached and a common musculo-fibrous tunic surrounded the

These measurements are crown-rump length taken with the neck straightened out until in line with the body.

552 RICHARD E. SCAMMON

two structures. A slender and abruptly curved cystic duct connected the gall-bladder with the posterior hepatic duct.


Fig. 8 Three sections showing the size of the gall-bladder in the pigeon embryo. A, embryo 6.8 mm. long (Minn. E. C. 591); B, embryo 8 mm. long (Minn. E. C. 554); C, embryo 62 mm. long. All X 50.

The lining epithelium of the bladder was of a low columnar type sinailar to that of the duct proper, and the lumen contained a small amount of granular precipitate (fig. 7). It is evident

DEVELOPMENT OF THE BILIARY SYSTEM* 553

from this specimen that the cystic anlage may persist in some cases mitil about the time of hatching and perhaps later. The structure is so small that it could hardly be detected by dissection. Attention has ah-eady been called to the great increase in the diameter of both hepatic ducts and the lack of corresponding growth of the gall-bladder. This is shown graphically in figures 8A, B and C which show longitudinal sections of the gall-bladder in embryos 6.7, 8 and 62 mm. in length respectively. These sections are all drawn at the same magnificatioL.

RAT

Helly ('01) while studying the development of the pancreas in the rat examined the hepatic anlagen in an extensive series of elnbryos ranging from 2.2 mm. to 10 mm. in length. He states that in no specimen could he find an indubitable trace of a gallbladder. He noted that in an embryo 2.8 mm. long the primitive ductus choledochus was of considerable caliber and that the ventral portion of its wall appeared somewhat thickened. But, as this thickening passed over directly into the sprouts of the hepatic trabeculae, he did not regard it as a clear anlage of a gall-bladder. Debeyre ('04), also working on the pancreas of the rat, studied a very complete series of embryos from 11 to 21 days old. He states definitely that he foimd no gall-bladder in an embryo 12 days old, and naakes no mention of the structure in his description of later stages.

The youngest rat embryos which I have examined were 1.75 mm. in length and 11 days old (Wistar Inst. Coll. Nos. 15346 and 15347) .* In these specimens the liver is already differentiated as a thickening of the ventral part of the foregut and anterior intestinal portal. The foregut is expanded a Uttle ventrally forming a shallow hepatic pouch. From this pouch extend the earUest hepatic trabeculae which appear as solid sprouts of epithelial cells. Sagittal sections show that the Uver is composed of two parts, an anterior, the true hepatic, from which the

My thanks are due to Dr. M. J. Greemnan, Director of The Wistar Institute, and to Dr. C. A. Heuser, for their kindness in loaning me these preparations for a considerable period.

564 RICHARD E. SCAMMON

trabeculae are arising, and a posterior which gives rise to no trabeculae and which becomes continuous with the wall of the anterior intestinal portal. There is no evident cellular differentiation between these two parts aside from that associated with the formation of the hepatic trabeculae and the presence of a larger number of mitotic figures in the pars hepatica. A median section of the hepatic anlage at this stage is shown in figure 9.

A specimen one day older and 3.36 mm. long (Wistar Inst. Coll. No. 15363) shows the liver anlage in the form of a distinct


Fig. 9 Sagittal section of the hepatic pouch of an Albino rat embryo 1.75 mm. long, II days, (Wistar Inst. Coll. No. 15346). X 400.

pouch sharply marked off from the gut anteriorly and posteriorly with the forward end somewhat recessed. A large number of looping and anastomosing trabeculae arise from the anterior part of the pouch and extend some distance into the surrounding mesenchyma (fig. 10). The posterior part of the pouch gives rise to no trabeculae. In the ventral wall of this portion just in front of the posterior end of the anlagen is a sUght swelling which is marked on the internal surface by a shallow pit (X, fig. 10). It extends but a little way on either side of the mid-line.

DEVELOPMENT OF THE BILIAKY SYSTEM 555

The arrangement of the cells in this swelling suggests that seen in taste-buds but the cell boundaries are not distinct. The nuclei are elongated and peripherally placed while those of the remainder of the hepatic pouch are rounded and more generally scattered. The cytoplasm is a little lighter than that of the remainder of the pouch. This structure may be the trace of a gall-bladder anlage. Its position corresponds to that of the gall-bladder in other manmials and to the thickening previously


Fig. 10 Sagittal section of the hepatic pouch of an albino rat embryo 3.36 mm. long, 12 days (Wistar Inst. Coll. No. 15363). X 400. X, possible cystic anlage.

described by Helly. The arrangement of the cells is distinct but not striking. The structure seems to be present in another embryo of the same age (Wistar Inst. Coll. No. 15362) which is cut in an oblique plane but only in sagittal sections would it be possible to be sure of it. Lewis and Thyng ('08) have observed well-defined pockets resembling those of intestinal diverticula in the hepatic anlagen of pig embryos, but none were found in specimens under 7.8 mm. in length. . This specimen just de 556 KICHARD E. SCAMMON

scribed is of so much earlier a stage that I do not think the two structures can be regarded as identical. I have found no diverticula, such as are described by Lewis and Thyng in other mammals, in the bile-ducts of later rat embryos. Debeyre ('04) has described numerous pancreatic tubules arising directly from the ductus choledochus in rat embryos, but these do not appear until the fifteenth day, and their position differs from that of the swelling described aboveOlder embryos examined show no trace of a gall-bladder. In embryos 5.4 mm. long the posterior part of the hepatic pouch has the form of a short wide duct bifurcated at its distal extremity. The lumen is large, particularly distally, and the cells of the ventral wall have peripherally placed nuclei but there is no cystic anlage. Other embryos (11, 13 and 18 mm. in length respectively) also show no trace of a gall-bladder. In all of these specimens the lumina of the hepatic and common bileducts are of unusual size.

SUMMARY AND DISCUSSION

The history of the gall-bladder is quite different in each of the three forms under discussion. In Petromyzon a complete biliary apparatus is formed and persists throughout the larval or Ammocoetes stage. At the time of the transformation of the larval to the adult form there is a total degeneration of both gallbladder and ducts. In the pigeon the gall-bladder is developed apparently in a perfectly normal way, and later, in the majority of cases at least, is completely lost. The duct to which it is attached, persists and grows to some size. This case is complicated, however, by the presence of a larger anterior hepatic duct which opens independently into the duodenum and is never associated with the gall-bladder. In the rat there is at most but a trace of a cystic anlage in very early stages and this soon disappears. I have observed no common factors which would account for the absence of the gall-bladder in all of these examples. In the rat it is possible that the rapid and early reduction in size of the yolk-stalk which leaves the foregut wall

DEVELOPMENT OF THE BILIARY SYSTEM 557

quite short antero-posteriorly may be a factor in inhibiting the development of the gall-bladder. In mammals generally, as compared with most lower forms, we find a distinct shortening of the liver anlage and a tendency towards consolidation of the cystic and hepatic portions. This condition is well exemplified in Bos tamiis as shown by Pensa's ('13) recent work. The rat may be but an extreme example of this consolidation due to the conditions above mentioned.

The occasional absence of the gall-bladder in man is not explained by these observations. Barring the cases of secondary obliteration of the bile-ducts, it is possible that there are in man two types of inhibition of the development of the biliary apparatus. In one the entire pars cystica of the liver anlage is suppressed. This type would be represented by the rare cases in which both gall-bladder and bile-ducts are absent. In the second only the gall-bladder fundament is lost either early or late in development and this type would be represented by the more common anomaly of absence of the bladder only.

The large size of the bile-ducts is noticeable in both pigeon and rat embryos. This seems to be a compensation for the absence of the usual bile reservoir in the form of a definite sac. A general dilation of the bile-ducts or their local expansion into chambers capable of containing a considerable amount of bile is often but not always found in animals regularly lacking a gall-bladder. This is frequently true in cases of congenital absence of the gallbladder in the human subject. Dilation of the bile ducts after cholecystectomy is also a common observation.

