mie: Cetaen sa seerieeeostt im 2 ew vee reese . * ee tee CS wee eee tae Sele oes : trop bets teeing trans anoe aie Se “es Digitized by the Internet Archive in 2009 with funding from University of Toronto http://www.archive.org/details/contributionstoe04carn CONTRIBUTIONS TO EMBRYOLOGY Votume IV, Nos. 10, 11, 12, 13 PUBLISHED BY THE CARNEGIE INSTITUTION OF WASHINGTON WASHINGTON, 1916 =—— = ,* CARNEGIE INSTITUTION OF WASHINGTON Pusuication No. 224 I ‘ | -g nw ‘ a - bOI . | Acs CONTENTS. AGE. No. 10. The human magma réticulé in normal and in pathological development. By FRANK- : TSDN EAN VUATIIE (ED) ACES) loner terete RC etree © crock cs ct cioré aiatSls cis icress Gasiace oft Sheuens 5-26 11. The structure of chromophile cells of the nervous system. By E. V. Cowpry (GLTIEWA os ds Oars ot OOOO es Feb eS CORY OOS a Ot Oe a ae a 27-43 12. On the development of the lymphatics of the lungs in the embryo pig. By R. S. IG NINN GHIADEG (CMOLULCS) Meee Meee ere ae Sec csa-e ae occ ee sane scuce cameras 45-68 13. Binucleate cells in tissue cultures. By CHarutes C. Mackin (4 plates, containing 7AD) GRD) en SBS S.crend. aoe WO MOOSE 6 a6 60 DOE ea OIC Si eae 69-106 3 i: nent 2e ama v4 ee a i CONTRIBUTIONS TO EMBRYOLOGY, No. 10. THE HUMAN MAGMA RETICULE IN NORMAL AND IN PATHOLOGICAL DEVELOPMENT, By FRANKLIN P. Matt. With three plates. THE HUMAN MAGMA RETICULE IN NORMAL AND IN PATHOLOGICAL DEVELOPMENT, By FRANKLIN P. MALtt. INTRODUCTION. Students of embryology are familiar with the jelly-like substance found in the human exoccelom, which varies much in appearance in different specimens. Some- times this substance is gelatinous, with delicate fibers; at other times it is mixed with granules; and, in extreme cases, it forms quite a solid body. I think it was Giacomini who pointed out definitely that the morphological appearance of the magma determines, with considerable certainty, whether or not the contained embryo is normal or pathological. Weare indebted to him for about a dozen papers on pathological embryology, a summary of which he published in Merkel-Bonnet’s Ergebnisse. In this summary the following statement is made: “Tn the early stages of development we can determine by the extent of the exoecelom and its contained magma whether or not the embryo under consideration is normal. A large ccelom, con- taining a rich magma, with its meshes sufficiently filled with a flaky precipitate to mask the embryo, is a certain sign of pathological development.” It is well known that the magma is least conspicuous in fresh specimens and becomes more pronounced after being hardened in alcohol or other preservative fluids. In recent years it is found that magma shows to the greatest advantage in specimens hardened in formalin; the fibrils are somewhat tougher, but the magma has usually the same appearance as in the fresh state. However, the experience of embryologists has been that the magma is more pronounced in pathological speci- mens, and for this reason it has been suspected that it does not exist in normal devel- opment. In fact, the illustration of the magma given by Velpeau in his monograph— in which he first uses the term ‘‘magma réticulé”’—is undoubtedly of a pathological specimen. A glance at the other plate which accompanies this handsome mono- graph shows clearly that most of the specimens he describes are decidedly pathologi- eal. During the 80 years which have elapsed since his time, embryologists, through comparative study, have been able to separate normal from pathological embryos with considerable precision; and in the abortion material, as collected in various laboratories, far over one-half of the specimens of the first 2 months of pregnancy are pathological, and in them we usually find a highly differentiated magma. How- ever, if normal specimens are studied with care, we find that they, too, contain some magma; therefore, magma must be viewed as a normal constituent of the human ovum. It has been shown by Keibel that there is marked magma within the exoccelom of monkey embryos. In specimens containing embryos 1.3 mm. and 5 mm. in length, he describes it as a flaky, reticular mass outside the amnion, and speaks of it = ‘ S HUMAN MAGMA RETICULE IN NORMAL AND PATHOLOGICAL DEVELOPMENT. as coagulum composed of reticular magma which had to be removed before the embryo could be seen. Undoubtedly he was dealing with normal specimens, thus showing quite conclusively that a delicate magma must be viewed as a normal constituent of the exoccelom. According to Keibel’s figure 7, the magma appears to be denser in monkeys than is usually the case in normal human specimens. How- ever, a very dense magma in human specimens invariably indicates, as was first demonstrated by Giacomini, that the ovum is pathological. The best account of magma réticulé is given by Retzius, who brought the subject up to 1890, and left it with the conclusion that magma réticulé is a normal constituent of the human ovum. The statements of the earlier embryologists, from the time of Haller, are mainly of historical interest; but these investigators were at times inclined to view magma as a ‘middle embryonic layer” of the ovum, and, again, they believed it to represent the allantois of lower animals. Retzius rein- vestigated the subject, taking into consideration normal as well as pathological embryos, and his conclusion is that magma is present in both kinds. His own words are as follows: “ Bei der Oeffnung des Chorionsackes der Eier des ersten und zweiten Monats sah ich, wie in der Finleitung erwihnt wurde, und dies oft, am besten nach kurzer Erhartung in Ueberosmiumsiure (von 14 Proc.) oder in Millerscher Lésung (gew6hnlich nach doppelter Verdiinnung) , in dem schlei- migen Inhalt, weleher zwischen dem Chorion und dem Amnion, also im subchorionischen Raume vorhanden war, diinnere oder dickere Fiiden und Striinge, die mehr oder weniger dicht von der dusseren Fliche des Amnion zur inneren Fliche des Chorion hiniiberliefen, um sich dort mit ihren Enden an den beiden Hiuten zu befestigen, indem sie sich oft an ihnen verbreiterten und in ihre bekleidende Schicht tibergingen. Diese Fiiden und Striinge, welche im frischen Priéiparate kaum sichtbar waren, traten nach der Behandlung mit den erwaihnten Fliissigkeiten deutlich hervor. In der Fig. 15 der Taf. XVIII habe ich ein solches Ei abgebildet. Das stark zottige Chorion (ch) ist geoffnet, und man sieht im subchorionischen Raume den Amnionsack (a) an weisslichen Striingen (m) aufgehingt liegen. “ Die Striinge, welche im unerhirteten Zustande eine schleimigfaserige Consistenz haben, sich aber ohne Schwierigkeit Stiickweise ausschneiden lassen und dann in Mikroskope eine deutlich faserige Structur darbieten, zeigen nach der Erhirtung einen ausgeprigt fibrilliren Habitus. In einer homogen, structurlosen Grundsubstanz treten Ziige echter bindegewebiger Fibrillen hervor, welche oft eine Hauptrichtung einschlagen, also ziemlich parallel verlaufen. Jedoch kommen auch viele sich kreuzende Fasern vor. Hier und da bemerkt man dickere Biindel verschiedenen Calibers welche aus dicht gedriingten Fibrillen bestehen. Es sind also fibrillir-bindegewebige Balken, welche durch eine homogene, zahlreiche einzelne Fibrillen enthaltende Intercellularsubstanz ziehen. Zwischen den Balken und Fibrillenziigen sieht man recht zahlreiche Zellen, welche theils und am meisten rundiich oder oval, theils auch spindelférmig sind und in ihrem oft reichlichten Protoplasma gréssere glinzende Korner enthalten. Diese Zellen liegen in der Grundsubstanz ohne besondere anordnung zerstreut, bilden also keine Scheiden o. d. um die Fibrillenbiindel. “Man hat es hier offenbar mit einem unreifen Bindegewebe zu thun, einem embryonalen mucdsen Bindegewebe, welches indessen in der Entwicklung zum fibrilléren Bindegewebe schon weit vorgeschriten ist,”’ THE MAGMA IN NORMAL DEVELOPMENT. We have now in the literature a detailed description of a number of young human ova, and, according to their clinical histories, some of them, at least, must be normal, ‘The classic specimen is that described by Peters, which came from a woman who had committed suicide. The specimen was hardened in situ in an approved manner, and was worked up and described under the best possible con- ditions. In it the ecelom is filled with a gelatinous substance, through which radiate HUMAN MAGMA RETICULE IN NORMAL AND PATHOLOGICAL DEVELOPMENT. 9 delicate bands of fibrils, among which appear scattered nuclei. Near the embryo there is a small space, the interpretation of which was very difficult at the time the specimen was described. Since Peters studied this specimen, the sections have been carefully reworked and discussed in a critical way by Grosser, who gives a new interpretation in two figures and states that the cavity of the ovum contains reticular magma which is partly made up of heavier strands of tissue accompanied by nuclei. In the neigh- borhood of the embryo there are two large spaces, lined with cells, which appear to be the primitive body-cavities. In his work on the comparative development of the embryonic membranes, Grosser describes this space in great detail and also gives us two new illustrations of the embryo in his plates 3 and 4. According to this authority these two body-cavities communicate by means of a slit-like canal just behind the umbilical vesicle (Grosser’s figure 31, plate 4). This interpretation of the Peters specimen shows that the cavity of the ovum is first filled with a free mass of reticular magma, after which the ceelom begins to form near the body of the embryo. As this cavity expands subsequently, it probably first destroys the more delicate strands of magma, leaving the heavier ones; thus in a short time the cavity of the ovum is lined by the endothelium of the ecelom, which also must cover the stronger bands of magma radiating as trabecule throughout this cavity (Grosser, pp. 78 and 79). Keibel explains the formation of the human ccelom as follows: “Tt is, however, not quite clear how the cavity traversed by scattered strands of mesoblast and lying between the yolk-sac and the chorion in the Peters ovum is to be interpreted. It may be sup- posed to represent the extraembryonic ccelom; but it may also be imagined that it has arisen from an extensive loosening up of the tissue, and not by a splitting of the mesoderm, and that the triangular space between the caudal extremity of the embryo, which is lined with flat cells having an epithelial arrangement, is the first primordium of the ccelom.”’ A condition similar to that found in the Peters specimen has been observed by Lewis in the Herzog specimen, which is of about the same stage of development. Lewis says (see his paper, p. 300) that there are occasional clefts in the mesoderm of the chorion of the Herzog embryo, but that they are of doubtful significance. His reconstruction shows a strand of mesoderm, more pronounced than in the Peters ovum, extending from the yolk-sac to the chorion and circumscribing 2 space on the ventral side of the embryo. Eternod has written several papers in which he describes the formation of the exoccelom and the fate of the magma réticulé. He says that it first fills the entire space between the primordium of the embryo and the chorionic wall. Later, larger spaces appear within the substance of the magma, leaving denser strands of magma fibrils to support the embryo within the gradually expanding chorion. In general this coincides with the opinions just cited. ’ The relation of the exoccelom to the magma is strikingly shown by Waterston in a section of a small embryo in situ. The space between the embryo and the chorion is filled with a dense mass of fibrils, into which the exoccelom is burrowing. Waterston’s figure 1 shows the relation of this cavity to the magma, and only near the embryo is the exoccelom lined with a layer of cells. When this figure is compared 10 HUMAN MAGMA RETICULE IN NORMAL AND PATHOLOGICAL DEVELOPMENT. with Grosser’s figure of the Peters ovum, it becomes clear that the two spaces in the latter are in reality the beginning of the exoccelom. The studies referred to above indicate that the space near the embryo is the primitive exoceelom and that the remainder of the so-called cavity of the chorion is simply the young normal ovum filled with delicate fibrils which communicate freely with the fibrils of the chorionic membrane. We have in our collection a young normal specimen, No. 763, containing an embryo anlage 0.2 mm. in length, which in veneral confirms the observations in the Peters ovum. A list of the normal speci- mens in our collection discussed in this paper is given in table 1. TaBLE 1.—List of normal embryos. | Men- i] Men- Ca L _ Dimensions strual Condition of | Cat. oe h Dimensions strual Gonditiontotedsesial No. of chorion. | age in magma, No : of chorion. | age in embryo | embryo. a : days. || days. | mm, mm. } mm. mm. 763 0.2 4X 2.2 60 Some reticular. 588 4 19X15xX 8 | 49 Strands of magma. 391 2 16 X14 X12 | 14 (?) Do. 136 | 4 14X11X 6 56 Reticular excessive. 779 2.75 16X14X12 40 None. 836 4 221811 36 (?) | Delicate reticular. 164 -3..5 LT IT KIO Ss Few strands 148 | 4.3 17 X14X10 | 34 Small amount of magma 463 | 3.9 17 X12X 7 | 48 Much reticular. around cord. 186 4 22 X22 X22 | 44 Do. 576 | 17 30 X30 X25 ... |Small amount of magma. 470 4 20 x13 34 Very few fibrils. Specimen No. 763 was removed from a woman who was the mother of 6 children, the oldest being 10 years old. She had had one miscarriage. During the year before the operation she suffered much from headache and backache, but otherwise her health appeared to be normal. When she was admitted to the hospital she com- plained of abdominal enlargement and there was some urinary disturbance. At the operation for rupture of the perineum the uterus was scraped out; subsequently the ovum was found in one of these scrapings. The fragments both of the mucous membrane and ovum appear to be normal. Unfortunately we have only a few of the sections of this valuable specimen, but these show that we are undoubtedly dealing with a normal ovum of the same stage of development as that described by Peters. The chorionic cavity is partly filled with mother’s blood, but there are some strands of reticular magma, with nuclei and protoplasm radiating through the blood. The specimen has been stained in hema- toxylin and eosin, which is not especially favorable for defining magma fibrils. The specimen described by Herzog is also undoubtedly normal, as it was obtained from a woman who was killed by a stab-wound through the heart. The large colored plate published by Herzog shows the specimen to be quite identical with that of Peters. It shows free cells in the ecelom, which contains no other for- eign substance, but a photograph (figure 24, published by Herzog) shows that the ccelom is filled by a very pronounced substance, reminding one very much of retieu- lar magma. The same is true of a specimen recently described by Johnstone. A colored photograph which he published shows quite distinctly a pronounced magma throughout the ecelom. (See, for instance, his figure 3.) This establishes definitely the presence of reticular magma in ova the size of the specimen of Peters. We have, however, the valuable specimen of Bryce and Teacher, which also shows the condi- HUMAN MAGMA RETICULE IN NORMAL AND PATHOLOGICAL DEVELOPMENT. Al: tion of the magma in an earlier stage. In this specimen the chorionic cavity is filled with a dense mass of fibrils, throughout which are scattered numerous nuclei, as shown in their plates 3 and 4. The specimen was not perfectly hardened and there is a small cleft between the chorionic wall and the mass of magma. As yet there is no exoccelom, showing that it is younger than the Peters specimen. More advanced stages of the condition of the magma are represented in the specimens described by Jung and by Strahl and Beneke. In the Jung specimen the cavity of the ovum is filled with a very pronounced magma, running together in stronger bands, as in our own normal specimen, No. 836, to be described later. The larger cavity Jung marks “exoccelom,”’ but it is not clear that this is lined with endothelium. From his large illustration one gains the impression that the speci- men is somewhat pathological, for it is of the same type as numerous specimens in our collection with embryos that are usually found to be pathological. Taking the illustrations given in Jung’s plates 1 and 2, the specimen again appears to be patho- logical, and I should be inclined to pronounce it such did not his plate 6, figure 17, show this same section on an enlarged scale, which gives a very sharp outline of dif- ferent embryo structures and scattered through them are numerous cells undergoing division. It would be impossible, with our present knowledge, to accept such sec- tions as coming from a pathological embryo. The specimen described by Strahl and Beneke is of about the same stage as the Jung specimen, although the magma does