658 RICHARD E. SCAMMON

BIBLIOGRAPHY

Brachbt, a. 1897 Sur le d^veloppement du foie et sur le pancreas de PAmmo coetes. Anat. Anz., Bd. 13. Brouha, M. 1898 Recherches sur le d^veloppement du foie, du pancreas, de

la cloison m^nt^rique et des cavit^s h^pato-ent^riques chez lea

oiseaux. Journ. de TAnat., T. 34. BujORi P. 1891 Contribution a I'^tude de la metamorphose de TAmmocoetes

branohialis^n Petromyzon planeri. Revue Biol, du Nord de la France,

T. 3. Debetre, a. p. p. 1904 Les bourgeons pancr^atiques tardifs. Thtee, Lille. Q5TTE, A. 1890 Abhandlungen zur Entwicklungsgeschichte der Tiere. Heft

V. Entwicklimgsgeschichte des Flussneimauges. Hellt, K. 1901 Zur Pankreasentwicklung der Saugethiere. Arch. f. mikr.

Anat., Bd. 57. y. KuPFFER 1893 Ueber das Pankreas bei Ammocoetes. Mtlnchener med. Abh.,

Reihe VII, Heft 5. Lanqerhans 1873 Untersuchungen ueber Petromyzon Planeri. Diss. Freiburg. Lewis, F. T., and Thtng, F. W. 1908 The regular occurrence of intestinal

diverticula in embryos of the pig, rabbit and man. Am. Jour. Anat.,

vol. 7. Nbstler, K. 1890 Beitrage zur Anatomie imd Entwicklungsgeschichte von

Petromyzon Planeri. Arch. f. Naturgeschichte, Jahrg. 52, Bd. 1.

Also: Diss. Leipzig. Pensa, a. 1914 Lo sviluppo del pancreas e delle vie biliari-extra epatiche in

Bos taurus.' Arch. Ital. Anat. Embriol., vol. 13. PiCQui, R. 1913 Recherches sur la structure et le d^veloppement du pancreas

chez Petromyzon. M^m. Soc. de la Zool. de France, T. 26.

Regarding The Length And Extent Of The Human Medulla Spinalis

Rollo E. Mccotter From the Department of Anatomy ^ Univereity of Michigan

Most of our modern text-books on human anatomy state that the spmal medulla is variable in length and in extent within the vertebral canal and that there is no definite relation between the length of the spinal medulla on the one hand and the length of the vertebral colmnn and the height of the individual on the other. It was with these well known facts in mind that the study of this series of 234 cases was undertaken.

The observations about to be reported were made on the cadaver in the dissecting room. The material had been prepared for dissecting piu^poses by the method described by Professor Streeter in volume 5 of The Anatomical Record. During the regular coiu'se in Gross Anatomy as given in this department the superficial and djeep muscles of the back are identified and removed thereby exposing the entire length of the vertebral canal. The neural arches are then removed. The cut pedicles of the vertebrae and the posterior root gangUa of the spinal nerves are exposed. The dural and arachnoid membranes are opened by an incision along the mid dorsal Une bringing into view the spinal medulla and the root filaments of the spinal nerves. The relation of the neural segments of the spinal medulla with the vertebrae is readily determined.

Because of the impossibiUty to determine with accuracy, macroscopically, the lowest point to which the nervous elements of the spinal medulla extend and the beginning of the filum terminale, the attachment of the lowest root filaments of the fourth sacral nerve was taken as the lowest point in all measurements. To avoid ei:ror the number of ribs and vertebrae were taken in all cases. The cadaver was placed in the prone position on a

559

THB ANATOMICAL RECORD. VOL. 10, NO. JULY. 1916

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LENGTH OF HUMAN MEDULLA SPINALIS 561

flat table top. The length of the spinal medulla here given represents the distance between the lower border of the foramen magnmn and the lowest filaments of the fourth sacral nerve roots.

By consulting table 1 it will be seen that these observations have been classified according to sex and race. In about 77 per cent of the white male subjects the lowest point of the spinal medulla was found to extend between the level of the upper border of the first and the upper border of the second lumbar vertebrae. In two cases the spinal medulla extended only to the middle of the twelfth thoracic vertebra and in two others it terminated on a level with the lower border of the second lumbar vertebra.

There is a gradual average increase in the length of the spinal medulla with the increase in extent down the vertebral canal. However^ spinal medullae of average length may terminate high in the canal, as in one of our series where a spinal medulla 44 cm. in length extended to the level of the middle of the body of the tweKth thoracic vertebra. Short spinal medullae may extend relatively low in the vertebral canal, as in a case where a spinal medulla 37 cm. in length extended to the level of the middle of the body of the first lumbar vertebra. As a rule the long spinal medullae extend relatively low in the vertebral canal. A spinal medulla 50 cm. in length terminated at the level of the lower border of the second lumbar, and one 52 cm. in length extended to the lower border of the first lumbar vertebra. It will be smn also that the length of the spinal medulla and the height of the individual bear no definite relation to one another.

Observations were made on a series of 25 females, in about 72 per cent of which the spinal medulla extended between the level of the upper border of the first and the upper border of the second lumbar vertebrae. As in the male series the short spinal medullae terminated as a rule at a higher level in the vertebral canal than the longer ones. In this group spinal medullae 38 cm. and 40 cm. extended to the level of the lower border of the first lumbar vertebra. As in the male series there was a gradual average increase in the length of the spinal medulla with its

562


ROLLO E. McCOTTER


extent down the vertebral canal and there also appeared to be no relation between the length of the spinal medulla and the height of the individual.

In the third group of 10 negro subjects the spinal medulla in 90 per cent did not extend below the level of the lower border of the first lumbar vertebra. As in the series of white subjects there is a gradual average increase in the extent of the spinal medulla down the vertebral canal with the increase in length of the former. A spinal medulla 42 cm. in length terminated at the level of the middle of the body of the twelfth thoracic vertebra, one 49 cm. in length reached to the level of the middle of the body of the twelfth thoracic vertebra, one 49 cm. long reached to the level of the middle of the first lumbar vertebra and a spinal medulla 47.5 cm. in length extended to the lower border of the second Imnbar vertebra.

TABLE II. Shows the relative lengths of medulla spinalis and vertebral column


NO. OF CASES


ATSRAGB LBNQTH OF VSRTI

AVBRAQB LBNGTH OF


CAL COLUMN


SPINAL CORD



cm.


em.


- 2


68


40.0


4


72


44.5


8


76


45.1


4


81


49.0


In 46 cases the length of the spinal column was also taken to determine if there was any definite ratio between the length of the spinal medulla and the vertebral axis. The length of the vertebral column was determined by taking the distance between the lower border of the foramen magnum, and the tip of the coccyx. By referring to table 2 it will be seen that the length of the spinal medulla is not in definite proportion to the length of the vertebral colmnn in these cases, but that short veilebral columns were always associated with short spinal medullae and that long vertebral columns with long spinal medullae.

By comparing the results of this investigation with those of earUer reports it will be seen by consulting table 3 that the average length of the spinal medulla in the male (44.79 cm.) in our series



Limits of variability

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563

564 ROLLO E. McCOTTER

is somewhat shorter than that given by Pfitzner and Moorhead; that the hmits of variability of cord lengths is greater and that the limits of variabiUty of cord levels in the vertebral canal are about the same as that of the earlier writers.

It will be seen, also that the average length of the spinal medulla in the female (41.8 cm.) is less than previously given and that the limits of variabiUty of cord lengths and levels are nearly the same.

Finally, we conclude from the foregoing:

1. That there is no relation between the length of the spinal medulla and the height of the subject although tall individuals usually have a long spinal medulla and short individuals a short spinal medulla.

2. That there is no definite ratio between the lengths of the vertebral axis and the spinal medulla. However, short vertebral axes always enclosed short spinal medullae and long colunms long spinal medullae.

3. The lowest point to which the spinal medulla was found to extend was to the level of the inferior border of the second lumbar. The highest level at which it was found to end was at the level of the middle of the body of the twelfth thoracic vertebra,

4. The average length of the spinal medulla for the male was 44.79 cm., for the female 41.8 cm. and for the negro 43.4 cm.

LITERATURE CITED

Moorhead, J. H. 1895 Jour. Anat. and Phys., vol. 29, pp. 48-52.

Pfitzner, W. 1884 Uber Wachsthumsbeziehungen zwischen Ruckenmark iind Wirbelkanal. Morph. Jahrb., Bd. 9, S. 99-116.

Thompson, A. 1895 Fifth annual report of the committee of collective investigation, etc. Jour. Anat. and Phys., vol. 9, pp. 40-48.