not seem to be so well pronounced. It is unequal in mass and has scattered through it delicate strands, as shown in their figure 63. In fact, the above-described specimen underlies also the diagram on the form of the coelom given by Strahl and Beneke on page 18 of their monograph. Magma of uniform consistency, as seen in the Bryce and Teacher specimen, soon arranges itself in bands, which gradually become more and more pronounced in older specimens. Between these bands are spaces filled with fluid, and those spaces near the embryo become lined with endothelium to form the exoccelom. There are other spaces between the exoccelom and the chorionic wall. The sharper bands of magma fibrils—well shown in our embryo No. 836 (plate 1, figs. 3 and 4)— apparently support the embryo and the wall of the exoccelom within the chorion. We have in our collection an excellent embryo, No. 391, which is a little larger than that described by Strahl and Beneke. This specimen came to us in formalin and was opened with great care. It was found that the embryo and appendages were suspended by means of numerous delicate fibrils which radiated from them to the chorionic wall. As the sections were stained with cochineal, the fibrils do not show in them, so that this description is based entirely upon the apearance of the uncut specimen. In general the specimen appears to be normal. Our specimen No. 779, somewhat older than the one just mentioned, appar- ently contains no magma. It also was hardened in formalin. The ovum is entirely covered with villi, which branch twice, are of uniform size, and appear to be normal. In the main chorionic wall there is a pronounced fold. The specimen was bent along the line of the fold, but the chorion was gradually dissected away with the aid of direct sunlight. The chorion is entirely lined by a smooth membrane, and contains a cavity which is filled with a clear fluid and which apparently contains 12 HUMAN MAGMA RETICULE IN NORMAL AND PATHOLOGICAL DEVELOPMENT. no magma. Within there is a clear, worm-like body, which is bent upon itself, with another body arising from the middle of the bend. Apparently this is a flexed embryo with the umbilical vesicle attached to it. The body is of uniform diameter, measuring less than a millimeter. We are probably dealing here with a normal embryo. In opening this specimen great care was taken not to touch the embryo, so as to avoid injuring it. The embryo was taken out and cut into serial sections. It contains 14 somites and is without limb-buds. The sections give the impression that the embryo is pathological. There are no data in the history of the case which bear upon this point; therefore, for the present we may view it as a normal specimen without magma—or, if the embryo is taken into consideration, as a pathological specimen with dissolution of the magma. Usually in pathological specimens the magma is greatly increased in quantity. No. 164 is a somewhat older specimen. It came to us from an autopsy, with the entire uterus, and the sections of it indicate that the embryo is undoubtedly normal. The only record of the magma which we now have is given by several photographs which were taken at the time we received the specimen. These show a few strands of reticular magma, without any granular magma, radiating from the embryo. The photographs were taken while the specimen was in formalin. The next specimen, No. 463, is somewhat more advanced in development and contains a flexed embryo, 3.9mm. in length. The ovum is covered completely on one side, and partly on the other, with vill 1.75 to 2.75 mm. long. On the partly covered side the villi leave relatively bare one area, centrally situated, measuring S by 4.5mm. Over it the villi occur only here and there, about 2 mm. apart, and are branched and apparently normal. On opening the ovum the reticular magma is found to fill the exoccelom. By carefully exploring with fine tweezers, an apparently normal embryo is seen with a yolk-sac measuring 3.5 by 4mm. The embryo has anterior limb-buds and at least three gill-slits which are visible externally. No note was taken at the time regarding the condition of the magma, but sections of the entire chorion show that there is a very decided reticular magma between the embryo and the chorionic wall. There is no granular magma. The magma is composed mostly of fibrils, of much the same appearance as those of mesenchyme. Between the network of magma fibrils are denser strands accompanied by cells. In the fresh state undoubtedly the denser strands would appear as fibrils, while the rest would be transparent and jelly-like. The specimen came from a woman who was perfectly healthy and had given birth to 2 children during the last 4 years. This was her first miscarriage, and there was no indication of uterine disease. Specimen No. 486, of the same degree of development as the one described above, is in a perfect state of preservation, but there is no history which would indicate whether or not the specimen is normal. However, the chorion is covered with villi about 3 mm. long, with a bare spot on one side about 4 mm. in diameter. The sections of the embryo do not show any attached fibrils of magma, but the chorionic wall, after hardening in alcohol, shows a decided layer of magma attached to it. No. 470 is an interesting specimen, as it was found floating in a mass of blood- clots, which were sent to the laboratory in formalin. The ovum is covered with HUMAN MAGMA RETICULE IN NORMAL AND PATHOLOGICAL DEVELOPMENT. 13 normal villi and contains a well-formed embryo within the amnion. It is apparently normal in every respect. No magma could be seen at the time, but drawings of the embryo subsequently made show delicate strands of fibrils forming a fuzzy layer around the umbilical cord and extending over the umbilical vesicle; undoubt- edly these are magma fibrils. This seems to be the normal condition for this stage and is verified in specimen No. 836, to be described later. Sections through the mass and the chorion, stained with carmine, show the magma as a granular mass; only at points is there any indication of fibrils. However, this mass resolves itself into the most definite fibrils when colored with Van Gieson stain, in Mallory’s stain, in hematoxylin, aurantia and orange G., or in iron hematoxylin. With Van Gieson stain the fibrils take on fuchsin color about as intensely as do the fibrils of the chorionic wall, with which they are continuous. The contrast obtained with Mallory’s stain is quite marked, as the endoplasm of the mesenchyme of the chorionic wall stains slightly blue, while the exoplasm and the fibrils of the magma réticulé remain unstained. This difference is not shown in sections stained in iron hema- toxylin, as all fibrils are colored intensely black. However, it does not come out with the Oppels-Biondi method or with hematoxylin and eosin or aurantia. As the fibrils of the magma are continuous with those of the exoplasm of the chorionic wall, which do not stain in Mallory’s connective-tissue mixture, they can not be considered as white fibers, and from their failure to stain in Weigert’s elastic-tissue mixture they are not elastic. As will be shown subsequently, they give the reactions of embryonic connective-tissue syncytium; and this is Retzius’s opinion regarding their character. In specimen No. 486 the fibrils of the magma are not accompanied by any nuclei; so they must be viewed as belonging to the cells of the chorionic wall, from which they extend to bind the chorion with the primordium of the embryo. Specimen No. 588 came from a woman who had 2 children living, aged 14 and 20 years respectively. Since the last birth she had aborted 11 times, and in the opinion of her physician all the abortions were due to mechanical means. This indicates that the specimen is normal. A figure of this embryo with strands of magma radiating from the umbilical cord and vesicle is shown in plate 3, figure 2. Specimen No. 136 is of about the same stage of development as No. 588, although the chorion is covered with poorly defined villi. For an embryo of this stage it is unusually small, and I have therefore listed it with the pathological specimens in my paper on monsters. A photograph of the ovum after it had been cut open shows that the chorion is completely filled with reticular magma, so that the embryo is practically obscured. A block of the whole ovum encircling the embryo was cut in serial sections. These show that there are strands of tissue accompanied by cells which form partitions in the exocceelom. The quantity of the magma appears to be somewhat excessive for a normal ovum of this stage of development. No. 836, a perfect specimen containing an embryo 4 mm. in length, settles definitely the condition of the magma at this stage of development (plate 1, figures 3 and 4). In this ovum the exoceelom, measuring 9 by 4 mm., contains a delicate spiderweb-like reticular magma, several of -the strands being considerably larger than the others. Most of this magma occurs between the yolk-sac and the amnion 14 HUMAN MAGMA RETICULE IN NORMAL AND PATHOLOGICAL DEVELOPMENT. and the adjacent chorionic wall where the fibrils are unusually abundant. This specimen was obtained from a hysterectomy upon a woman, 25 years old, for a fibrous tumor of the uterus. She had been married 4 years, this being her first pregnancy. There were no special symptoms bearing upon the case, excepting the discomfort which accompanied the tumor of the uterus. Her last menstrual period had been delayed, and as it had been more profuse than usual she believed that she had had a miscarriage; otherwise, everything appeared normal. This was con- firmed by a careful examination of the specimen, which showed it to be normal in eyery respect. The uterus was opened by the surgeon at the time of the operation, but fortunately the site of the ovum was not injured. The specimen was sent to the laboratory immediately, where it was fixed by Dr. Evans, who made the fol- lowing record: “The specimen consists of a myomatous uterus which has been opened (apparently in a midline anterior incision) so as to disclose an abundant deciduous endometrium thrown into large folds. At the upper posterior surface of the uterus an oval mass, about 25 by 20 by 20 mm., projects. Itisa sae and is covered with a rather smooth membrane (decidua reflexa), beneath which tortuous vessels are apparent. On one side the sae (the implanted chorion) is adherent to the uterine mucosa (decidua vera). With a sharp scalpel the entire mass was dissected away from the uterus and brought under a binocular microscope in warm salt solution. The middle portion of the free surface was opened carefully, beautiful villi being found, and then the delicate wall of the chorion was divided. Within, a transparent young embryo and its umbilical vesicle were seen, the embryo appearing to be about 5mm. in length. Through this opening in the chorion, warm (40° C.) saturated aqueous solution of HgCl., containing 5 per cent glacial acetic acid, was gently introduced and the entire mass placed in 500 c.c. of this fixation fluid. The main body of the uterus was dissected from the myomatous nodule and fixed in 10 per cent formalin, the site of the implanted ovum being marked by a short wooden rod.” The fixed and hardened specimen had undergone a readily appreciable shrinkage from the condition seen in warm salt solution. All of the tissues were beautifully preserved. The implanted ovum, covered with the decidua capsularis, measures approximately 22 by 18 by 11mm. The adjacent decidua parietalis is thrown into large folds, which are themselves marked by numerous tiny elongated crack-like depressions, as well as by more circular pit-like apertures. The relatively smooth but irregular surface of the decidua capsularis is marked here and there by very conspicuous, small, oval pits, which may attain 0.5 mm. in diameter. The four flaps of this coat at its highest point, where it was opened directly over the middle of the ovum, are rather smooth on their inner surface arfd stand apart from the subjacent chorionic villi (intervillous space) to which they were originally adherent. The villi are about 2.5 mm. in length and possess one or two large branches and many ‘‘stub-like” tiny bulbous ones on the main stem. The villi are uniformly distributed in the small area exposed. With a slender sealpel the ovum was care- fully divided under the dissecting microscope, the embryo and yolk-sac being visible. The yolk-sac appears to be almost 2 em. in diameter and the embryo is surrounded by its amnion, its head (visible from above) being about 3 em. in length and showing the fourth ventricle covered by a transparent ependyma. 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On the metabolism and action of nerve cells. Brain, vol. 28, pp. 506-526. Scurroxocororr, J. J. 1913. Die Mitochondrien in den erwachsenen Nervenzellen des Zentralnervensys- tems. Anat. Anz., Bd. 43, pp. 522-524. Saarnow, A. V. 1906. Ueber die Mitochondrien und den Golgischen Bildungen analoge Strukturen in einigen Zellen von Hyacinthus orientalis. Anat. Hefte, Bd. 32, pp. 143-153. Tasuriro, Suiro, and H. S. Apams. 1914. Comparison of the carbon-dioxide output of the nerve fibers and ganglia in Limulus. Jour. Biol. Chem., vol. 18, pp. 329-334. Van Durme, M. 1907. Les mitochondries et la methode de Sjévall dans l’ovogenése des oiseaux. Ann. de Méd. de Gand, vol. 87, pp. 76-86. Wetts H. Gmeon. 1907. Chemical Pathology. 549 pp. Philadelphia, W. B. Saunders Co. CONTRIBUTIONS TO EMBRYOLOGY, No. 12. ON THE DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. By R. 8. CunnINGHAM. With five plates. CONTENTS. PAGE. Methods... <5eae eee ee een eared hs yh Oe Fee Cee eee eee 50-52 Vessels arising from the left duct... . . oot a ee he eee tlh ak Bol Tio Bf 52-64 Lymphaties' ofthe: bronchi: 7 sof uentja diay s Se Aas 2 eRe ee COR Tanae 63 Lymphatiesoisthewyems2 iy ees eto. soe nee nt cro ae yee ee ees eee 63 Lymphaticssotthe pleural sj. s.< c.g es oo acter See cnn = csc Mane rene eek nore 63 SumMMAlyon sin seuss oe reas eee e Hig ee Meee oe eae ye See ee 64-66 Bibliographycecae ct Sore ee oes Ss ns dbs ese s aad Aas en eee ee 66 Wxplanation: Of Plates.) sce Scere ba 2 cocis HE oie ise oper Wiha Orn ery see one eee 67-68 ON THE DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS TN THE EMBRYO PIG. By R. 8S. CUNNINGHAM. From an analysis of the literature on the development of the lymphatic sys- tem, it is clear that there is a general agreement among recent workers that the mammalian lymph-sacs precede the lymph-vessels in the time of their appearance, and hence constitute what may be called a primary lymphatic system. This system consists, in mammals, of 8 sacs: 3 paired, the jugular, the subclavian, and the posterior iliac lymph-sacs; and 2 unpaired, the retroperitoneal sac and the eys- terna chyli. The further development of the lymphatic system—that is, the formation of the thoracic ducts and the peripheral vessels—has been discussed at length by numerous workers during the past decade. These workers have been grouped into two general schools: the one holding that the lymphatics grow by a centrifugal sprouting of pre-existing endothelium, the other believing that these vessels are formed by a coalescence of numerous isolated spaces developing in the mesenchyme. According to the centrifugal theory, briefly stated, the sacs arise from the veins and are joined together by vessels that sprout out from their endothelial walls. Thus the thoracic duct arises from both the retroperitoneal sac and the left jugular sac, and the two elements unite somewhere between the two points of origin. Supporters of the centrifugal theory claim that the secondary lymphatic system (the capillary bed) arises by the sprouting of the endothelial walls of the sacs and of the right and left thoracie ducts. These sprouts invade the organs and, becoming progressively more complex, assume the adult form of the lymphatic system. The supporters of the multiple-anlagen theories (whether they believe in coalescing tissue- spaces, multiple venous origins, or degenerating veno-lymphatics) agree in claiming that lymphatics do not grow by the centrifugal sprouting of the pre-existing endo- thelial walls. It is not my intention to review here all the various theories that have been advanced, but only to call attention to the two general views, in order to correlate my findings with them. A very thorough discussion of these two views, as well as a comprehensive review of the literature, may be found in the Ergebnisse der Anatomie und Entwickelungsgeschichte, 1913. (Dr. F. R. Sabin, Der Ursprung und die Entwickelung des Lymphgefissystems. ) Though primarily concerned with the problems of origin and the method of erowth of the lymphatic vessels, the supporters of both theories have aided in establishing the morphology of the primary system and have laid the foundation for the further