STUDIES ON CELL DIVISION IN THE ALBINO RAT (MUS NORVEGICUS, VAR. ALBA)

II. EXPERIMENTS ON TECHNIQUE, WITH DESCRIPTION OF A METHOD FOR DEMONSTRATING THE CYTOLOGICAL DETAILS OP DIVIDING CELLS IN BRAIN AND TESTIS

EZRA AI^LEN

The Philadelphia School of Pedagogy and the Zoological Laboratory of the Univer eiiy of Pennsylvania

ONE TEXT FIGURE AND TWO PLATES

(X)NTENTS

Introduction: the purpose of the experiments 565

The scope of the experiments 566

Experiments on fixatives 567

The fixative which proved best 560

Results of dehydration experiments 570

The efficacy of the drop method 570

An automatic apparatus for dehydrating and washing 571

The value of anilin oil as a substitute for the higher alcohols 574

Results of clearing experiments 575

• Range of oils tried 575

The best clearing oils 575

Detailed account of the technique as finally developed 576

Discussion of the results of the technique 577

Cytological 577

Volumetric 580

Conclusions 583

Bibliography .♦ 585

INTRODUCTION

This paper deals with a series of experiments on technique by which a method has been developed for demonstrating the details of mitosis in the central nervous system and testis of the albino rat without producing distortion of the other parts of the tissues. The work was begun because of difficulty experienced

565

566 EZRA ALLEN

in positively identifying dividing cells while conducting experiments on influencing the rate of cell division in the cerebellum of the albino rat. In the external granule layer of a sick individual seven days old, I found a large number of peculiar cells which oftentimes resembled certain phases of dividing cells. The tissue had been fixed in Carnoy's fluid and treated according to the method previously used in my study determining the duration of cell division in the central nervous system of the albino rat (Allen '12). At Professor McClung's suggestion I attempted to secure a technique which would insure the cytological details desired.

King CIO), after a thorough and exhaustive set of experiments on various standard fixatives for brain tissue, showed that for general histological purposes Ohlmacher's fixing fluid followed by double imbedding with celloidin and paraffin gave the best results. This method, however, fails to prevent the chromosomes from fusing, and consequently was inadequate for the purpose in hand.

My experiments grew to a series of tests dealing with each step in the preparation of sections, finally resulting in a method which not only clearly differentiates dividing cells in all stages, but also prevents fusing, or 'clumping,' of the chromosomes, a distinct gain in the technique for mammalian tissue. Other workers in the Zoological Department of the University of Pennsylvania are experimenting on other tissues. The results of their work will also be published, so that this paper forms one of a series on the problems of technique.

THE SCOPE OF THE EXPERIMENTS

It is needless to record all the details of the work done or of the results obtained. The general plan and scope of the work are as follows: While a few experiments were made on a pure trial and error basis, with the hope that something desirable would result, most of them were carefully planned along the line of modifying approved methods. The success of each experiment was measured by two criteria: (1) the isolation and definition of the

CELL DIVISION IN THE ALBINO RAT 567

chromosomes and (2) the 'general fixation.' The term 'general fixation' covers the points of shrinkage or swelling of the whole mass, and the degree of distortion of one part or of one tissue. A good general fixation dep'^ribes the condition of a preparation which has suflfered only flight changes in total volmne, either by shrinkage or swelling, and in which the individual parts maintain their normal relationship of volume and structure; that is, the connective tissues are not torn, the limiting membranes retain their size and smooth outUnes, while the cells show no vacuoles about them nor cjrtolysis of any kind.

For a long time it looked as if excellence of general fixation would have to be sacrificed to distinctness of the chromosomes, so that I was particularly pleased when the best chromosome differentiation obtained was accompanied by the best general fixation. A comparison of figures 2, 9, 10, 13 and 15 with figures 4, 7, 12, 14 and 17 respectively will show the contrast between poor and good preparations according to the standards just described.

EXPERIMENTS ON FIXATIVES

The first line of investigation followed was that of modifying the proportions of constituent parts of Bouin's fluid, since this fixative had proved so excellent for general purposes. The proportions are given in table 1. The first set of experiments was with the first six modifications shown in table 1. One promising fluid appeared in modification no. 2. It failed, however, upon further trial to yield constant or fully satisfactory results, though it seems to be a^better fixative for albino rat tissue than Bouin^s combination.

After thorough trial of the picro-formol-acetic combinations as far as no. 14 in the table, the following fluids were usedrFlemming^s, Worcester's, Lavdowsky's, Carnoy's (often called van Gehuchten's), Ohlmacher's, King's potassium-bichromate-aceticsublimate, and Tellyesniczky's. Urea was added to each in varying proportions for studying its effects; fixatives were made up in normal salt solution; different temperatures — 0°C., room and 30 to 40°C. — and different fixation periods were tried with any

568


EZRA ALLEN


TABLE 1

Shovoing the variolic combincUiona of picric add, formol, acetic acid, corrosive Bvblimate, chromic add and urea employed. Fluids are measured in cubic centimeters; solids, in grams; the percentages are thus indicated


SOLUTION


PICBIC

acid;

SATU BATBD

AQUBOUS

SOLUTION


POBMOL


GLACIAL

Acmnc Aao


COBBO SIVB 8UB LIMATB

saturated;

AQUBOUS SOLUTION


salt; crystals


urba; crystals


CHROMIC

acid; crystals


WATER


Bouin'


75


25


6 No. 1


75


15


6 2


76


10


10 3


76


15


10 4


75


10


5 6


76


6


5 6


' 75


10


10


5



7


50


10


10



26


8


90


6


5



0.9


0.5 9


75


10


10



0.9


1.5 10


75


15


10



0.9


1.5 11


75


10


10 0.1



100


12


76


10


10 1.5



300


13


75


25


6 4.0



300


14


75


25


5



0.9


0.5 15


76


25


5 2.0


1.5



16


75


20


3 1.0


1.0



17


75


5


1 1.0


0.5



18


75


20


7 0.3


0.5 1 Lee ('13).

fluid which seemed hopeful on first use. Various strengths of formalin, with and without sugar, and different combinations of acetone and formaUn were also tried. See table 2.

A second promising fluid resulted from the addition of a little dilute chrome-acetic mixture (one of the parts of Flemming's fluid) to the Carnoy fluid. The general fixation was excellent and the dividing cells in the brain showed better cytological detail than with anything else tried up to that time. This combination was therefore tested. The constituents were mixed in different proportions; and different temperatures, periods of fixation and quantities of urea tried out. Chromic acid yielded better and better results upon the chromosomes as its percentage was increased, but the mitotic figures were still not satisfac CELL DIVISION IN THE ALBINO RAT


569


TABLE 2

Showing some of the various combinations of fluids used based upon either formol, acetone or Carnoy. The measurements are the same as in table 1


SOLtJTION


PORMOL

(NBtl t»al)


ACIJTONB


ALCOHOL (100 PBB

CBNT)


CHLOBOPOBIC


GLACIAL

ACBTIC

ACID


CHROMIC

acid;

CBT8TAL8


CORBO nvB

8ITBLI matb;

CBTSTALS


ubba;

CRYSTALS


suoab;

CBTSTALS


WATBB


No. 1 1 McClendon/


10



2.0


88


2


10


1


1.0


90


3


10



0.5


90


4


1



1.0


98


5


2



1.0


98


6


5



1.0


94


7


2



48


50


8


1


90



10


9


10


50


1.0


40


10


5


50


1.0


40


11


10



50


45


12


5



50 . 1.0


45


Carnoy 13 60 60


30 30


10

5


1.0



14 98 5.0


2


15 60


30


10


1.5



2 16


5



60


30



1.5



2



2


17 60


30



1.5



2



2


Ohlmacher 80


15


5



Saturation


tory for accurate work when the concentration had reached a point in which the rest of the tissues were torn and distorted worse than the results of Flemming's fluid shown in figure 13. Some of the combinations used are shown in table 2, nos. 13 to 17. Formol and acetic acid both swell tissues. For this reason formol was substituted for the acetic acid in several trials, only one of which is shown in the table (no. 16, table 2).

Finally one half of a seven day brain was put into the combination of chromic acid and Carnoy's fluid which had given the best results, and the other half in some Bouin's fluid to which I added at the same time some chromic acid and urea. The half placed- in the latter fluid showed all the good points of general

570 EZRA ALLEN

fixation gotten by Bouin's fluid with the addition of well diflferentiated chromosomes, while the other half was useless in comparison. Further trials of the new fixative (no. 15, in table 1) yielded practically constant results and later when appUed to the testis gave the material from which figiu-es 12, 14 and 17 were made. The exact proportions used appear in table 1, no. 15. This fixative will henceforth in this paper be spoken of as the picro-formol chrome-acetic in urea fixative, or for convenience 'B-15.'