study of the development of the system as a whole. If the centrifugal 47 48 DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. theory is correct, it is clear that it should be possible to follow the growth of lym- phaties from the sacs into any organ or group of organs. It should also be possible to demonstrate in progressively older stages constantly increasing lymphatic zones and decreasing non-lymphatic zones. The development of the lymphatics of the skin, of the intestine, and of the lung has now been studied in this manner. In 1904, Dr. F. R. Sabin demonstrated that the skin received its lymphatic supply from the two jugular sacs and the two iliac sacs. From each of these sacs a group of radiating vessels invade the skin and form there a close-meshed plexus. These four plexuses gradually increase in size and finally unite, so that the entire skin is supplied with lymphatics. The differentiation which takes place varies with the location and depends upon the adaptation which the vessels must make to the other structures. Continuing the work of Baetjer (1908) on the retroperitoneal sac, Heuer (1909) studied the development of the intestinal lymphaties by the injection of this sac. He observed and described progressive changes in the intes- tinal supply, finding more complex injections possible in each older stage. He inter- preted these results to mean that the lymphatics had not extended beyond the point which his injections reached and. that the region beyond this point constituted a non-lymphatic zone. There is, therefore, a primary and a secondary lymphatic system. The former consists of a series of sacs formed from the veins and connected by the right and left thoracic ducts. The secondary system consists of the peripheral vessels, which are held by some to be outgrowths from the sacs and by others to be formed in situ. With regard to the development of these peripheral vessels, only those of the skin and the intestine had been studied. There was need, therefore, for the study. of the other abdominal and the thoracic lymphatics. This work was begun to establish a clearer conception of the development of the secondary system. In presenting this study, I do not claim to have found any new evidence as to the mode of growth of lymphatics. This work supports the centrifugal theory in the same manner as does that of Heuer (1909); and it is certain that the theory is sufficiently well established to serve as a basis for this work. It is the object of the present paper to follow the gross morphological changes in the development of the lymphatic vessels of the lung from the primary stage to the adult form. It is desired to indicate the general lines of growth and the various stages which the system passes through in the course of its development. No attempt has been made to study the finer structure of the vessels or the mode of growth. It is important to note that complete injections are very difficult to make, and that it is also difficult to be certain whether the injection in a particular specimen is complete or not. Therefore it isnot claimed that any of the injections are com- plete; and the limits of the lymphatic and the non-lymphatic zones at any stage are defined in a general manner, depending on the comparison of a number of specimens. The lymphatic supply of the lungs develops from three sources: the thoracic duct, the right thoracie duet, and the cephalad portion of the retroperitoneal sac. In 1913, Sabin remarked: “The right lymphatic duct curves ventralward and grows to the heart and lungs.’ This is the only statement which I have been able to find DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. 49 in the literature regarding the development of the cardiac and pulmonary lymphatics from the right duct, or the morphological fate of the right duct in mammals. In the same report attention was called to the fact that the lung-vessels can be injected from the retroperitoneal sac, but this was not studied further at that time. The right duct grows primarily to the heart, just as the left grows to the aorta, this asymmetry depending upon that of the cardio-vascular system, according to the general rule that the principal lymphatic trunks follow the large blood-vessels, and grow with the greatest rapidity where the blood-supply is most abundant. In the beginning I wish to lay emphasis upon the fact that the lung lymphatics develop partly from the retroperitoneal sac, and to call attention to the fact that these vessels persist in the adult as part of the permanent drainage of the lung, and hence may be of importance in pathology. On account of the complexity of the development of the lung lymphatics, it has seemed best to present this work, not by describing and figuring a series of progressively more complex specimens, but by describing the development as a consecutive growth and illustrating with those preparations that may seem best to clarify the text. However, as a matter of ref- erence, the following table of periods has been arranged, in order to offer a brief outline of the complexity at varying stages. These stages are selected with regard to the more important principles of growth and are as follows: (1) The downgrowth of the two ducts, completion of the primitive system, and the first vessels to the trachea and lungs. Embryos 2.3 to 3.5 em. (2) The migration of the heart; the coalescence of the cardiac drainage with that of the lungs, by the formation of the tracheal plexus and the plexus on the arch of the aorta; the growth of the vessels in the lung from the earliest sprouts along the bronchi to the primitive pleural plexus, and the early marking-off of the connective-tissue septa; and the growth up from the retroperitoneal sac through the ligamentum latum and the anastomosis in the primitive septa into which the vessels grow. Embryos 3.5 to 4.5 em. (3) The completion of the primary lymphatic system; that is, when the entire organ is supplied , and the further development is in an increasing complexity of the plexuses already present, incident to the increase in the size of the organ and its assumption of mature activities. During this period the formation of the valves and nodes begins. Embryos 4.5 to 7 cm. (4) The remainder of the development is considered a period, as it is, in reality, an adaptation of the system already present to the increasing needs of the organ. This includes the differentiation of the drainage-lines and the final development of the nodes. In describing the development of the lymphaties of the lung, the growth of the left duct down to the aorta, of the right duct to the heart, and the formation of the primitive tracheal plexus and the early vessels to the lungs from both ducts will be considered first; the further development of the tracheal plexus, together with the changes incident to the descent of the heart, will follow; then the origin of the vessels from the retroperitoneal sac and their growth up through the ligamentum pulmonale into the lungs will be considered. After the anastomoses of the two sets of lymphatics, the lung will be considered as a whole, inasmuch as the further development is symmetrical for the entire organ, with the exception of the final lines of drainage and the development of the nodes. I wish to express here my indebtedness to Professor F. R. Sabin for her con- stant advice and assistance throughout this work. Also I wish to thank Mr. James F. Didusch and Miss Flora Schaefier for the illustrations. 50 DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. METHODS. The injection method has been principally used, but it has been supplemented and supported by evidence from both single and serial sections. The collection of pig embryo of the Anatomical Laboratory has been at my disposal, and I have also studied a number of especially prepared series. Many of the series have been of embryos in which the blood-vessels have been injected, and this has materially aided in their interpretation; in fact, in all the especially prepared series the blood-vessels were injected. All these embryos were fixed in Carnoy’s fixing fluid, consisting of 6 parts of absolute alcohol, 3 parts of chloroform, and 1 part of glacial acetic acid. The method of fixation is as follows: Place the embryo immediately in the fluid and allow it to remain there 6 to 8 hours; then transfer directly to 70 per cent alcohol; dehydrate by ascending grades of alcohol with 2 per cent difference until 95 per cent is reached; then change to absolute. This gives excellent fixation with very little shrinkage. The stains used were Ehrlich’s hematoxylin and a mixture of eosin, aurantia, and orange G. The injection masses used were india ink, a saturated solution of prussian blue, a 5 per cent aqueous solution of silver nitrate, and an aqueous suspension of lamp- black. The india ink and prussian blue give about the same results, except that the specimens injected with prussian blue are more easily studied after clearing, as the ink renders them more opaque. The india ink, however, flows more easily and hence the injections are more nearly complete. The silver-nitrate injections are easiest to analyze and give beautiful preparations, but its caustic action prevents the finer vessels from filling, so that only the larger trunks are injected; however, it furnishes an extremely valuable method of following the principal drainage-lines at different stages. The lampblack is the mass which gives the most nearly complete injections, but unfortunately it precipitates in fine flakes and gives a feathery appearance to the specimen, thus rendering it difficult to use for illustrating. It will be necessary to review the methods used in injecting the various stages, as they differ considerably and are of especial importance in interpreting the results. The earliest injections were made by filling the jugular sacs from the superfictal plexuses and then gently moving the embryo. I have succeeded in injecting the early vessels to the trachea and the lungs in only a few pigs less than 3 em. long, beeause the injection mass usually follows the path of least resistance, which is into the jugular vein. In injecting embryos between 3 and 6 em. in length, three general methods have been employed: (1) The best and by far the easiest method of obtaining good preparations of the left part of the tracheal plexus is to inject through the retroperitoneal sac in the manner described by Heuer (1909); but this seldom gives good preparations of any of the vessels of the lung except those of the lower lobe. However, this method has been of particular importance in following the lymphatics up from the retroperi- toneal sac to the pr ysterior poles of the lower lobes. 2) One may inject the tracheal plexus, especially the left part, by plunging the needle deep behind the aorta and injecting cerebralwards; the right plexus is some- times filled also, and often the vessels of the left lobe of the lung. DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. 51 (3) Finally, the vessels of the lung are best injected by a puncture just ventral to the trachea (the tracheal plexus) and behind the arch of the aorta. Here the tracheal plexus is always extravasated, but the lung-vessels fill up nicely. The embryos older than these mentioned, that is, longer than 7 em. (or after the valves are formed), are much more difficult to inject, and this difficulty increases with further development. The method employed has been to inject directly into the connective-tissue septa of the lung and to continue the injection slowly until there is some extravasation at the point of puncture, when a part of the lung sur- rounding the area of extravasation is well injected. This method has been very satisfactory in all specimens that were obtained very soon after the removal of the uterus; most of the injections were made while the heart was still beating. In order to study the relations between the blood-vessels, bronchi, and lym- phatics, multiple injections had to be made. Various combinations were employed. In some, the lymphatics were injected together with veins and arteries; in others with either veins or arteries alone. Again, the lymphatics and the bronchi were injected; and in still others the lymphatics were combined with either veins or arteries. In these multiple injections prussian blue, india ink, and carmine were used, the lymphatics being injected with either the blue or the ink. The specimens in which three systems were injected were difficult to clear, unless only the large bronchi and blood-vessels were filled. In order to trace the vessels more accurately, many of the injected lungs were embedded in paraffin and cut in thick serial sections (100 to 5004); these were mounted in balsam but not stained. Other lungs were cut at 10 to 20u and stained similarly to the series already referred to. All measurements of embryos refer to crown-rump diameter and were taken before fixation, as is customary in this laboratory. The illustrations are labeled “C. R. —’; this refers to the crown-rump measurement. In 1906, Flint published his study on the development of the lungs in the pig, and his work has been taken as a basis of the general structure of the lungs, especially with reference to the development of the bronchi and blood-vessels. He reviewed all the important literature on the embryology of the mammalian lung, studied the lymphatics in sections, and briefly summarized their structure and distribution at various stages, but he did not attempt to inject them. I have been able to confirm most of his observations. However, he labored under the difficulty of having neither reconstructions nor injections. He gives a short summary of each stage, and of these summaries I quote the more important parts: Stage 3 cm.: At the root of the lung a few dilated lymphatics may be noted near the bronchi and pulmonary vessels; however, they have not grown beyond this point into the substance of the lung wings. Stage 5 cm.: From the root of the lung the lymphatics have gone some distance into its sub- stance. They have thin walls composed of young fibrils lined with endothelium with occasional valves. They are confined, however, to the immediate neighborhood of the main bronchi and their chief subdivisions. Stage 7 cm.: The most interesting change, however, lies in the further growth of the lymphatics, which in the earlier stages are found in the root of the lung in the neighborhood of the pulmonary vessels and the large bronchi. As they grow in, they accompany these structures for a distance; 52 DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. da then approaching the end branches they leave them and run in a plexiform manner midway between the bronchial tubes until they reach the pleura. This gives the lung now an indefinitely lobulated appearance in which the periphery of the simple lobule is indicated by the lymphatic vessels and the center by the bronchi. The lymphatics are lined with flattened endothelium; their walls are formed by the young connective-tissue fibrils, and here and there valves are beautifully shown which, in general, point away from the pleura. Stage 13 em.: The lymphatics, forming a plexus around the bronchial veins and arteries at the root of the lung, accompany them towards the periphery, giving off branches to the interlobular spaces en route. * * * On reaching the periphery of the lung they leave these structures and pass out, as in the preceding stages, to the pleura. They have a plexiform arrangement and may be traced at times into the substance of the lobules. This course may be observed in the deeper lobules of the ung as well as in those on the surface under the pleura. Stage 19 em.: In general the relations of the lymphatic system have not changed. Stage 23 em.: At 23 em. the first evidence of the submucous lymphatic system is seen in the stem bronchi. It may, however, be found earlier, but the vessels are difficult to follow. It would seem thus that we have in the pig’s lung, besides the lymphatic plexuses accompanying the bronchi, arteries, and veins, an interlobular system which Miller has been unable to find in the human lung. Injections pointing to such a relationship he has interpreted as artefacts. If Miller’s conclusions prove correct, then the lymphatics of the human lung must develop, so far as the interlobular system is concerned, in some other way. I quote at length from Flint because he alone, of the many workers on lung lymphatics, has approached the subject from the embryological side. As I have said, Flint was seriously handicapped by having only sections from which to draw his conclusions. He was especially struck by the prominence of the vessels lying in the interlobular septa, and attempted to explain their apparent change of course (7. e., from the bronchi to the septa) by the theory that the density of the tissue was greater around the bronchi and vessels and that the lymphatics chose the path of least resistance. He did not call attention to the relation of the veins to this point in the development of the lymphatics, which will be discussed later, but emphasized the fact, so amply shown by injections, that these interlobular vessels grow much more rapidly than the vessels around the bronchi and arteries. It will be necessary hereafter to discuss the work of Miller on the adult lym- phatic system, in connection with the later stages; therefore it will suffice to refer here to the statement which Flint discussed in the quotation given above. Miller has called attention to the fact that the terminal vein lies in the periphery of the lobule and that the lymphatics accompanying the vein communicate with those of the pleura. He cites Councilman’s (1900) description of the interlobular vessels, but does not claim to have found the same vessels. I think that these different views will be reconcilable when we have followed the development of the lymphatics through the various stages that lead to the adult form. The literature on the lymphaties of the adult mammalian lung is very large, and for a comprehensive review of it the reader is referred to the papers of Miller (1893, 1896, 1900, 1902, 1911). It seems needless to discuss it more at length here. THE VESSELS ARISING FROM THE LEFT DUCT. As has been said, the lymphatics of the lungs arise partly from the two thoracic ducts by sprouts, ‘These vessels grow to the mesenchymal wall of the trachea and form there a plexus which sends vessels down into the lungs. Other vessels grow directly into the lungs. DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. 53 The thoracic duct, as has been shown by Sabin (1913), Baetjer (1908), and Kampmeier (1912), is complete—that is, it connects the jugular sae with the retro- peritoneal sac—in a pig embryo 2.5cm. long. Very soon after this the first evidence of the pulmonary supply may be found. I have obtained partial injections at 2.8 em., and have found some small vessels in serial sections at 2.6 em.; so it is evident that these sprouts are either formed from the thoracic duct as it grows down or very soon after the primary system is completed. About midway between the jugular anastomosis and the arch of the aorta the thoracic duct leaves its position lateral to the trachea and bends dorsalward to lie near the dorso-lateral border of the esophagus. In this position it comes down behind the arch of the aorta. This transition is shown by Sabin (1913, figures 12 and13). Just at the point where the duct begins to bend dorsally the earliest sprout to the lung is formed. At this point a single large vessel buds off from the thoracie duct and passes down over the arch of the aorta to reach the hilum of the lung. This vessel unites with the vessels that grow up from the thoracic duct just caudal to the arch of the aorta and forms the lower part of the tracheal plexus. This vessel usually persists in the adult as one of the drainage trunks from the hilac nodes to the thoracic duct. It is shown in figure 5, plate 1, and figure 2, plate 4, marked with an asterisk. From the region of the thoracic duct, where this vessel buds off to a point about the level of the aortic arch, a number of other vessels are formed very soon afterwards. These vessels arise very close together and grow across to the lateral wall of the trachea, where they anastomose and form the primitive left tracheal plexus; they lie in the undifferentiated mesenchymal tissue that surrounds the tracheal lumen. These lymphatics have formed a plexus by the time the embryo has reached a length of 3 em. From this plexus vessels grow across the trachea to anastomose with other vessels from the similar plexus on the opposite side; other lymphatics grow up the trachea and form a coarse-meshed plexus around it. This is the anlage of the adult supply of that structure. But the most important of the branches of this plexus, as far as the present work is concerned, are those from the lower part. These pass down the trachea and, being joined by other vessels that leave the duct near the arch, pass up over the bifureation and into the lung. The left tracheal plexus is shown in figure 5, plate 1, and figures 1 and 3, plate 2. Here must be noted the fact that the plexus of the left side supplies the greater portion of the ventral surface of the trachea and forms the largest part of the great sheet of lymphatics around the primary bronchi. Later these vessels anastomose freely with those from the right side. It is important to call especial attention to the difference in the richness of the supply of the dorsal and the ventral surfaces of the trachea. There are vessels that grow to each from the left plexus, but a much greater number pass to the ventral surface than to the dorsal. Thus the plexus formed from the two lateral groups is much more closely meshed on the ventral surface, and from it is derived the greater part of the lung supply. Over the bifur- cation there is a very complex group of vessels, and these form tubes around the principal bronchi as they grow on into the lung. Below the level of the hilum several vessels, three or four in number, grow up from the thoracie duct and its plexus surrounding the aorta, to join with the large D4 DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. vessel which has been described as the first to the lung and which comes over the arch to reach the hilum. These vessels from the duct below the hilum form a plexus with the vessel from above, as has been described. It is well known that the thoracie duct is double below the level of the arch of the aorta and that the two divisions are connected by numerous anastomostic vessels (figure 1, plate 2). This system is the anlage of the vessels that surround the aorta in the adult. This relation has been figured by Heuer (1909). One of the lymphatics that pass up from below to join the first vessel from the thoracic duct above leaves the duct near the diaphragm and is consequently very conspicuous in injections of this region. Heuer has figured this lymphatic as one that goes to the heart, a conclusion entirely justifiable from the general appearance of the injected specimen. Figure 1, plate 2, is from a dissected embryo 4 em. long, in which the lymphatics were injected from the retroperitoneal sac. The thoracic duct and part of the left tracheal plexus are injected, and the extension of the plexus down on the bronchus is also shown. Below the arch may be seen some of the vessels that grow up to meet the branch from above. These vessels have been cut off, with the arch, to expose the tracheal plexus. The double duct is also shown, the more ventral element being the one figured by Heuer. The pulmonary vessels reach the hilum when the embryo is about 2.8 em. long, and ean be seen in sections at 3 em. (see figure 1, plate 1). The lung-tissue is at this time very slightly differentiated mesenchyme, containing the early bronchi and blood-vessels. For a further description of the structure of the lung at this stage see Flint (1906). These early lymphatics are grouped in an irregular manner in the hilum of the lung and may be found at 2.9 and 3 em. in sections. But I have not been able to inject them earlier than 3.3 and 3.5em. Figure 1, plate 1, is of a section from an embryo 3 cm. long, in which the blood-vessels were injected while the embryo was still living. The lymphatics are shown as a few dilated spaces (blue) in the hilum. These vessels are beginning their invasion of the lung-tissue while the tracheal plexus is forming. It is necessary, however, to complete the deseription of this plexus before considering the portion of this study which relates to the lung proper. ‘The development of the vessels within the lung-substance will be considered after the formation of the right lymphatic plexus has been described. It is important, however, to note here that all the vessels to the left lung come from the closely united group of vessels on the trachea and around the aortic arch, as has been described. This will be studied in relation to the first vessels to the lung on the right side, which will next be considered. On the right side the development is, in general, similar to that on the left, but differs in a few particulars, chiefly relating to and in consequence of the asymmetry of the vascular system. The right duct is primarily to the heart, or perhaps to the vena cava, since it follows that vessel to reach the cardiac base. But while the heart supply is at first only from the right side, the vessels to the lung and the trachea develop at about the same time. The right duet grows caudalward parallel to the thoracic duct to the point where the vena cava arches ventralward to reach the heart, ‘There it divides, and one branch follows the posterior wall of the vena cava to reach the cardiac base, while the other passes into the hilum of the lung. The DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. Be ~~ cardiac division, after reaching the base of the heart, along the posterior wall of the vena cava, passes around the bulbous arteriosus to reach the anterior surface of the heart, where it divides to form the primitive pericardial plexus. By intro- ducing a canula dorsa! to the vena cava and injecting towards the heart, I was able to fill this plexus in a pig 3 em. long. At this stage it extends about one-fourth of the distance from the base to the apex of the heart. Figure 13 in Volume V of the Johns Hopkins Hospital Reports, Monograph Series (Sabin on ‘‘ The Origin and Development of the Lymphatic System’’), shows the right duct near the heart in an embryo pig 2.5 em. long. In that paper attention was called to the fact that the duct grows towards the heart and that it probably represents the cardiae supply. The second of the two terminal branches of the right duct passes down parallel to the dorsal wall of the trachea in about the same general position as that occupied by the duct above the point of division. Thus it might seem proper to consider the lung division as the more fundamental of the two, as it appears to be the continua- tion of the undivided duct. However, the heart branch is probably the more fun- damental and the earlier of the two, since it is a general principle in the growth of lymphatic trunks for the principal vessels to follow the larger blood vascular channels. Hence we consider the left duct as primarily aortic and the right as primarily cardiac in distribution. This vessel enters the hilum of the lung and breaks up into a few branches that are grouped around the bronchi and blood-vessels as on the left. The nature of the grouping and the further development are similar on the two sides, and hence both will be considered together. There is, however, an interesting difference between the two upper lobes, which is dependent upon the relation of the aortic arch to the hilum on the left. On the right the lung is distinctly higher (7. e., nearer the neck) than on the left, because on the latter side the aortic arch lies in the groove made at the juncture of the upper lobe with the trachea. Thus the vein to the upper lobe on the left passes close to the bronchus under the aortic arch, while on the right it is well above the bronchus. This allows more freedom in the lymphatic growth on the right, so that the vessels to the upper lobe come down directly into it instead of growing back from a single group, as they do on the left. It must be understood that the stage referred to is between 2.5 and 3 cm., when the heart is still higher than the bifurcation. Later the heart passes still farther down into the thoracic cavity, and these differences disappear as the cardiac and aortic relations to the lung begin to assume their adult form. There is, however, one very important effect of this asymmetry; the lymphatics of the right duct pass directly into the lung, while those of the left must course up over the arch of the aorta and the bifurcation of the trachea to reach the lung-tissue. This has been mentioned briefly before. It is clear that the principal supply of the bronchi, and therefore, ultimately, of the lungs, comes from the left duct. This is in large measure the result of the asym- metric relations of the heart and aorta. The development of the first vessels to the trachea and lungs on the right side will next be described in detail. From the heart limb of the right duct a few vessels arise and grow down over the vein to the upper lobe on the right side; after crossing the vein they enter the lung near the hilum and divide into several branches, some 06 DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. of which anastomose with those mentioned above as growing down into the hilum of the lung from the pulmonary limb of the right duct. Other vessels turn outwards along the bronchi and veins and grow into the lung-tissue of the upper lobe. This process will be described later. Along the right duct, cephalad to the division into the two branches, other vessels are given off; some grow down to anastomose with ascending branches lying along the tracheal wall and coming from the vessels described above, while others erow to the tracheal wall at varying positions along the section lying between the jugular anastomosis and the bifurcation, corresponding somewhat to the vessels on the other side, with which their branches anastomose, forming the tracheal supply. The earliest injection of the lymphaties of the right side were at 2.8 and 2.9 cm. Figure 2, plate 3, shows an embryo of 3 em., where the injection was made into the right sac, which illustrates the relative position of the vessels to the upper right lobe and the limb that follows the vena cava to the heart. This drawing is diagram- matic and does not show the different vessels to the lobes on the right side, though some of them were injected. The left duct is shown without any branches. In figure 1, plate 2, the right tracheal plexus is represented. Though it is very incomplete, it shows the general form of the plexus and its relation to the similar plexus on the other side. The right tracheal plexus, in its simplest form, consists of a few vessels which are beginning to anastomose along the lateral wall. These anastomoses become more and more complex and numerous until, along the right side of the trachea, a plexus somewhat similar to that of the other side is formed. They differ, however, in that on the right there is no aortic arch to complicate the form. Therefore the plexus is a simple sheet-like group of vessels which lie along the lateral wall of the trachea, but do not extend up over the ventral surface of the bifureation, except by a few anastomosing vessels. It anastomoses freely with the larger plexus from the other side on the ventral surface of the trachea, and later the combined plexuses lose their individuality and appear continuous. In the meantime the two tracheal plexuses have begun to anastomose. This will next be described. Between 3.3 and 4.5 em. the two tracheal plexuses anastomose by means of numerous vessels which grow around the trachea, both dorsally and ventrally. Above the level of the aortic arch these connecting vessels are far less numerous than below, where the two are merged into a sheet-like plexus that surrounds the trachea and passes down into the lungs as tubes of vessels surrounding the bronchi. Above the bifurcation the dorsal surface of the trachea has fewer vessels than the ventral, while the two original lateral plexuses are much more closely meshed, representing the anlagen of the two lateral groups of lymph nodes of the adult. From the close-meshed plexus on the left side of the trachea just at the bifur- cation a group of lymphaties pass up over the left stem bronchus and sweep across to the right bronchus, forming the upper group of vessels lying on the bronchial wall. These grow down on the side and anastomose with the vessels coming down from the plexus on the right side. Thus it will be seen that the left supply is a more important part of the general origin than the right, supplying, as it does, all of the left lung and part of the right. ~I DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. 