DEHYDRATION

The well recognized necessity of a very slow change from a fluid of one density and osmotic pressure to another which differs in these respects, in order that cjrtolysis may be avoideid, led to a consideration of how this change might be easily and effectively accomplished. Professor McClung while working on grasshopper testis found that if the higher grades of alcohol were added drop by drop better results were obtained than by transferring the tissue from water to 35 per cent, 50 per cent, 60 per cent alcohol, etc. To accomplish this purpose he used the principle of capillarity in a cotton cord or a fine glass siphon. This method, however, when appUed to more than two or three cubic centimeters of fluid did not insure mixing, the Ughter liiquid remaining on top of the heavier. Even when the Ughter Uquid is introduced through a tube to the bottom of the heavier, it rises immediately and accumulates on top of the heavier in an independent layer and remains there for days, as is readily shown by introducing colored alcohol in the manner described. I have found that a flow of air gently and rapidly mixes the fluids of different density. The ciurent may be obtained from a pressure bottle when compressed air is not available in the laboratory. The apparatus devised for this purpose is shown in textfigure 1." It consists of two bottles of equal volume, one for water and the other for air pressures, marked W.B. and PB. One of these is of the aspirator type. The remainder of the apparatus is a supply bottle for storing the alcohol or other replacing fluid, S,B.y also fitted with bottom stopcock; a container

CELL DIVISION IN THE ALBINO RAT


571


for the tissue, C, fitted with a siphon opening from the side, S; a jar for receiving waste; glass and rubber tubing for connections as shown; corks and screw clamps or stopcocks of small bore, as indicated in the figure.

The size of the water and pressure bottles should be two or more hters, in order that Uttle attention need be given the supply of air. A variety of sizes of the container is desirable, since a small amoimt of tissue needs only a small amount of fluid. For two or three pieces of 0.5 cc. volume a vessel of 30 cc. total


/='H



^SSSSl Drying bottle


capacity is ample. Shell vials or shallow stender dishes are very suitable. One should avoid narrow, tall containers as handling the tissue in them is difficult. The supply bottle for alcohol may be large or small, depending upon the quantity of fluid needed. Where large quantities of tissue are being dehydrated or changed, several supply bottles will be convenient, one for each kind of fluid that is in use.

If aspirator bottles are not available, ordinary bottles fitted with siphons through their corks may be substituted.

The connections should be made as shown in the diagram. The cork for P.B. fits tightly; that for C is loose-fitting, or else the

572 EZRA ALLEN

hole for one of the tubes is slightly larger than the tube, to allow for escaping air. The cork for S.B. must also fit loosely.

To operate: after seeing that all connections are adjusted, fill the water bottle W.B. and open its stopcock slightly until the water begins to drop into the pressure bottle. .As soon as the air pressure in this bottle overcomes the pressure of the fluid in C, bubbles will emerge from the mouth of the air tube and pass to the top of the fluid. A steady stream of air instead of bubbles is better, and may be produced by drawing out the end of the air tube into almost a capillary and clamping its rubber tube almost shut, thus forcing the air to accumulate under pressure in P.B.

Next start the alcohol dropping at the desired rate. The air and alcohol will continue to flow imtil their supply is exhausted in each case or until stopped by the operator. The siphon will remove the excess of fluid, so that the concentration of the alcohol is steading rising in C The waste jar must be large enough to acconamodate the excess fluid.

A two-Uter bottle for W.B. if filled will nm all night at the rate of a drop a second. When the water has nm over into the pressure bottle it is poured back into the water bottle and thus used repeatedly.

When the apparatus is used for higher alcohols or for oils, the air should be dried by passing it through a tube containing calcium chloride or through sulphuric acid. The latter method is easily accompUshed by a gas washing bottle, shown at one side in the diagram.

As a concrete illustration, suppose that the tissue is to be raised from water to 75 per cent alcohol. It is placed in the container C and covered with the fixing fluid or with whatever fluid it may be in at the time. The air and alcohol are set flowing as previously described. Sometimes a difficulty appears in connection with the^ stopcocks or pressure clamps. Stoppage will occur in the rubber tubing so clamped after the clamp has been on for some days. Consequently the clamp must be moved occasionally. The stopcocks need turning wide open occasionally and then readjusted to the desired rate of flow.

CELL DIVISION IN THE ALBINO RAT 573

The rate of dropping is affected by the pressure of the water above the stopcock in the water bottle, this pressure varying directly with the quantity of water. A httle experimenting will usually suffice to regulate the flow satisfactorily.

If the tissue is very deUcate and is seen to be moving about in the container, the rate of air admission may be slowed or a glass sUde interposed between the air and the tissue. This should fit loosely enough to allow of free interchange of liquids about its edges but to prevent rapid currents at the same time. Another excellent scheme is to put the tissue into a smaller bottle, the sides of which have been perforated with numerous holes. This receptacle prevents the air currents from forcibly disturbing the tissue while at the same time it permits very free circulation of the fluids.

The siphon may be adjusted to discharge constantly, once it has started, or intermittently. If its discharge is at the same level as the point where it leaves the container (as shown in the cut), the flow will be constant. If the descending arm is longer than the ascending arm, thus lowering the discharge point, the flow will be intermittent.

By measuring the amount of water in the container at the beginning, one can readily calculate how much alcohol of a given strength will be required to bring the fluid in the container up to 70 per cent, or any other percentage desired. This quantity can be put into the supply bottle S.B.

The percentage of alcohol to be used at first varies somewhat with conditions. If the tissue is small compared with the volume of fluid in the container, 95 per cent may be used. On the other hand, if the quantity of fluid is small compared with the tissue it is better to start with 50 per cent, or 70 per cent and continue this until the tissue is in 50 per cent when 95 per cent may be run in. The new fluid may be introduced with constantly increasing speed as its concentration rises in the container.

The same apparatus is very convenient for washing out picric acid or corrosive sublimate, as the current of aii* hastens the process considerably, since the agitation constantly removes the layer of fluid which exudes from the tissue, and the interchange

574 EZRA ALLEN

is thus not dependent solely upon diffusion of fluids. If desired, alcohol of the same strength may be kept running into the container from the supply bottle; or the fluid in the container may be changed at intervals.

For passing to the higher alcohols and clearing oils, the same apparatus is employed in the same way as for bringing the tissue up to 70 per cent.

THE USE OF ANILIN IN PLACE OF THE HIGHER ALCOHOLS

While carrying on these experiments at Woods Hole, Dr. E. E. Just called my attention to Suchannek's ('90) use of distilled anilin oil as a substitute for the higher alcohols, and spoke of his own success in its use on heteronereis eggs. I tried it after 75 per cent alcohol. It produced no appreciable change in the volume of the tissue even if left in it for twenty-four hours, and I have since employed it entirely except after a Flemming fixation. Tissue so fixed is turned very black by it, even though previously very thoroughly washed.

Anilin does not mix with paraffin but is readily followed by any clearing oil., I have had no difficulty with its use. On the other hand, it seems to improve the tissue for sectioning and staining. By one distillation (the distillation point is 184°C.) anilin becomes a clear, pale-yellowish fluid. This keeps well if stored in the dark. The Kahlbaum anilin comes a pale red and can be used without distillation, but may darken the tissue somewhat.

King ('10) found that double imbedding prevented shrinkage of the nerve cell body. She says:

When a brain that has been fixed in Ohlmacher's solution is imbedded in paraffin after being cleared with chloroform or with any other substance commonly used for this purpose, there is invariably a shrinkage of the cell body, .... and a condensation or vacuolization of the cytoplasm. If, however, the brain is imbedded in celloidin-paraffin, . . . . there is no shrinkage evident anywhere in the cell.

referring to large cells in the cerebral cortex. I am confident that the use of anilin obviates this difficulty, and consequently avoids the shrinkage due to the higher alcohols essential to the

CELL DIVISION IN THE ALBINO RAT 575

use of celloidin, and furthermore gives perfectly flat sections. The photographs in figures 3 and 4 are typical of the appearances produced by my method of treatment. Neither large nor small cells show vacuolization of cytoplasm nor shrinkage of the nucleus.

CLEARING

Xylol, as is well known (Lee '13) shrinks tissues considerably. I experimented with chloroform, oils of cedar, cassia, wintergreen and bergamot. Each produced less shrinkage than xylol. The effects in each case were measured on the whole mass of tissue, on the smoothness of outline of the individual tubules of the testis, and on nerve cells. The least shrinkage occurred with bergamot; wintergreen is a close second. Cassia (cinnamic aldehyde) seemed excellent for the testis, but since this does not mix with paraffin it must be followed by a paraffin solvent. It has the advantage of mixing with 85 per cent alcohol freely, and it is therefore useful after Flemming to avoid the higher alcohols. The pure synthetic preparations of all the oils are better than the natural products. The following illustrations serve to show the extent of shrinkage during the clearing process.

cc.

Volume of brain when put into xylol . 65

Volume of brain 2} hours later 0.60

Volume of brain 23 hours later 0.50

The loss in this case was 23 per cent. In some cases it was as high as 25 per cent.

The effects of bergamot oil were compared with those of a special cedar oil which has the merit of mixing with alcohol in any proportion. I passed the halves of the same cerebellum through these oils respectively, and then measured ten undeveloped Purkinje cells from each preparation, carefully selecting those cut in the same way. Drawings were made with the aid of the camera lucida. The mean diameter of the ten fixed in bergamot oil was 9 units; that of the ten fixed in the special cedar oil was 8.1 units, a loss of 10 per cent. The diameters of the latter were consistently smaller throughout.

TBS ANATOMICAL RECORD, VOL. 10, NO. 9

576 EZRA ALLEN

Testis fixed in Flemming seems less susceptible to change in volume at this stage in the technique than when fixed in one of the picro-formol-acetic combinations, an indication that this fixative (Flemming) has brought the tissue more nearly to its limit of shrinkage.

DETAILED STATEMENT OF THE METHOD

Formula for the fixative. (B-15; table 1, no. 15)

cc.

Picric acid, saturated aqueous solution 75

Formol 25

Glacial acetic acid 5

In this fluid dissolve at time of fixing 1.5 gram of chromic acid in crystals, and then 2 grams of urea in crystals.

Fixation. Heat the fixative to 37° or 38°C., place the tissue in it and hold at that temperature. Pieces of brain 0,5 cc. in volume fix in one hour; pieces of young testis of the same or less volume require a httle longer. The adult testis should be given two to three hours, depending on the age of the animal.^ The external membranes of the tubules and the connective tissues become, with age, progressively less easy to penetrate. Plenty of fixing fluid is used. It is agitated several times during the fixation. To secure quick penetration of the testis it is necessary to cut the outer covering (the tunica albuginea), and in addition snip the tubules apart with scissors.

A low temperature paraffin bath is convenient for keeping the fluid at the desired temperature.

Dehydration. The fluid and contained tissue are cooled to room temperature and slow, gradual dehydration begun with alcohol as described previously (p. 578). The rate of dropping should be about one drop per second unless the quantity of fluid is very small, when it should be less rapid.

After this fixative, it is desirable to bring the tissue up to 75 per cent within an hour (for pieces 0.5 cc. in volume) if the tissue is soft; harder tissues need a little more time.

The young mature testis is better for the study of spermatogenesis than the old.

CELL DIVISION IN THE ALBINO RAT 577

Since the picric acid must be washed out, fresh 75 per cent alcohol to which a few drops of a saturated aqueous solution of hthium carbonate has been added is placed on the tissue in the supply bottle, and the flow of air started very slowly. By agitation and constant drainage, the time for washing out picric is lessened to one-third that required when the tissue simply soaks in the same solution, which is renewed from time to time. Very small pieces will wash in a few hours. Large pieces must be left until no yellow appears in the fluid.

It is desirable to keep the tissue in alcohol as short a time as possible. As noted in table 3, the shrinkage that occurs in getting the tissue into this grade of alcohol is considerably more than occurred in the fixative.

The anilin oil is started dropping slowly.' It is heavier than alcohol so that a stronger current of air may be needed to insure quick mixing. The eye will readily detect the flow of air necessary. When nearly pure aniUn is reached, the tissue is left in pure anilin until it is clear Uke amber.

Clearing. The oil follows by the same method. The tissue should be changed once after being placed in the pure oil, as it is quite essential that all of the aniUn be removed, or poor infiltration by paraffin results.

Infiltration with paraffin. This is accomphshed by warming the oil and tissue sUghtly and adding to it every ten minutes a few drops of melted paraffin, which is then thoroughly mixed with the oil by a pipette. This is continued until the mixture is 85 per cent or 90 per cent paraffin. It is then transferred to the melted paraffin. I use one melting point only — 52° or 55°C.

If bergamot oil has been used, at least four changes of paraffin should be made, the tissue remaining in each a half hour, and in the fifth paraffin at least one hour. Testis requires longer. I cannot see that twelve to twenty hours in paraffin of 55°C. injures brain or testis.

Imbedding. This is done in the usual way.

578 EZRA ALLEN

RESULTS OF THE METHOD DEVELOPED

Cytological. As stated in the introduction, the object of the experiments was to obtain a method which would positively differentiate dividing cells in the brain of the albino rat. This object was attained. In addition, the same method has given the most satisfactory preparation of the testis yet obtained for the study of spermatogenesis, as simultaneously with the work on the brain tissue the testis was also tested. On this latter material cold Flemming's fluid (0°C.) to which was added 2 or 3 per cent urea gave some very admirable results, but of many testes fixed only two came out well, and these were good only in a few places, while the tubules that showed chromosomes in good condition for study were invariably badly torn, as is illustrated in figures 13 and 15. Figure 10 was made in order to show that this torn condition is general and not confined to one or two places. All stages of spermatogenesis are shown in the tubules of figure 10. It is evident that any conclusions relative to chromosomes drawn from tissues so badly distorted should be fully controlled by a preparation in which no such distortion is to be seen. It is beUeved that figure 14 illustrates such a preparation.

In the Flemming-fixed material, the early prophases seem more sharply defined, as if the chromatin were compressed into more of a dense thread than in the picro-formol-acetic preparations. It is likely that the latter fixation preserves them in a more nearly normal condition. A^Tien chromic acid and urea are added to the picro-formol-acetic mixture the metaphase chromosomes of both spermatocytes are held in some way from clumping, just as they are in the best Flemming preparations, while the prophase threads are not hardened down appreciably. The chromatin maintains its diffuse character between the chromomeres. It may be that on this account the study of these prophase chromosomes will be easier in Flemming-fixed material if one desires to differentiate a single chromosome in the large number possessed by rat and some other mammals, but for the study of the structure of the individual chromosomes, it would seem that the

CELL DIVISION IN THE ALBINO RAT 579

B-15 preparation is decidedly preferable. I shall have more to say upon this subject in the paper bearing upon the spermatogenesis of the albino rat.

In this paper, however, I wish merely to show the possibilities of isolating chromosomes in the metaphases of spermatocytes by the addition of urea to Flemming's fluid and fixing at 0°C. Figure 15 is to be contrasted with figure 16, where the chromosomes are so clumped as to be hopelessly unresolvable with the best microscopic combinations available. Figure 17, prepared by the B-15 fixative, does not show its full worth in this figure. I had no sUde of that preparation which showed several metaphase conditions in polar view, and was compelled consequently to use lateral views at this magnification, trusting that the higher magnification shown in figures 14 and 14 a would bear witness to the real excellence of the preparation. Furthermore only a few cells in figure 17 focussed at the same level, while many did in figure 15. The excellence of general fixation comes out clearly in both figures 14 and 17.

In this connection, attention is called to the apparent excellence of fixation in figure 11. This contrasts favorably with figure 10. Examination of figure 16, however, which is made from the same sUde as figure 11, shows the clumped condition of the chromosomes and some spaces about the cells. Neither of these conditions is manifest in figure 17. The spaces near the center of this last figure are the normal lumen of the tubule.

Consider now the cytological details of the brain tissue as illustrated in the figures. The superiority of the general fixation shown in figures 3 and 4 over that shown in figure 2 is manifest. The spaces about the cells in figure 2 do not appear in tissue fixed by B-15 and subsequently treated by the method described in this paper. Tissue fixed in Carnoy's fluid, in formalin, in alcohol and in Ohlmacher's fluid show these spaces throughout. Figures 3 and 4 are typical of the preparations by the method which I have employed in connection with the picroformol-chrome-acetic-urea fixative. No such spaces appear about cells of any size. Large cells have been purposely chosen for the figures, as they would show such spaces most strikingly if present.