5 It is of importance to note here that the heart is migrating downwards (1. e., caudalwards) during this period, and, by the time the embryo has reached 4.5 em. in length it has come to lie almost directly over the hilum of the lung. Hence the vessels that formerly ran in a long course from their point of origin in the heart limb of the right thoracic duct to reach the upper lobe and the hilum of the lung have become a part of the common tracheal plexus, and the formerly distinct duct to the heart has also been absorbed by the plexus over the bifurcation. The cardiac vessels then (at 4.5 em.) drain directly into the plexus over the hilum of the lung (figures 1 and 3, plate 2). This relation remains in the adult in the drainage of the cardiac vessels into the mediastinal nodes and the union of the efferent trunks of these nodes with those from the hilum of the lungs. Here must be mentioned, though not bearing particularly on the lymphatics of the lungs, the connection between the right and the left ducts. In specimens of about 3.5 to 4 em. in length, I have regularly found a vessel arising from the dorsal part of the right tracheal plexus and joining the thoracic duct behind the aorta. As has been said, it seems best to consider the vessel to the heart as the continuation of the right thoracic duct; hence this vessel must be considered, as was the one to the lung, as a part of the collateral supply. The lung, as has been stated. also derives lymphatics from another source the cephalad portion of the retroperitoneal sac. These vessels are growing into the lung during the period when those already described are differentiating, but it seems best to postpone the discussion of this portion of the pulmonic supply until we have studied the early changes that take place in the lung itself, following the invasion by the vessels already described. The desirability of this is evident when it is remembered that the vessels from below must follow a similar course in the lung, with the exception that this course is reversed, due to the fact that these vessels invade the lung through the pleura instead of the hilum, and must reach the other supply through the interlobular septa, to be described later. At 3 em. there are two primary bronchi and two veins on either side, one of each to each upper lobe and one to each lower lobe. From these the secondary branches are beginning to form. From 3 cm. to 5 em., these secondary branches are developing rapidly and are very large in comparison to the size of the lung. The arteries are very much smaller, and the veins are somewhat larger than the arteries, but much smaller than the bronchi. It is of great importance to note the relations of these structures to each other during this period. Flint has studied their develop- ment very thoroughly, but he does not call attention to the fact, so important with reference to the lymphatics, that the developing vein is separated as widely as possible from the bronchus with which it is morphologically associated. The artery, on the other hand, follows the bronchus very closely and is distributed with it to the center of the developing lobule. The two primary branches of the pulmonary vein lie close to the corresponding bronchi. This is, indeed, as far separate as is possible, since there is almest no lung-tissue at this period, while the secondary vessels which may be considered the terminal branches lie about equidistant from the two adjacent bronchi. The arteries follow the bronchi more closely. This fact is of the greatest importance in the development of the lymphatics and also in the relation of the veins to the periphery of the lobule in the adult, as has been shown by Miller (1900). 5S DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. As the lung increases in size and the veins and bronchi which we have termed secondary give off other branches, these in turn become the terminal ones and assume the relations that have been described. The others are, by the increasing amount of lung-tissue, forced closer together. Thus it is seen that it is only the terminal veins that occupy the position described; that is, pass along the periphery of the lobule. In the pig there is considerable connective tissue forming definite lobules in the adult lung; and these septa, bounding as they do the area supplied by terminal bronchi, divide the lung into a large number of irregular cones or pyra- mids, which have the bronchus and artery in the center and the veins passing along the periphery until close to the apex, where they enter veins of the next larger size. For further discussion of this arrangement see Miller’s article (1900). As we have seen, a few dilated lymphatics are found in the hilum of the lung at 2.9and3 em. These are the first branches from the vessels that are forming the plexus on the trachei and bronchi already described. The bronchi, as has been said, are surrounded by lymphatics which follow them into the lung-tissue; and, as secondary bronchi are formed, lymphatics from these plexuses branch off to follow them. The primary veins lie very close to the corresponding bronchi at this stage, and are accompanied by a few lymphatic trunks which arise from the same general plexus that covers the bifureation. These vessels anastomose very richly with those of the bronchi, and, close to the point where the trachea divides, they merge together. We have seen that the secondary veins lie midway between the adjacent bronchi, and represent the outer border of the primitive lobule of the developing lung. Along these veins the lymphatics grow towards the pleura; they are derived both from the plexus that follows the primary vein and from the vessels that sur- round the primary bronchi. The lymphatics from the bronchial supply join those from the vein, and the combined group passes along the vein, spreading out on either side to form a sheet, until the vessels reach the pleura. Flint observed these sheets of lymphatics, but thought that there must be some difference in the density of the tissues to account for their leaving the bronchi to run midway between. He did not recognize the relation between the veins and the lymphatics. It will be clear, when it is remembered that the smaller branches of one vein spread out fan-like to meet those of the other vein, that the sheets of lymphaties lying between the bronchi are directed by the veins as well as the separate lymph-vessels directly associated with them. In this manner the true primitive lobules are formed by the interpolation of a sheet of rapidly growing lymphaties between the bronchial tubes. It is along the distal margin of these plexuses that the pleural marking begins. When these vessels reach the pleura there is a marking-out of the characteristic coarsely-meshed plexus, each interspace corresponding to the sheet beneath (figure 3, plate 1). It must be remembered that these vessels, growing as they do very rapidly, reach the pleura very early, and hence the pleural plexus is developing while the above-mentioned interlobular plexuses are forming. We have so far described only the formation of the large parallel plexuses shown in figure 1, plate 4, figure 2, plate 5, and figure 1, plate 3. But the formation of veins in other planes directs the growth of the lym- DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. 59 phatics, so that with each bronchus there are several veins and several sheets of lymphatics developing. Thus the series of cone-shaped or pyramid-shaped lobules are surrounded by plexuses of lymphatics. Along these plexuses the differentiation of the connective-tissue layers takes place, for, when the lymphatics invade these areas, there is only an undifferentiated tissue, which is characteristic of the lung. Flint suggested that the lymphatics followed the bronchi for a certain distance and then turned away midway between them, because of some relative difference in the density of the tissues. It is quite impossible to observe the relation to the veins in uninjected sections, and consequently this point was not discussed in relation to the problem of the question of tissue density. Notwithstanding this phase of the development which Flint was unable to follow, there still remains considerable probability in his suggestion. The fundamental reason for the direction of growth is as yet entirely a mystery, but there seems to be little doubt that the principal lines of lymphatic development are along the larger blood-channels; and, in general, the veins are chosen, though the left duct may be considered as following the aorta. The much slower-growing lymph-vessels on the bronchi follow each branch out towards the periphery. The primary bronchus is surrounded by a very close-meshed plexus, which consists of a large number of vessels; in cross-section one can count from 50 to 75. However, this number is very greatly reduced on the secondary bronchi, each of which has four or five trunks following it. These are closely bound together by anastomosing collaterals. With reference to the secondary bronchi, almost the same series of events occur as given above for the primary ones. These secondary bronchi are likewise marked off by interlobular septa in which the lymphatics develop more rapidly than along the bronchus whose lobule they mark off. The lymphatics around the bronchus give off small vessels near each branch of the bronchus, and these pass across to join the plexuses that surround the area of the lobule (figure 1, plate 3). As the new-formed bronchi grow larger they are, in turn, followed by two or three lymphat- ics, which end, as did those around the secondary bronchi, by passing over to join the septa or, if close to the pleura, the vessels there. These lymphatics that pass from the bronchial system to join those in the septa follow the branches of the veins which bend in from the septa to reach the capillary bed of the arterial tree. These persist in the adult as the vessels that pass from the bronchus to the vein and thence to the pleura (figure 2, plate 1). We will consider now the lymphatics that grow up from the retroperitoneal sac into the caudal pole of the lower lobe. In 1906 F. T. Lewis described, in rabbit embryos, a lymphatic sac just median to the mesonephritie vein. Baetjer (1908) showed that it arises from the ventral surface of the large vein which connects the two Wolffian bodies (embryos 17 to 23 mm.); Heuer, following Baetjer, found that numerous lymphatic sprouts arise from this sac and invade the intestine through the mesentery. This sac supplies lymph-vessels to the stomach, the liver capsule, the Wolffian bodies, and the repro- ductive glands. The lower pole of the lower lobe of the lung is continuous with the mesentery in the early stages. As the embryo develops, this connection becomes a thin band 60 DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. of tissue that passes down behind the diaphragm to end in the tissue around the aorta; it corresponds to the ligamentum pulmonale in the human. It is through this prolongation of the lower lobe that the lymphatics from the retroperitoneal sac grow up to reach the lung. These vessels arise from the cephalad portion of the sac and pass up behind the dorsal wall of the stomach to enter this long posterior or lower pole of the lung (figure 2, plate 4). There are three or four vessels that grow out from the sae and up into the lung; these are closely associated with those that pass to the diaphragm and, in adult life, join with them just before reaching the nodes into which they drain. They pass upward and divide, on reaching the lung, into two groups, one of which passes up over the diaphragmatic surface and the other over the outer or lateral surface of the lower lobe. The anlage of the hgamentum pulmonale is connected not only with the lower pole of the lung, but also with the median border of the lower lobe. Thus the lymphatics grow directly up about one-third of the way to the hilum in this medial extension of the ligament, and from there sweep out in a fork-like division which produces the two plexuses on the two borders of the lung (figure 3, plate 5). I have injected these vessels at 3.4 em-; but I think that they reach the lung border a little earlier. From the two plexuses described above vessels grow into the lung in exactly the reverse order to that followed by those developing from the hilum. They grow in just where they will meet the veins, and along these form the septal plexuses, exactly similar to those described above. These rapidly anastomose with the other lymphaties, and, by the time the embryo has reached 4 em. in length, the entire lung is uniformly supplied. It is very pertinent to inquire why the lymphatics that reach the lung from below select these points for the invasion of the deeper tissue of the lung. However, when it is recalled that the lymphatic vessels which lie in the mesenchymal tissue (the pleural anlaga) are very large in proportion to the other structures and that the budding vessel would be in direct relation to the outgoing veins, it is easily understood that exactly the same causes must be acting here as those which direct the growth from above. So here, as above, the position of the veins controls the direction of growth. Of course, the plexuses on the two surfaces become more complex as the lung is invaded and follow the same steps as the pleural supply in general. As has been said, there are branches along the pleura, and these anasto- mose with the other pleural vessels, so that the supply becomes general. The drainage in the early stages—that is, before the formation of the valves—is probably divided; the flow of lymph might be to the retroperitoneal sae via the vessels that grow up from that structure, or to the thoracic ducts through the tracheal plexuses and the vessels accompanying the veins and the bronchi. We have seen how the lymphaties grow into the lung-tissue and there form two distinet groups, and how one of these rapidly reaches the pleura and there forms the characteristic plexus-pattern marking off the boundaries of the lobules; also how the vessels grow into the posterior poles of the lower lobes and anastomose with the system from above, which follows the veins in the connective-tissue septa. DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. 61 Now, it will be well to review briefly the state of the development of the lung lymphatics at the time that the primary system is complete—that is, in 6 em. embryos. At6cm. the lymphatics around the trachea form a close-meshed plexus near the bifurcation, extending down into the lung around the bronchi. Above the bifurcation there are only a few connecting vessels on the ventral and dorsal surfaces of the trachea, but the two plexuses on the lateral surface are very close-meshed. From the left plexus the principal supply of both lungs is derived, but there are numerous vessels passing down into the right lung from the right plexus, and the two are closely bound together, especially near the bifurcation, where they have fused into one plexus. The vessels surrounding the bronchi follow them towards the periphery, giving off branches to the venous tree at every division of the bronchial tree. Each smaller bronchus derives its lymphatic supply from the plexus that accompanies the parent bronchus. These vessels are very difficult to inject. Accompanying the primary divisions of the pulmonary vein there is another group of vessels that is closely bound, by anastomoses, to the lymphatics around the principal bronchi (figure 4, plate 1). Along each of the tributary veins vessels pass to the pleura and spread out in the region that has been described as the septa between the lobules. Each of these dividing sheets anastomose with other sheets and with the pleural vessels. The vessels derived from the retroperitoneal sac are continouus with those derived from the two ducts; there can be determined no line of differentiation either within the lung-tissue or on the pleural surface. The poste- rior pole is connected with the retroperitoneal sac by three or four vessels that pass down in the fold of tissue that precedes the ligamentum pulmonale (figure 3, plate 5). The pleural plexus has begun to form within the gross markings that we have described as corresponding to the connective-tissue septa. These vessels are very superficial and are not connected, at this time, with the deeper vessels. The further development is chiefly due to the multiplication of the lung units and the increase in volume of the interbronchial tissue. As new bronchi are formed, new groups of lymphatics bud off from the plexus that accompanied the parent bronchus and follow the new-formed structure towards the periphery. These lymphatics leave the bronchus and pass to the venous group when they reach the region where the air-sacs are developing. As the lung-tissue differentiates further and further, the larger veins become more closely associated with the bronchi and only the terminal vessels are peripheral with reference to the lobule. This brings about the relations that are found in the adult, where the principal veins and bronchi are closely associated, while the terminal ones have the same relative positions that have been described for the developing structures. The arteries in early stages lie very close to the bronchi and are associated with the plexuses that follow that structure. As these blood-vessels increase in size the bronchial plexus differentiates into two parts, following the arteries and the bronchi. This is accomplished by the growth of vessels around the arteries, and, as the artery increases in size, the two plexuses become entirely distinct, but are still connected by numerous anastomotic vessels. 