680 EZRA ALLEN

In these cells the characteristic fibrillation of the larger protoplasmic process appears distinctly. Fibers are also distinct between the cell bodies. The chromatin masses in the nucleus are more deUcately treated than in the other fixations.

The equal action of B-15 throughout the tissue is illustrated in figures 6 and 7. The latter figure shows the outer part of the external granule layer, or where this layer Ues on the outside of the cerebellum, while figiu-e 6 shows the same layer deep within a sulcus, as at /, figure 1. The cells in each region seem alike. Those in figure 9, on the contrary, are markedly different from those in figure 8. Figm-e 9 shows the outer cells and figure 8 the deep-lying ones. The appearance in figure 9 is characteristic of Carnoy, alcohol and Ohlmacher preparations, which therefore prove very unsatisfactory fixatives if one desires to study this layer of cells throughout.

The demonstration of dividing cells in photographs was found very difficult for several reasons. One is that the chromosomes are very small and numerous, and that they do not form a flat equatorial plate. A photograph of any equatorial plate would therefore appear to show some fusing. In fact, if one wishes to be absolutely sure of the number of chromosomes, the cell must be sUghtly incUned to the plane of section, since in a strict polar view one or more of the chromosomes may be entirely hidden by another chromosome. Figm-e 5 shows a dividing cell in early anaphase, in which some of the chromosomes may be seen separately at this focus. In the preparation they are perfectly distinct from each other. In figure 6, one of the daughter nuclei of a dividing cell in late anaphase is seen (D. c. in lower part). This stage is the most critical one in the fusing of the chromosomes. In all other fixatives they mass into a solid body at this stage. In this figm-e, however, they will be seen as distinct, although too small to be isolated in the photograph. The dividing cell in the upper part of this figure is out of focus, but it can be seen that its edge is not as hard and smooth as the dividing cell in figure 8, which is typical of all other fixatives except B-15.

CELL DIVISION IN THE ALBINO RAT


581


SHRINKAGE DUE TO FIXATION

Several brains were weighed to show the change in volume due to fixation. The data appear in table 3. The method of weighing is as follows: as soon as the brain is removed it is dipped for a few seconds into the fixative, then removed and drained on filter paper. It is then placed in a weighing bottle and the weight of brain and bottle determined. The brain is then removed and

TABLE 3

Showing loss in weight of different-aged brains in the fixing fluid which gave the, best results, viz., picro-formol chrome-acetic urea. See table 1, no. 15


NUMBKB or BAT


SBX


KGE IN

DATS AFTER

BIRTH


NUMBER

OP HOURS

IN FIXING

FLUID


WEIGHT IN

GRAMS

BEFORE

FIXATION


WEIGHT IN

GRAMS

ArTER

FIXATION


LOSS OF

WEIGHT IN

FIXING

FLUID


PBRCBNT AGB LOSS OF

WEIGHT IN

FIXING

FLUID


133


d^


2



0.2860


0.2640


0.0220


7.6


119


9


3



0.2992


0.2770


0.0221


7.4


117


&


7



0.6605


0.6024


0.0581


8.8


118


9


7



0.6799


0.6206


0.0593


8.7


135


&


8



0.9433


0.8969


0.0464


4.9


136


9


8



0.9271


0.8871


0.0400


4.3


137


d^


25


li


1.3590


1.2759


0.0831


6.1


138


9


25


u


1.2848


1.2031


0.0817


6.4


139 •


&


90


2


1.7927


1.6598


0.1329


7.4


139 48


1.7927


1.4398


0.3529


19.6


140


&


63


2


1.6050


1.4852


0.1198


7.4


140 48


1.6050


1.3611


0.2449


15.2


the bottle weighed before cleaning. The difference between the first and second weights is taken as the brain weight. If the brain is weighed as it comes from the animal, its condition is not the same after being in the fixing fluid, as some of the fluid will remain on its surface. This may be sufficient, even after draining, to counterbalance the loss of weight due to the action of the fixative.

The specific gravity of B-15 is about that of the brain. Any loss of weight while the tissue is in the fluid is therefore due to loss of water. King (^10) found that the loss of weight during fixation in Ohlmacher's fluid varied from 8 per cent to 15 per cent, when left in the fixing fluid from 1 to 6 hours. The 8 per cent

582


EZRA ALLEN


loss was on the brain left in 6 hours. She concludes that length of time in the Ohlmacher fixing fluid does not make much difference in the shrinkage. Her method of calculation, however, was to weigh the brain after fixation and compare this weight with the computed weight for a rat of this size and body weight. The percentage losses of weight shown in table 3 run from 4.3 to 8.8 per cent when the tissue is left in the fixing fluid one to two hours, but jump to as high as 19.6 per cent when left in 48 hours. Age seems to make Uttle difference, if any, when fixation time remains the same.

SHRINKAGE DUE TO ALCOHOL

It is well known that alcohol shrinks tissue. Table 4 shows that this is considerable if the tissue remains in the alcohol long, running as high as 17.6 per cent when left in 48 hours. These figures show the desirability of passing the tissue through alcohol as quickly as possible.

Table 4 shows also the total loss in weight due to fixing fluid and alcohol up to 60 per cent. These figures run on the whole a Uttle less than those given by King (40) for Ohlmacher fixation followed by 48 hours in 70 per cent alcohol, these running from


TABLE 4


Showing typical losses in weight of brains in alcohol after fixation as described in table S, and the total loss of weight due to the effects of the fixing fluid and the alcohol, this loss increasing with the length of time the tissue is left in each fluid


NUMBER OF RAT


8EX


A0£ IN DAYS AFTER BIRTH


NUMBER OF HOURS IN ALCOHOL


WEIGHT IN

• RAMS WHEN THE ALCOHOL

WAS STARTED DROPPING


WEIGHT AT

END OF THE TIME INDICATED IN COLUMN 4


LOSS

OP

WEIGHT


PERCENTAGE LOSS or WEIGHT


TOTAL LOSS

or WEIGHT

IN FIXING

FLUID AND

ALCOHOL

(60 PER

CENT)


TOTAL PBHCENTAGB

LOSS or

WEIGHT


133


&


2


2


0.2640


0.2360


0.0280


10.6


0.0500


17.4


133 48


0.2640


0.2294


0.0346


13.0


0.0666


23.6


135


C^


8


2


0.8969


0.8780


0.0189


2.1


0.0650


6.9


135 48


0.8969


0.7458


1511


16.8


0.0975


20.7


136


9


8


2


0.8871


0.8664


0.0207


2.3


0.0607


6.5


136 48


0.8871


0.7309


0.1562


17.6


0.1962


21.1


137


cf

25


48


1.2759


1.1046


0.1713


13.6


0.2544


18.7


138


9


25


48


1.2031


1.0145


0.1886


15.6


0.2703


21.1

CELL DIVISION IN THE ALBINO RAT 583

16 per cent to 30 per cent, but most of them being in the neighborhood of 21 per cent. There is probably, then, very Uttle difference, if any, between the shrinkage effects of these two fixatives and methods of preparation up to 70 per cent alcohol. Some rough areal measurements of mesial views of brains indicate that the chief shrinkage is due to alcohol in the method described in this paper, taking the whole technique from fixation through paraffin to the slide. Some substance for replacing alcohol is therefore a decided need in technique.

CONCLUDING REMARKS

I have searched the literature for some mention of the use of chromic acid with a picro-formol-acetic combination, but find none. It would seem that my special contribution to microscopic technique as a result of these experiments is the fixative and the method of agitating fluids automatically as described in this paper.

I am greatly indebted to Professor McClung for constant criticism and advice; also for as constant encouragement when attainment of the desired result seemed hopeless. From him came the suggestion to add to the fixatives some urea, which had proved efficacious in some experiments carried on by Miss Eleanor Carothers at his direction. As an account of these experiments will be printed later by Professor McClung, no further reference need be made in this paper.

The Zoological Department of the University of Pennsylvania has provided me with laboratory faciUties at Woods Hole and has placed its exceptional photographic facilities in the University at my disposal.

Professor Donaldson has shown a cordial interest in this work and his encouragement and advice have been very helpful. Wistar Institute has suppUed the rats, the number of which passed the hundred mark, and has also provided equipment for carrying on these experiments.

584 EZRA ALLEN

GENERAL CONCLUSIONS ON TECHNIQUE

For cytological work, the slightest gain at any point in the technique is worth working for.