62 DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. In the meantime, the vessels of the pleura, which at from 5 to 6 em. we have seen beginning to form the true pleural plexus, continue to proliferate, and thus form a fine-meshed plexus in the pleura between the blocking-off of the lobules. The completion of the primary plexus is shown in figure 3, plate 1. This is the surface of the lung in a pig embryo of 6 em. with the pleural vessels injected. Each of these uninjected areas represents a primary lobule, and the surrounding lymphatics mark out the connective-tissue plexuses. Figure 1, plate 5, illustrates one of the primary lobules, and the close-meshed plexus is the true pleural supply. It is still seen to be connected with the deep vessels of the septum. Here and there one finds vessels passing from the terminal bronchi to the sur- face, in the lobule proper, to join with the fine-meshed plexus of the pleura. These pass around the air-cells, but are never found on their walls, and, uniting with the terminal vessels of the end veins, pass to join those in the pleura. These are the vessels described by Flint (1906) as seeming to dip down into the lobules from the pleura; these, he said, he could follow only a little way into the lobule. This is easily understood from the information gained from injections, for the vessels around the bronchi can not be seen in uninjected specimens, and consequently those which remain patent in sections seem to end abruptly in the midst of a lobule, whereas they in reality connect with those following the bronchi and terminal veins. The lymphaties that follow the terminal bronchi leave them just before the atria are reached and cross over to join the lymphatics which follow the veins. The lymphaties which accompany the veins pass to the pleura just where the veins bend to reach the center of the lobule. Flint first observed the submucous plexus of the bronchi and trachea in embryos 23 em. long. It was surprising that injections did not reveal this plexus very much earlier. I have not been able to demonstrate any lymphatics in the submucosa before the embryo reached a length of 19 em. This plexus develops, as do all the secondary plexuses, by the outgrowth of vessels from the primary one and their coalescence to form the new group. This process has been carefully studied by Heuer in the formation of the mucosal plexus in the intestine. The submucosal plexus is complete just before birth and consists of numerous fine vessels that lie just beneath the bronchial epithelium. From this plexus numerous vessels pass down between the cartilaginous rings and join the lymphatic trunks which follow the bronchi, as has been described. In those bronchi having no cartilaginous rings there is only the one group of lymph-vessels to be found, and these have already been described. The lymphaties of the adult lung were first described by Olaf Rudbeck in 1651-1654 (quoted from Miller, 1900). Since that time numerous workers have studied these vessels. In 1900 W.S. Miller reviewed the literature very thoroughly, and it will, therefore, be unnecessary to repeat that here. Muller studied the lymphatics in the lungs of adult cats and dogs by injecting them from one of the pleural vessels. He divided the lymphaties into four groups, as follows: \. The lymphatics of the bronchi. C. The lymphatics of the arteries. 3, B. The lymphatics of the veins. D. The lymphaties of the pleura. DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. 63 The lymphatics of the bronchi.—Miller describes two sets of lymph-vessels asso- ciated with those bronchi which have cartilaginous rings and only one with those which have no rings. In the former the two sets are connected by vessels that pass between the rings and join the trunks situated on the outer side of these structures. These trunks drain the lymphatics that accompany the smaller bronchi and empty into the nodes which are situated at the hilum of the lung. While there are several lymphatics accompanying the larger bronchi, only three are to be found with those nearer the air-sacs. These end by leaving the terminal bronchus just before it ends in the atria; one of them passes to the artery, while the other two join the lymphatics of the vein. The lymphatics of the veins.—There is a single group of vessels that extends from the terminal vein to the hilac nodes. Along the larger veins there are several vessels, but the terminal ones are accompanied by only one or two lymphaties. Anas- tomotic vessels pass from the bronchial lymphatics to join those of the vein at each branching of the bronchial tree. The lymphatics that accompany those veins which go to the pleura join the pleural lymphatics. The lymphatics of the arteries —The lymphatics which accompany the arteries are very similar to those of the veins, with the exception that none of them pass to the pleura. The lymphatics of the pleura.—There is only one plexus in the pleura, and this drains through several large trunks to the nodes at the hilum. There are anasto- moses with the lymphatics of the veins, as has been said, but the drainage probably does not pass through these. Miller put his canula into a large pleural vessel and injected towards the hilum. After some time the deep lymphatics, as well as those of the pleura, were filled. He thought that the injection mass backed up into the deep vessels from the nodes at the hilum, since both the sets of vessels drain into the same nodes. Miller does not confirm the findings of Sappey (1874) and of Councilman (1900) with regard to the interlobular lymphatics. Sappy thought that it was wrong to divide the lung lymphatics into superficial and deep groups on account of the rich anastomosis of these vessels. He thought that the lobules were surrounded by lymphatics which formed networks between the adjacent lobules in mueh the same manner as the blood capillaries do around the air-saes. Councilman divided the deep lymphatics of the lung into two sets, the bronchial and the interlobular; the latter he interpreted as very important in infections. While Miller does not agree with these observers in regard to the interlobular lymphatics, he does describe anastomoses between the lymph-vessels of the venous radicles and those of the pleura, and he emphasizes the peripheral location of the veins. It might well be that the vessels which Sappey and Councilman found in the interlobular septa were the lymphatics of the veins, since they did not have very accurate methods for the differentiation of these structures. It becomes more difficult to reconcile Miller’s findings with those of Flint and the results of this study. Both Flint and I have found distinct groups of vessels in the interlobular connective tissue in embryo pigs. These groups of vessels are directed in their growth and location by the position of the veins, but are not limited in their distri- 64 DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. bution to the venous trunks. The fact that so careful an observer as Miller does not find these lymphatics in the septa suggests the possibility that the assumption of mature activities in some way brings about the atrophy of all of the interlobular lymphaties except those that accompany the veins. Again, this plexus may be peculiar to the pig. It seems necessary that this question must remain unsettled until studied by some method other than simple injection. The question of the drainage of the lung lymphaties is of exceptional interest and importance; and while we must depend, for the final settlement, upon physio- logical methods, there is much evidence available from morphological observations. In the larger vessels on the bronchi, the veins, and the arteries there are valves which point towards the hilum. This is assumed to be very good evidence that the flow is in that direction. No valves have been described in the lymph-vessels which accompany the smaller bronchi, veins, and arteries. Hence it can not be stated whether the lymph flow, in the lymph-vessels of the veins, is towards the pleura of the hilum; and, in like manner, the flow in the bronchial vessels might be either towards the hilum or towards the veins and arteries. With regard to the vessels on the pleura, all of the lymphatics above a certain regional level of the lower lobe drain either towards the mid-line and then course up in the pulmonary ligament to end in the nodes at the hilum, or pass by direct paths to these nodes. Those below this level drain to the nodes lying in the mesentery of the lesser curvature of the stomach. Some of these drain as do those above—towards the median and pass down in the ligamentum pulmonale—while others pass directly down from the posterior pole. This group of vessels which pass to the preaortic nodes drains about one-third of the lower lobe of the lung. This varies considerably; in some specimens as much as half of the lung has been found to drain in this direction. This peculiar drainage of the lower lobes seems especially important from the bearing that it may have on the pathology of the lungs. It has long been known that the diaphragmatic vessels drain to these nodes, but there is no connection between these vessels and those of the lung proper. The lymphatics that pass through the pulmonary ligament apparently drain only the pleura; but, as has been shown, the deep lymphatics anastomose with those of the pleura, and therefore it seems possible for substances to pass from the lung-tissue to the preaortic nodes. What bearing this may have upon the pathology of the lungs or of the abdomen remains to be settled. SUMMARY. The lymphatics of the lungs are derived from three sourees—the right and the left thoracic ducts and the retroperitoneal sac. In embryos 2.6 to 3 em. long, vessels bud off from the thoracic duct and grow across to the trachea, forming there a plexus that gradually extends over the ventral surface of the trachea, and especially down over the bifurcation. From this plexus vessels pass into both lungs and into the pleura. The right thoracie duct divides, in embryos about 2.5 em. long, into two vessels. One passes to the heart, while the other breaks up to form a plexus on the right lateral wall of the trachea. Some vessels from this plexus pass down into the hilum DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. 65 of the right lung, while others anastomose with the plexus from the left side, which extends up over the trachea. The development of the lymphatics within the lung depends upon the division of the vessels into two groups—those associated with the veins and connective-tissue septa, and those associated with the arteries and the bronchi. The former grow very rapidly, and following each of the branches of the pulmonary vein, pass to the pleura. There are at first only two or three lymphatics with each vein. In the early stages the terminal veins lie about midway between the adjacent bronchi, and in this plane a sheet of lymphatics develops from the vessels surrounding the veins and passes to the pleura, where they mark out the boundaries of the distribution of each bronchus. These vessels anastomose with those that grow direct to the pleura from the plexus on the trachea. The bronchial vessels develop more slowly and at first are to be found only around the larger bronchi. As these structures increase in size and number,. the lymphatics surrounding the main bronchi send vessels to the smaller ones and these form a plexus around each of the bronchi, so that the bronchial tree is sur- rounded by a continual series of branching tubes made up of lymphatic vessels. From every point of division of the bronchi, lymphatic vessels pass to the lymphaties of the veins; those around the terminal bronchus leave it near its ending in the atria, and pass to join the lymphaties of the veins or septa, or, more rarely, those of the pleura. Lymphatics also arise from the retroperitoneal sac and grow up posterior to the diaphragm to enter the lower pole of the lower lobe of the lung. These vessels form a plexus on the median surface of the lower lobe, and send branches both to the pleura of the other surfaces and into the lung along the veins. Plexuses develop here as with those that come from above and the two groups soon anastomose. The further development consists in the multiplication of the plexuses on the bronchi and blood-vessels, following their continued differentiation. As the lung increases in size, the larger veins become approximated to the bronchi and only the terminal ones are separated from them; these lie in the periphery of the lobule. Connective tissue is formed along the sheets of lymphatic vessels, and these become the septa of the lung, containing a definite set of vessels which develop from the early vessels following the veins. The lymphatics accompanying the veins remain connected with those of the bronchi and septa. The common plexus surrounding the artery and bronchus is separated into two individual plexuses, incident to the increase in size of the artery; however, these continue to have anastomosing branches. The vessels of the pleura mark out the early connective-tissue septa, but later there develops a fine-meshed plexus between these larger vessels, which is not connected with the vessels of the lung-tissue. The valves begin to form at about 6 em. and, in general, point away from the pleura. None, however, have been found in the smaller vessels which accompany the terminal bronchi. In the adult tnere are lymphatic vessels accompanying the bronchi, the arteries and the veins; these anastomose freely. There are also vessels in the connective- tissue septa which drain chiefly into those around the veins, and, to some extent, 66 DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. into those of the bronchi and arteries, near the point where the vein and the bronchus separate to take their relative positions with relation to the lobule. There are numerous anastomoses between the deep vessels and those of the pleura, but prob- ably most of the flow is towards the hilum. All the deep vessels, together with the greater number of the pleural vessels, drain into the nodes at the hilum; but the vessels of the lower half of the pleura of the lower lobe drain through several vessels to the preaortic nodes. and behind the diaphragm. These vessels pass through the ligament of the lower lobe BIBLIOGRAPHY. Baerser, WALTER A.: On the origin of the mesenteric sac and thoracic duct in the embryo pig. Amer. Jour. Anat., Philadelphia, 1908, vu. Crark, A. H.: On the fate of the jugular lymph sacs, and the development of the lymph channels in the neck of the pig. Amer. Jour. Anat., Philadelphia, 1912, rx. Crark, E. R.: Observations on living growing lymphatics in the tail of the frog larva. Anat. Record, Phila- delphia, 1909, m1. An examination of the methods used in thé study of the development of the lymphatic system. Anat. Record, Philadelphia, 1911, v. . Further observations on living growing lymphatics: their relation to the mesenchyme cells. Amer. Journ. Anat., Philadelphia, 1912, x11. CouncitMan, W. T.: The lobule of the lung and its relations to the lymphatics. Journ. Boston Soc. Med. Science, Boston, 1900, tv. CRUIKSHANK, W.: The anatomy of the absorbing vessels of the human body. London, 1790. Detemere, G., P. Porrrer, and B. Cuneo: The lymphatics. 1904. Furnt, J. M.: The development of the lungs. Anat., Philadelphia, 1906, vr. Hever, G. J.: The development of the lymphatics in the small intestine of the pig. Amer. Jour. Anat., Philadelphia, 1909, rx. Kampmerer, O. F.: The development of the thoracie duct in the pig. Amer. Jour. Anat., Philadelphia, 1912, x11. Kern, E.: Anat. of the lymphatic system. London, 1875, 11. Lewis, F. T.: The development of the lymphatic system in rabbits. Amer. Jour. of Anat., Philadelphia, 1906, v. McCuure, C. F. W.: The development of the thoracic duct and right lymphatic ducts in the domestic cat. Anat. Anz., Jena, 1908, xxx11. Masecaont, Paun.: Vasorum lymphaticorum corporis humani historia et ichnographia. Senis 1787. Amer. Jour. Miter, W.S8.: The structure of the lungs. 1893, vu. The lymphatics of the lungs. 