Very gradual change of fluids, agitation of the fluids during changing, and slow infiltration appear essential in order to get the best results from any fixative.

The addition of a low percentage of urea to fixing fluids results in sharpening the chromosomes and preserving the structure of the achromatic nuclear material. It may help penetration of the fluid.

Picro-formol-acetic mixtures are more effective when used at about 38°C. Cold is detrimental.

Flemming's fluid is more effective if used at 0°C. or a few degrees lower.

Flemming's fluid is of no value as a brain fixative at any temperature. It at times (if urea is added) isolates metaphase and anaphase chromosomes in spermatocytes somewhat better than any other fixative tried (except B-15), but is hard on the rest of the tissue and shrinks heavily.

Anilin oil is an excellent substitute for the higher alcohols.

Xylol shrinks tissue more than the vegetable oils.

Special conclusions applicable to the albino rat

B-15 is the only fixative tried which clearly differentiates brain chromosomes. It is superior to all others as a general fixative for developing and adult testis and central nerve tissue. It insures cytological detail in the testis better than any other.

CELL DIVISION IN THE ALBINO RAT 585

LITERATURE CITED

Allen, Ezra 1912 The cessation of mitosis in the central nervous system of the albino rat. Jour. Comp. Neur., vol. 22, pp. 547-568.

King, H. D. 1910 The effects of various fixatives on the brain of the albino rat, with an account of a method of preparing this material for a study of the cells of the cortex. Anat. Rec, vol. 4, pp. 213-244.

Leb, a. B. 1913 Microtomists' Vade Mecum.

McClendon, J. F. 1913 Preparation of material for histology and embryology. Anat. Rec, vol. 7, pp. 51-59.

SucHANNEK, 1890 Technische Notiz betreffend die Verwendung des Anilinoels in der Mikroskopie sowie einige Bemerkungen zur Paraffineinbettung. Zeit. f. w. Mikr., Bd. 7, pp. 156-60.

EXPLANATION OP PLATES

The figures are photographed from material fixed in five different ways. They show the essential differences between poor and good preparations.

Plate 1 illustrates the effects of fixatives upon the brain: Plate 2, upon the testis.

Modes of preparation. Figures 1, 3 to 7, 12, 14 and 17 wore prepared by the method described in this paper. Figure 2 was fixed in Carnoy's fluid plus 1 per cent chromic acid and 2 per cent urea (table 2, no. 15), and carried through by the method described herewith. Figures 8 and 9 are from tissue fixed in Carnoy's fluid, dehydrated and double-imbedded as described by King ('10). Figures 10, 13 and 15 are from tissue fixed in Flemming's fluid and carried up by the method described in this paper. Figures 11 and 16 are from tissue fixed in Bouin's fluid and carried through by the method described in this paper. Sections were cut at 6 to 10 micra and stained with Iron Haem. and Or. G.

Photographing. Figures 1, 10, 11 and 12 were made with low-power projection lenses; figures 15, 16 and 17, with Zeiss 4 mm. apoc. obj. and Zeiss No. 4 projection ocular; figures 2 to 9, 13 and 14 were made with Zeiss 2 nmi. oil immersion apoc. obj. and Zeiss No. 4 projection ocular. All were reduced for publication to the magnifications indicated below.

PLATE 1

EXPLANATION OF FIGURES

1 Sagittal section through cerebellum and medulla of 7-day rat. Ext. gr. layer is the region of actively dividing cells, magnified portions of which appear in figures 5 to 9. Only a small portion of the medulla is shown. X 15.

2, 3 and 4 show typical effects of different fixation upon cells in the medulla. Tissue is from 7-day rats. X 600.

2 Fixation: Carnoy plus 1 per cent chromic acid and 2 per cent urea.

3 Fixation: Picro-formol chrome-acetic in urea ('B-15').

4 Fixation: same as figure 3.

5 Dividing cell at inner edge of external granule layer of 7-day cerebellum shown in figure 1. Fixation by B-15. X 1200.

6 to 9 illustrate the external granule layer of the cerebellum of 7-day rat under different fixatives. The contrast is between the surface and the deep-lying portions. X 600.

6 Deep-lying tissue. Fixation: B-15.

7 Surface layer. Fixation: B-15. From same section as figure 6.

8 Deep-lying tissue. Fixation: Carnoy.

9 Surface layer. Fixation: Carnoy. From same section as figure 8.


686

CELL DIVISION IN THE ALBINO RAT

BZBA ALLBM


PLATE 1


587

PLATE 2

EXPLANATION OF FIGURES

10 Testis fixed in Flemming's fluid. X 24.

11 Testis fixed in Bouin's fluid. X 24.

12 Testis fixed in B-15. X 24. This tissue is from a younger animal than those shown in figures 10 and 11.

13 Same preparation as shown in figure 10. From adjoining slide. X 600.

14 From same slide as shown in figure 12. X 1200.

14a This figure shows the cell directly above it in figure 14 at a diflferent focus. X 1200.

15 From same slide as shown in figure 10. X 300.

16 From same slide as shown in figure 11. X 300.

17 From same slide as shown in figure 12. X 300. The chromosomes appear at some disadvantage in this figure compared with those in figure 15. The reasons for this are two: (1) they are lateral views in figure 17 as opposed to polar views in figure 15; (2) the cytoplasm is much better preserved in figure 17. A third reason may be suggested: they are less shrunken than in figure 15.


588

CELL DIVISION IN THE ALBINO RAT

EZRA ATXEN


PLATE 2


i REFERENCE MODELS OF THE ABDOMINAL VISCERA

H. D. SENIOR From the Department of Anatomy ^ New York University

THREE FIGURES

It is a common experience that students are frequently handicapped in the study of visceral anatomy by imperfections in the material allotted to them. The occurrence of better viscera on neighboring tables, although contributing alternative objects for study, is often a source of further discouragement.

Quite apart from the numerous cases in which the lungs are more or less disorganized by chronic disease, and the pleurae or peritoneum rendered useless for study by adhesions, there is often difficulty in obtaining sufficiently good preservation. Unfortunately, even after thorough injections with a fluid of good hardening capacity, organs do not become sufficiently resistant to retain their form unless the material is moderately fresh when injected. Delay in the delivery of cadavers not uncommonly leads to practical disintegration of such perishable organs as the pancreas and suprarenals.

A sufficient number of well preserved extra specimens with healthy viscera, prepared in various ways for study, are of great advantage for general reference. Such preparations, with replaceable viscera, etc., are used in many schools, and in a great measure obviate the difficulties above referred to.

We have employed this method, but have abandoned it since the reference preparations require frequent replacement when handled by a large number of students. A sufficient quantity of young or middle aged material, free from chronic disease, is not available for the purpose. During the past few years, we have used casts of the viscera in place of reference preparations, and have found them extremely satisfactory and instructive.

We began by casting the abdominal organs of a well-developed f ormahn-hardened male who died of double pneumonia at the age of 42. The first casts were made by members of the staff.^ Later we were fortunate in interesting a very skilled moulder who reproduced our originals and cast the remaining parts directly from our dissections.

Kidney and colon by Mr. J. Globus of Cornell University Medical School; kidney and spleen by Prof. W. C. Smith of Wake Forest Medical College; stomach and liver by Mr. G. Noqu6, preparateur in this College; pancreas and duodenum by the writer.

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592 H. D. SENIOR

The latter include the bladder, pubic region, and abdominal wall. The complete set consists of all the abdominal organs of this subject with the exception of the jejunum and ileum. We call it ^'Bellevue Model No. 1.^'

The casts, which are made of a light and durable plaster composition are not used for formal instruction. Two or three sets are constantly in the laboratory, and the students are left to use them as they see fit. We have made it a point to refrain from indicating the uses to which the casts may be put. It has, therefore, amused us somewhat to notice the students at first regarding them with a sort of mild curiosity, and later, as they go deeper into the subject, using them with increasing frequency and interest.

The quality of dissection has by no means deteriorated, in fact it has improved. Reference to the casts, in cases of difficulty, leads to a quicker and surer elementary knowledge of the part dissected and we find that the average student passes naturally to an intelligent understanding of the more interesting and essential relations involved. We have recently used casts of the lungs of a formalin-hardened female. Also a cast of a dissection comprising the diaphragm, the posterior wall of the pericardium, and the respiratory passages, etc., from the same subject. It has seemed to us that these have led to a better understanding of the thorax as a whole.

Hitherto the maker of the casts has sold uncolored sets, at a very moderate price, to several other anatomical departments. These have, in some cases, been painted, as ours have, and appear to have given satisfaction. Experiments in the technique of casting in colors, which are now meeting with success, will soon enable the maker to sell colored casts. These will be about double the price of the uncolored sets but the difficult and unsatisfactory task of painting will be avoided and there will be no tendency, as hitherto, to loss of color from wear.

All inquiries regarding price, etc., should be addressed to this department. Orders should be addressed to G. Von Bouchaute, care of Department of Anatomy, University and Bellevue Hospital Medical College, 338 E. 26th Street, New York.

In making the dissection of the abdominal wall great care was exercised in retaining the movable parts accurately in position. All departures from the common arrangement of structures may be taken as representing those found in the original.

Figures 1, 2 and 3 represent the model reduced about three diameters. The following notes will ex^plain the topography and anomalies of the dissections used for the casts represented .

The transverse section passes through the body of the 10th thoracic vertebra, the 10th costo-vertebral articulations, and the neighboring parts of the 10th ribs; also through the 9th ribs. At the plane of section a right intercostal artery is arising from the aorta, and the vena azygos is receiving a right intercostal vein. The vena hemiazygos and

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594 H. D. SENIOR

REFERENCE MODELS OF ABDOMINAL VISCERA 696

596 H. D. SENIOR

the thoracic duct are also cut in this section. The longitudinal sections pass through the 9th, 10th and 11th ribs.

The lateral lumbo-costal arch is absent on both sides, leaving an interval between the pars costaUs of the diaphragm, and the portion of the pars lumbalis arising from the medial lumbo-costal arch. The anterior surfaces of the 12th ribs appear in the interval.

The ischio-cavernosus and transversus perinei superficialis muscles have been removed from the left side to expose the crus penis and the inferior fascia of the urogenital diaphragm. The superior fascia of the latter structure is convex towards the pelvis, and is seen, perforated for the urethra, between the levatores ani muscles immediately below the arcuate ligament.

The inferior phrenic, the 2nd and 3rd lumbar, and the 12th thoracic arteries are shown on the left side only. There is a communication between the obturator vein, as it is entering the obturator canal, and the external iliac. The 12th intercostal and part of the ascending lumbar vein appear on the left side.

The structure lying between the vena cava inferior and the aorta, opposite the 3rd lumbar vertebra, is a lymph node. A lymphatic vessel passes upward from it to join the cysterna chyli which is hidden by the aorta.

The nerve immediately succeeding the 12th thoracic represents the conjoined ilio-hypogastric and ilio-inguinal, it passes towards the anterior superior spine. The lateral cutaneous nerve of the thigh is more medially placed than usual, and divides into two branches above the anterior inferior spine. The femoral and genito-femoral nerves are conjoined to form a single trunk the origin of which extends from the first lumbar ventral ramus to the fourth. The genito-femoral becomes free just lateral to the place of origin of the inferior epigastric artery. All these nerves are symmetrically placed. The 11th intercostal is shown on the left side only.

A small portion of the sympathetic trunk, and a trunk-ganglion, appears on the left side just medial to the psoas muscle.

THE DEMONSTRATION OF CENTERS OF OSTEOBLASTIC ACTIVITY BY USE OF VITAL DYES OF THE BENZIDENE SERIES

p. G. SHIPLEY AND C. C. MACKLL^

From the Departmenl of Anatomy, Johns Hopkinn University, Baltimore, Md,

The sites of osteoblastic activity have hitherto been studied in two ways: by use of microscopic sections, and by the examination of gross specimens in which the areas showing bone formation have been revealed by making the surrounding tissues transparent. Of the socalled 'clearing methods/ the best known and most used are those of Schultze and Spalteholz, the first of which depends for its success on the relative opacity of accumulations of calcium salts, and the latter on the post mortem staining of bone by solutions of alizarin. Therefore they are deficient since they shed no light on the beginning of the ossification process, and they do not make visible the areas in which it is taking place until extensive calcification of the locus has occurred.

If an azo dye (e.g., trypan blue) be administered to a very young animal the bones are stained quickly and very intensely with the vital color. The staining is so rapid and selective that the growing osseous system is colored blue black throughout its entire extent, whereas the organs (the liver, spleen, etc.), which are so deeply stained after the same treatment in the normal adult, are in these young animals in a great measure excluded from participation in the storage of the dye. The bones, if dissected out and cleared, show distinct epiphyses and apophyses sharply outlined within their cartilaginous capsules, and separated by intervening cartilage from the diaphyses. In preparations of this kind, the various centers of osseous development may be readily seen and studied.

The method pursued to attain this end is briefly as follows: Young animals are given a single large dose of a sterile 1 per cent solution of trypan blue. The site of election for the exhibition of the dye is the peritoneal cavity, because the superficial veins are very tiny, and the subcutaneous injection of large amounts of fluid favors pressure necrosis. That the different stages in the process of ossification may be as sharply cut as possible, it is best to administer a large quantity of trypan blue solution at once, rather than to give the same amount of dye in divided doses. Concentrated solutions should be given to avoid the mechanical difficulties involved in the administration of large quantities of fluid. The animal is sacrificed forty-eight hours after staining and the tissues are fixed by injection of a 10 per cent solution of neu 597

598 p. G. SHIPLEY AND C. C. MACKLIN

tral formalin through the blood vessels, followed by immersion in the same solution for a period of from twenty-four to forty-eight hours. The bones are then washed thoroughly, and hardened in ascending grades of alcohol, after which the soft parts are dissected away. The bones are now dehydrated in absolute alcohol, and, after immersion in benzol for twenty-four houps\ are cleared in oil of wintergreen according to the method of Spalteholz. Oil of wintergreen is an excellent clearing agent and causes little or no shrinkage in the tissues even after long immersion in the fluid. These preparations are conveniently studied with the aid of the dissecting or binocular microscope.

Bones which have been decalcified previous to clearing may be used for study in the same way, and afterward embedded in paraflSii directly from oil of wintergreen and sectioned ; or bones previously cleared without decalcification may be returned through absolute to 95 per cent alcohol, decalcified and redehydrated, embedded and cut.

In bones which have been decalcified before clearing, the osteogenetic foci are as distinct though not as opaque as in bones which have not been treated with acid. The loss in opacity is due to the extraction of a small amount of stain which goes hand in hand with the solution of the calcium salts. It is sometimes of advantage in these specimens to introduce as a contrast a very light eosin stain.

The pictures seen in examining these bones are very beautiful. The epiphyseal and apophyseal centers, colored deep blue, are seen embedded in their transparent cartilaginous matrices, separated from the growing end of the diaphysis by a disc of modified cartilage which is readily recognized by the striated appearance which is given it by the arrangement of its cells in parallel columns. The growing ends of the diaphysis are much more darkly stained than the remainder of the bone's shaft; this phenomenon can be readily seen in the advancing ventral ends of the ribs and in the ends of the digital diaphyses adjacent to the epiphyses.

It may be well to emphasize here the fact that stained areas in these bones are not necessarily the so-called primary ossification centers. The stain is deposited in the sites, where, at the time of the exhibition of the dye, active ossification is in progress, and these loci of osteoblastic activity may or may not be identical with the ossification center of the bone or epiphysis under observation. Nowhere is the difference between the primary ossification center and the focus of active growth brought so sharply to the attention of the observer as in the membrane bones of the skull. If one of these bones is examined, it is apparent even to the unaided eye that the vital stain is deposited in a heavy line along the rapidly growing edge of the bone, while the primary ossification centers are seen to be almost entirely without color.

The unique feature of this method is that it demonstrates clearly the different ossification areas even before the process of deposition of calcium salts has occurred in any marked degree. In centers where, by the older methods, the new bone is almost or quite invisible (on account

CENTERS OF OSTEOBLASTIC ACTIVITY 599

of the slight amount of calcium salt present) this method brings out the locus of ossific activity very distinctly.

A large field for the application of this method is suggested by the appearance of the bones which have been examined. In the skuU, for instance, graphic demonstrations of the growth of membrane bones and closure of the sutures are afforded. In the base of the skull the various anlagen are easily separated through the sharpness of the contrast between the stained new bone and the intervening masses of cartilage. In the long bones, the epiphyses and apophyses stand out with diagrammatic clearness.

The physiological and histological aspects of the vital staining of bone with trypan blue are now being considered, and will be published in the near future.