1896, xu. - Das Lungenlippchen, seine, Blit- und Lymph- gefiisse. Archiv. fiir Anat. und Physiologie, Leip- zig, 1900. . Anatomy of the lungs. Reference Handbook of the Med. Sciences, 1902, 575-586. Lymphoid tissue of the lung. Anat. Record, Phil- adelphia, 1911, v. PApPENHEIM, —: Sur les lymphatiques des poumons et du diaphragme. Compt. Rend., 1860, xxx. Porrter and CHArpEy: Treatise of human anatomy. Saprn, F. R.: On the development of the superficial lym- phatics in the skin of the pig. Amer. Jour. Anat., Baltimore, 1904, mm. . The lymphatie system in human embryo, with a consideration of the morphology of the system as a whole. Amer. Jour. Anat., Philadelphia, 1909, rx. . Der Ursprung und die Entwickelung des Lymphge- fassystems. Ergebnisse der Anatomie und Ent- wickelungsgeschichte, Wiesbaden, 1913, xx1. . The origin and development of the lymphatic sys- tem. The Johns Hopkins Hospital Reports, Mono- graphs, new series, 1913, v. P. C.: Anatomie, physiologie, pathologie des vaisseaux lymphatiques. Paris, 1874. Srkorski, J.: Ueber die Lymphgefiisse der Lungen. tralbl. f. Medicin. Wissensch., 1870. TricuMan, L.: Ueber Lungenlymphegefiisse. Anz. d. Akad. d. Wissensch., in Krakau, 1896. von Wrrricu, W.: Ueber die Beziehungen der Lungenal- veolen zum Lymphsystem. Mitth. Anz. d. Kénigsberger Phys. Laborat., 1878. Wywopzorr, —.: Die Lymphgewege der Lunge. Medicin. Jahrbucher, 1866, x1. Jour. Morph., Anat. Anz., Jena, Saprey, Cen- Wiener Fic. Fic. Fic. Fic. Fig. 5. Fic. Fie. 3 Vic. Fic. Fic. Fie. 4 Fic. ¢ EXPLANATION OF PLATES. PLATE 1. . Diagram of transverse section of left lung of an embryo pig 3 cm. long, in which the blood-vessels were injected through the umbilical artery with india ink. The lymphatics appear as dilated spaces (blue). The sec- tion is 204 thick and is stained with hematoxylin and eosin, aurantia,and orange G. 55. Ao, aorta; T, trachea. 2. Diagram of section through lobule of lung of an embryo pig 7 cm. long, in which the lymphatics were injected with india ink through the left tracheal plexus. The veins were slightly injected by the rupture of a lymphatic vessel into a vein near the hilum. The section is 1004 thick and is unstained. 47.5. A, artery; B, bronchus, V, vein; Pl, pleura. . Surface of lung of an embryo pig 6 em. long, in which the lymphatics were injected with india ink through the left tracheal plexus. The section was taken from the ventro-lateral surface of the left lower lobe and is about 2004 thick and is unstained. 29.4. PL, primary lobule. . Longitudinal section of lung of an embryo pig 6 cm. long, in which the lymphatics were injected with india ink through the left tracheal plexus. The veins contain some blood pigment. The section is 4004 thick and is unstained. X33. V, vein; B, bronchus. Diagram of left tracheal plexus in an embryo pig 6 cm. long, in which the lymphatics were injected through the thoracic duct. Cleared by Spalteholz method. Note that part of the vessel marked with an asterisk (*) has been removed in dissecting the body-wall away. This vessel is the one described as the first to the lung. X15. *, first vessel to lung; 7, trachea; Th D, thoracic duct; L T P., left tracheal plexus; Ao, aorta. PLaTE 2. . Dissection of an embryo pig 4 em. long, in which the lymphatics were injected with prussian blue through the retroperitoneal sac. The heart, aortic arch, left lung, and the body-wall have been removed. Cleared by the Spalteholz method. X19. Th D, thoracic duct; R Th D right thoracic duct; R T P, right tracheal plexus; L T P, left tracheal plexus; C L, cardiac lymphatics; Ao, aorta; B, bronchus; Oe, esophagus. . Section of a small area of the lung of an embryo pig 7 cm. long, in which the lymphatics were injected with prussian blue through the retroperitoneal sac. Drawing to show the relation of the peri-bronchial lymphatics to the wall of the bronchus. Section is 20x thick and is stained with hematoxylin and eosin, aurantia, and orange G. X93. 3B, bronchus. . Dissection of an embryo pig 4 em. long, in which the lymphatics were injected with prussian blue through the retroperitoneal sac. The left lung, the arch of the aorta, the pulmonary artery, and the body-wall have been removed. Cleared by the Spalteholz method. The left tracheal plexus is shown as a solid blue mass because the meshes are so close that they could not be analyzed in the drawing. X15. Th D, thoracic duct; R Th D, right thoracic duct; Ao, aorta; L T P, left tracheal plexus; B, bronchus. . Longitudinal section of upper lobe of right lung of an embryo pig 6 em. long, in which the lymphatics were injected with prussian blue through the retroperitoneal sac, and the veins were injected with india ink through the pulmonary vein. The section is 4004 thick and is unstained. Cleared by the Spalteholz method. X39. PLaTE 3. - Small block of an embryo pig 15 em. long, in which the lymphatics were injected with prussian blue by punc- ture of aninterlobular septum. The arteries were injected with india ink through the pulmonary artery. Cleared by the Spalteholz method and mounted in balsam. The specimen was mounted at a convenient angle to best show the interlobular septum; unfortunately, it was jarred out of position while being drawn and hence the group of lymphatics in the septum is shown bent to one side. X40. PI, pleura; A, artery; J LS, interlobular septum. . Diagram of a dissection of an embryo pig 3 cm. long, in which both the right and left jugular sacs were injected and, from them, the right and the left thoracic ducts respectively. India ink was used. The body-wall, heart, and left lung have been removed. Cleared by Spalteholz method. 30. Th D, thoracic duct; R Th D, right thoracie duct; V C S, vena eava superior; Ao, aorta; P A, pulmonary artery; C L, car- diac branch of the right thoracic duct. . Diagram of a section of the right lung of an embryo pig 6 cm. long. This is the same specimen from which figure 4, plate 2, was made; the part of the section shown in that figure is indicated by an X. X20. 67 a » 7 dat : — = 4 . Se - : eh |. iy 68 DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. ; > 2h: 3 = PuaTe 4. : “iy Fi. 1, Longitudinal section of the left lung of an embryo pig 5 em. long, in which the lymphatics were injected with — prussian blue through the retroperitoneal sac. The veins have some blood pigment in them. The section is 4004 thick and is unstained. X22. V, vein. ppd el : Fic. 2. Dissection of an embryo pig 4 cm. long, in which the lymphatics were injected with prussian blue from the — retroperitoneal sac. The right lung, esophagus, and body-wall have been removed. The stomach was pulled to the left side of the embryo in order to expose the retroperitoneal sac. Cleared by the Spalteholz method. X20. Ao, aorta; L L, left lung; Th D, thoracic duct; D, diaphragm; R P S, retro- peritoneal sac; *, first vessel to the lung. ; ee Puate 5. ) a ay Fig. 1. Surface of lung of an embryo pig 23 cm. long, in which the lymphatics were injected with prussian puncture of an interlobular septum. Cleared by Spalteholz method. The interlobular septum cated by a very large lymphatic trunk. 28. J LS, interlobular septum. ‘ Fic. 2. Longitudinal section of the upper portion of the lower lobe of the left lung of an embryo pig 5 cm. long, in which the lymphatics were injected with prussian blue through the retroperitoneal sac, and the veins have retained a little blood pigment. The section is 4004 thick and is unstained. 57. V, vein; — A, artery; Pl, pleura; B, bronchus. F Fic. 3. Lower portion of left lung of an embryo pig 5 cm. long, in which the lymphaties were injected with india ink through the retroperitoneal sac. Cleared by the Spalteholz method and mounted in balsam. X28. PLATE 2 CUNNINGHAM Campbell Ari Companr J. F. Didusch fecit PLATE 3 CUNNINGHAM SHH Ff. Th. D icay ak ((egnrcnal J. F. Dicusch fecit CUNNINGHAM By PLATE 4 2(C. R. 4m) Fig. 1 (C. R, 5em) g. Pio J. ¥. Didusch fect a A \ CONTRIBUTIONS TO EMBRYOLOGY, No. 13. BINUCLEATE CELLS IN TISSUE CULTURES. By CuHarzes C. MAcktin. Four plates, containing seventy figures. 59 PAGE. The binucleate cell—Continued. Introduction): (osc ees ie ecatace 71 (c) Origin: Method: (1)! Dheoreticallie ascn sence... (a) Preparations of the cultures: (2) Observations: (CU Mieswerandiamedine. tateccticweees sc 71 LAVINg 2 Se s< sn eee (2); @Gultnvationye. ee ane kava ecclesia. 2 72 Pixeds . Sacceka, aan (b) Observations: (3) Mechanism’, .o455.0 nee (1) Living: (d) Fate: Continuous observation.............. 73 (1) Observations: AAU ONT Coke eo 74 Living and fixed.......... (2) Fixed .. Fee ee a tA 75 Nuclear Fragmentation ............... Wha puerta eee acc n <1,012. Figs. 11, 12, 13. These figures show a simple degree of nuclear fragmentation. They were found in a culture in which the cells were otherwise apparently normal. In figure 11 the nucleus is constricted at one end; the larger portion contains two nucleoli of unequal size and irregular shape. In figures 12 and 13 the con- striction is farther advanced. No. 14, Lewis (see fig. 2). 1,012. Fic. 14. Prophase of mitosis. Nuclear membrane and nucleoli are disappearing and skein is forming; cell not yet Fie. 15 Fic. 16 Fig. 17 rounded. No. 14, Lewis (see fig. 2). This, and the four figures which follow it, represent the process of mitosis in a mononucleate cell. All drawn from the same preparation. 1,012. . Metaphase. Cell rounded and compact; processes drawn in; cytoplasm granular and stains very densely with hematoxylin; definite spindle with equatorial plate of chromosomes. 1,012. . Anaphase. The chromosomes have separated and the remains of the spindle may be seen as faintly defined streaks connecting the two aggregations of chromosomes; cytoplasm still densely granular and darkly staining, the entire cell contracted; cell-processes small and thread-like. 1,012. . Early telophase. Chromosomes less clearly marked, the chromatin masses breaking up. No evidence of nuclear membranes is to be seen. The cytoplasm is dividing, as shown by constriction about the equator. Markedly granular and darkly staining protoplasm. 1,012. 105 Fig. Fic. ‘ Fig. ¢ BINUCLEATE CELLS IN TISSUE CULTURES. aate telophase. The two daughter cells are seen, separated and spread out thinly; protoplasm stains much more lightly; nuclei well defined and contain coarsely granular chromatin. In each daughter nucleus the beginning of a nucleolus is to be seen; but is very small compared with the size of this body at maturity. 1,012. . Late prophase of mitosis, in cell probably of connective-tissue type; two centrospheres at opposite poles; nuclear membrane has disappeared; spireme well marked; mitochondria short and thick, No. 42, Lewis (see fig. 1). Figures 20 and 21 are from the same preparation. 1,032. . Early prophase in a nucleus showing beginning direct unilateral fission; skein forming, nucleoli disappearing; centrosphere still single, situated in the fissure; mitochondria becoming shorter and thicker, and are intermediate in these respects between those seen in figure 8 and those of figures 19 and 21; nuclear mem- brare has almost disappeared. Cell of connective-tissue type. 1,032. . Late prophase in a nucleus undergoing direct unilateral fission. Skein has formed and nuclear membrane has disappeared; one centrosphere is to be seen in the cleft, and there is some indication of a second on the opposite side of the nucleus, in the area devoid of mitochondria. 1,032. . Prophase in a binucleate cell. Early stage. Skein is forming; membrane and nucleoli are disappearing. Some chromatin has become segregated in the area of contact between the two parts. The method of fixation and staining does not permit of the centrosphere and mitochondria being seen. No. 14 Lewis (see fig. 2). 1,012. . Prophase in a binucleate cell of connective-tissue type; somewhat later stage than figure 22. In each nuclear portion there has been formed simultaneously a skein and the nuclear membrane has disappeared. The chromatin material of the combined double nucleus will form a single equatorial plate of chromosomes, as in figure 67. Only one centrosphere, containing two centrosomes, is seen in the preparation, situated at one extremity of the fusing nucleus, it having come from the interval between the nuclear parts. It is thus probable that the spindle will form parallel with the long axis of the fusing nucleus. Mitochondria are short and thick. No. 18, 21: 2:14, Lewis. 7-day chick heart, grown for 2 days in Locke (0.25 per cent dextrose); osmic-acid vapor and iron hematoxylin. 1,032. Puate III. Fias. 24-35. A series of drawings from a living connective-tissue cell made at 15-minute intervals for 2? hours. The nucleus at the start was elongated and notched at one side. It was seen to take various forms, and ended as two separate nuclear parts. The series thus shows direct nuclear fission. It will be seen that the centrosphere is contained within the unilateral cleft, and when the nucleus ultimately divides the centrosphere is situated between the parts of the nucleus; mitochondria stream across the interval separating these two parts. The nucleolar bodies undergo interesting changes. The nuclear outlines, position of nucleoli and centrosphere, the cell outlines, and principal features of the cytoplasm were sketched in freehand from direct observation of the living cell. The drawings were afterwards retouched by reference to fixed preparations. Small circles represent fat globules, and short threads mitochondria. c, figure 24, marks the centrosphere. 5-day chick heart; 57 hours’ cultivation, from No. 7, 9:1: 15, in Locke (0.5 per cent dextrose) with extract of chick embryo. about 900. Fias. 36-47. Fragmenting nuclei showing probable effect on form of nucleus of prolonged growth in unchanged media; outlines of nuclei very irregular, each has a number of lobes; in some cases separation of these lobes has taken place. Culture shows other evidences of degeneration. No division of the cytoplasm following division of the nucleus was observed. Drawn from various cells selected from No. 23, 12: 1:15 (Lewis). 5-day chick stomach in Locke (0.5 per cent dextrose). Zenker; Mallory connective-tissue stain. Culture grown for 6 days in the same media. 1,012. c Fias. 48-58. A collection of nuclei of irregular form, grown in media containing aleohol; centrospheres are sketched in to show their characteristic relationship. Same preparation as figure 10. 1,500. Fic. 59. A regular paired nucleus from the same preparation as that from which the series 48-58 was drawn. Some of the nuclei have escaped distortion. 1,500. Puate IV. Fics. 60-70. A series of camera-lucida drawings from a single living binucleate cell of the connective-tissue type, which was observed continuously for 8 hours. At the beginning there were two separate nuclear parts, with one centrosphere; the parts combined to form a single mitotic figure, and the successive stages of mitosis are seen in figures 66 to 70. The ultimate result is two separate mononuclear cells, each con- taining a single centrosphere. The series brings out the fact that the parts of the “double” nucleus are not independent so far as their reproductive capacity is concerned, but in cell division they combine and act asa single nucleus. c, figure 60, represents the centrosphere. 7-day chick heart, grown for 19 hours when the observation commenced. Locke (1 per cent dextrose) with extract of chick embryo. Culture of March 15, 1915. 1,500. MACKLIN " , of on PLATE i! MACKLIN 19 MACKLIN 6.4. fea PLATE iil 10.30 p.m. 31 C.C.M. del. PHOTO LITH_ BYAHOEN & £0. GALTIM ORE, MD. MACKLIN PLATE | C.C. M. del. PHOTO LITH_ BY AHOEN BED. BALTIMORE MO CONTRIBUTIONS TO EMBRYOLOGY VotuME V, No. 14 PUBLISHED BY THE CARNEGIE INSTITUTION OF WASHINGTON WASHINGTON, 1917 CARNEGIE INSTITUTION OF WASHINGTON : PusuicaTion No. 225 PRESS OF GIBSON BROTHERS WASHINGTON CONTRIBUTIONS TO EMBRYOLOGY, NO. 14 THE DEVELOPMENT OF THE CEREBRO-SPINAL SPACES IN PIG AND IN MAN By Lewis H. WrEEp il Ol SOOTY A aD SHOTT aa aoe ¥ i " rat fee eathaaies k e ev ant | a | Kew WD UKA OO Le a wae Ve (TP apyeel pe CONTENTS. Rep ENETOCUCCORY co ote olere fs nie'ofealstire craic, «sas ave IS Meview Of literature: m te home ; eee a. rier | i : : ately ad -_ < ‘ ia a 4 * fothawy ane edt % oypdome Se a sb Ate hak et] then, tamed ‘i.oeet Am alt ‘ +. aleesten ged ie a) ms s«elirntgiawed Aneeig? oe ja Cy aall, Deh) Hogh rat ot ' ane lortpemei oy £2 - ane (2) i aor ee we wes) a os ‘ Ub eakade wiaG ceniant’ ore off ‘ fas Grow = Ses) t 1M eae Ale ra oT : : oar geese Af og $ ae * o iv: Gee pce aw abn ta . seein hel ey THE DEVELOPMENT OF THE CEREBRO-SPINAL SPACES IN PIG AND IN MAN, By Lewis H. WEEp. I. INTRODUCTORY. Probably no field in embryology has been less explored than that relating to the meninges. Our knowledge of the transformation of the perimedullary mesen- chyme into the three fully developed membranes about the cerebro-spinal axis has been largely of a crude sort, with gross generalities based on inexact or incomplete evidence. The present work was undertaken in the hope that by a study of the various stages in the development of the cerebro-spinal spaces there might be gained some knowledge which would afford a basis for a conception of this dynamic metamorphosis. Many of the problems centering around the development of the meningeal spaces have recently been expounded by Cushing®).* Not only do we lack knowledge as to the method of differentiation of the primitive mesenchyme, but we know little about the establishment of the circulation of the cerebro-spinal fluid. When do the chorioid plexuses begin to secrete? When does the venous absorption of the fluid take place? When does the perivascular system begin to remove waste products from the cerebral tissue? And also, what factors play a part in the formation of the subarachnoid and subdural spaces? These questions, some of which it is hoped the present study will answer, relate to the field of physiological anatomy. Consideration of the subject, however, serves to convince one that they must be investigated coincidently with the stages of morphological differentiation; for it may readily be conceived that the physiological use of the meningeal spaces may precede any morphological differentiation of the three membranes, nor indeed is it unlikely that one of the active causative factors in the metamerphosis concerns this filling of the mesenchyme about the nervous system with fluid. This study, therefore, has been anatomical, but with a broader scope than purely morphological studies would have afforded. Not only has it dealt with the morphological differentiations about the nervous system, but throughout the inves- tigation the relationship of these structures to the possible presence of cerebro- spinal fluid has been considered. As the problem developed it was projected more and more into the difficult realm of “tissue spaces.’’ Interest in these spaces largely concerned their physiology, but many points of correspondence between structure and function were found. In some measure this work is a development of an earlier study of some of the anatomical and physiological problems of the cerebro-spinal fluid, carried out in the laboratory of Dr. Harvey Cushing at the Harvard Medical School. *The figures in parentheses refer to the bibliography at the end of this paper. 8 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. I]. REVIEW OF LITERATURE. In order fully to understand the problems which confront one in the study of the embryonic cerebro-spinal spaces, a comprehension of the stage to which investi- gations have brought our knowledge of these fluid-pathways in the adult is neces- sary. It is with this purpose that the adult relationships are here considered. The inclusion of this material may be pardoned, for it will be seen that unanimity of opinion has by no means existed in regard to any of the problems concerned in the circulation of the cerebro-spinal fluid. Modern anatomical knowledge of the meninges dates from the work of Axel Key and Gustav Retzius?%). These Swedish investigators, in their excellent mono- graph published in 1875, first conclusively demonstrated the anatomical continuity of the spinal and cerebral subarachnoid spaces. But for years after their publica- tion appeared, a physiological continuity between the subdural and subarachnoid spaces was argued for by many observers, notably by Hill@#). Gradually, however, workers in this field have reached the opinion that the subarachnoid spaces (the interrupted but continuous channels between arachnoidea and pia) are functionally the channels for the cerebro-spinal fluid. Between the intra-leptomeningeal and the subdural spaces no anatomical connection exists; physiologically there may be some mode of fluid-passage. Thus Hill" states that either by filtration or through actual foramina fluid passes readily from one space to the other. Quincke'4®), from observations on animals, somewhat similarly premised a connection between the two spaces, but only in the direction from subdural to subarachnoid. His experi- ments, based on the results of the injection of cinnabar granules, are open to criti- cism as indicating a normal passage-way for the fluid; for, as he has recorded, an intense phagocytosis of practically all of his granules occurred. More modern con- ceptions of the subdural space treat it as a space anatomically closed, lined externally by a polygonal mesothelium. Less error is introduced if it be regarded as analogous in many respects to well-known serous cavities rather than as an essential portion of the pathway for the cerebro-spinal fluid. The question of the absorption or escape of cerebro-spinal fluid from the sub- arachnoid space has claimed the attention of many workers. Since the original conception that the meningeal coverings were actually serous cavities, anatomical investigations have furnished many new views. Key and Retzius, by spinal sub- arachnoid injection of gelatine masses colored with Berlin blue, demonstrated an apparent passage of the injection fluid into the great cerebral venous sinuses through the Pacchionian granulations (die Arachnoidzotten). Their observations were made on a cadaver and the injections carried out under fairly low pressures (about 60 mm. of mercury). A lesser drainage of the fluid into the lymphatics was also shown. Since the view advanced by Key and Retzius of the absorption of cerebro- spinal fluid, the general trend has been away from the idea of an absorption into the venous sinuses. Quincke’s'46) observations, made on lower animals after the sub- arachnoid introduction of cinnabar granules, really offer some substantiation of REVIEW OF LITERATURE. 9 this theory, but the failure to find the great Pacchionian granulations in infants and in the lower animals caused many workers to reject utterly the conception of the Pacchionian granulations as the functionally active mechanism for the fluid escape. Physiological evidence, however, advanced by Hill(24) from intraspinous injec- tion of methylene blue, indicated that the major escape of the cerebro-spinal fluid was into the venous sinuses of the dura, while a slow and minor absorption took place along the lymphatic channels. Ziegler”), with potassium ferrocyanide intro- duced into the cerebro-spinal space, likewise found that the venous absorption was much greater and more rapid than the lymphatic. Reiner and Schnitzler48) with the same agent detected the ferrocyanide in the jugular blood-stream after injection. With olive oil these investigators found a similar venous absorption, but with a slowing of the venous blood-stream. Lewandowsky’3?), also using ferrocyanide, found this salt in the urine within 30 minutes after its subarachnoid injection. Spina2), from observations on freshly killed and living animals, presented somewhat similar evidence of a major venous and lesser lymphatic absorption. Cushing’) suggested a valve-like mechanism of escape of the fluid, his hypothesis being based on the findings after the introduction of mercury into the meningeal spaces. Several theories concerning the absorption of cerebro-spinal fluid into the blood- vascular system have more recently been offered. Mott‘), from a study of dilated perivascular and perineuronal spaces, has advanced the idea of fluid-escape by way of the perivascular system into the cerebral capillaries. Dandy and Blackfan{®), from an analysis of their evidence, consider that the chief drainage of the fluid is into the capillaries of the pia-arachnoid. Opposed to this conception of a major drainage of cerebro-spinal fluid into the blood-vascular system is the view cham- pioned by Cathelin, that the lymphatic drainage is the chief method of fluid-escape. Cathelin’s contention of a veritable circulation of the fluid has not received support from other workers. Thus it will be seen that since the work of Key and Retzius the trend of opinion has been away from the view that the Pacchionian granulations carry the cerebro- spinal fluid into the venous sinuses. In the earlier investigation) carried out in the Harvard Medical School the problems of this fluid absorption were attacked in a somewhat different manner than by previous workers. True solutions of potassium ferrocyanide and iron- ammonium citrate, such as have been used in the present investigation, were injected into the spinal subarachnoid space under pressures but slightly above the normal. The animals (dogs, cats, and monkeys) were kept under anesthesia during the period of injection, which was usually continued for several hours. Complete filling of the subarachnoid channels was secured by this technique, provided the injections were continued for a sufficient length of time. At the conclusion of the experiment the foreign solution was precipitated in situ and blocks were carried through for histological purposes. Many of the anatomical findings in this work carried out as outlined are of interest in the present problem. The complete correspondence of the spinal and 10 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. cerebral subarachnoid spaces as demonstrated by Key and Retzius was amply veri- fied. The normal return of the cerebro-spinal fluid to the general circulation by way of the arachnoidal villi into the great dural sinuses was demonstrated. These villi are projections of the arachnoidea through the dural wall, prolonged directly beneath the vascular endothelium of the venous sinuses. Furthermore, columns of arachnoid cells were found, normally affording fluid channels in the dura. In addition to the major escape of cerebro-spinal fluid into the sinuses a lesser drainage was also demonstrated, slower than the primary drainage, out along certain of the emergent nerves into the lymphatic system. No evidence whatsoever was obtained in support of any of the theories of a drainage of cerebro-spinal fluid into either the leptomeningeal or cerebral capillaries, nor could an anatomical valve-like mechanism along the great sagittal sinus be demonstrated. The process of escape of cerebro- spinal fluid from the arachnoid villus unto the great sinus appeared to be a simple one of filtration or of diffusion. Another of the problems concerning cerebro-spinal fluid, which has been of interest to anatomists and physiologists, is the source of the fluid. Haller?!) and Magendie®), to whom the greatest credit for work on this subject must be given, believed it to be the product of the leptomeninges. Faivre >) in 1853 and Luschka(4) in 1855 were the first to suggest the chorioid plexuses as the elaborators of this cireumambient medium. Since then the view has been generally accepted that these villous structures do give origin to the fluid, but the early evidence was based wholly on the glandular character of the plexus. Cappelletti® and Pettit and Girard(9) offered more definite proof of this relationship by the introduction of pharmaco- logical agents which affected the rate of production of the fluid. These latter authors recorded definite histological changes in the cells of the plexus when influenced by these drugs, indicating, in conjunction with the changed rate of production of the fluid, an undoubted relationship of the chorioid plexus to the fluid elaboration. Since these early investigations many observers—Findlay“@”, Meek®87, Mott@), Pellizzi‘?), Hworostuchin(?®), and others—have studied the histology of the chorioid plexus with reference to its function as an elaborator of the cerebro-spinal fluid. In addition to the elaboration of the fluid by the chorioid plexuses, increments are furnished by the nervous tissue itself. This elimination from the nervous system occurs by way of the perivascular spaces. In the previous work referred to'55) it was found possible to inject the entire perivascular system by continuing a physiological injection of the spinal subarachnoid space, and subsequently causing an extreme cerebral anemia. By this procedure an injection of the system to its termination about the cerebral capillaries and nerve-cells could be secured. From this and other evidence the view was advanced that the cerebro-spinal fluid was derived from a dual source—in part from the perivascular system and in greater part from the chorioid plexuses. This view had already been advanced, but on rather insufficient grounds, by Mestrezat®) and by Plaut, Rehm, and Schottmuller™4), Recently Frazier™®) has signified his acceptance of this conception of the source of the fluid. REVIEW OF LITERATURE. 11 Such, then, is the basis for our present understanding of the meninges, in regard to their characteristic morphology and particularly their functional relationship to the cerebro-spinal fluid. Without a consideration of the cireumambient fluid morpho- logical studies of these membranes would be incomplete, for in order to understand the meninges knowledge concerning the cerebro-spinal fluid is necessary. THE COMPARATIVE ANATOMY OF THE MENINGES. Sterzi®%) has published a comprehensive report of the comparative anatomy of the spinal meninges. From his studies he has advanced hypotheses, supported by observations on a limited number of fetuses, regarding the development of the human meninges. On account of the interest of this subject in relation to the present dis- cussion a brief summary of Sterzi’s work will be here included. In the acrania there is no special envelope of the central nervous system, but rather a fibrous sheath corresponding to the meninges of higher forms. This fibrous sheath is largely made up of circular fibers, except in the median ventral line, where there occurs a ventral ligament of longitudinal fibers. In cyclostomes, how- ever, there is found a single “primitive meninx’’—vascular and composed of white and elastic fibrils coursing in a longitudinal direction. Some of these fibrils traverse the perimeningeal spaces (filled with star-like cells, with some fatty tissue) and are attached to the inner surface of the vertebre. This same general plan of a single “primitive meninx”’ is likewise found in fishes (elasmobranchs, teleosts, etc.); the membrane here is often pigmented and follows closely the external architecture of the spinal cord. The perimeningeal space is filled by mucus in elasmobranchs, but in teleosts this is replaced by fat. For the most part there are found dorsal and ventral ligaments and two lateral ligaments. The next stage in the development of a more complete form of spinal covering is found in the urodele amphibia. A “primitive meninx,” formed of two layers, often artificially separated from each other, replaces the simpler meninx of cyclo- stomes and fishes. Of the two layers in this membrane the external is thin and free from pigment; the inner, strongly pigmented, adheres to the spinal cord. The meninx is perforated by the denticulate ligaments. In amphibia (Anura) Sterzi found the first evidence of a “secondary meninx,”’ corresponding to the pia-arachnoid. Surrounding this membrane, but separated from it, is the dura, thin and transparent; between the two meninges is the intra- dural (subdural) space. The dura lies in the peridural space. The spinal pro- longations of the endolymphatic canals lie in the dorsal part of the peridural space. Both the dura and the ‘“‘secondary meninx”’ continue outward along the roots of the spinal nerves and along the filum terminale. Embryologically the perimedul- lary mesenchyme is differentiated into these two meninges in the Anura. This arrangement of the two meninges in Anura is followed out in reptiles. The dura, thin as in the amphibia, is covered by endothelium and is vascular. The “secondary meninx”’ possesses laterally the denticulated ligaments and ventrally the ventral ligament. Both the peridural and intradural spaces are very small. 12 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. Likewise in birds Sterzi was able to differentiate only two meninges—the dura and the “secondary meninx.’’ These membranes are quite similar to those of reptiles. The “secondary meninx” has acquired three layers—an outer endothelial covering, a middle vascular layer, and an inner membrane closely adhering to the cord. This is a distinct approach to the three meninges of mammals. An intra- dural (subdural) space covered by endothelium can be easily made out. The development of these avian meninges concerns a differentiation of the perimedullary mesenchyme. The arachnoid, according to Sterzi, first appears as a definite membrane in mammals (marsupials and placentals). In marsupials this arachnoid has become well differentiated and the pia mater possesses denticulated and ventral ligaments. A transformation of the extradural portion of the denticulated ligaments unites the dura to the endorachis. In perissodactyla the differentiation of the three meninges (particularly of the arachnoid) is incomplete. The arachnoid is separated from the pia mater by a peculiar tissue which contains numerous lymphatic lakes, forming the intra-arachnoid spaces. No intradural (subdural) space is apparent, due to the approximation of dura and arachnoid. The subdural space is clothed by endo- thelial cells; these can not be made out in the intra-arachnoid spaces. The dura is surrounded by a fatty pad. According to Sterzi the augmentation of the intra-arachnoid (subarachnoid) space is the distinguishing characteristic of the meninges of carnivora. This increase takes place at the expense of the peridural space. As Sterzi developed the knowledge of the comparative anatomy of the lower forms—of the transition from the primitive meninx of the cyclostomes to the three membranes of mammals—the possible correlation of this analogy to the embryo- logical development in mammals became apparent. He extended his observations to human beings and to human fetuses. His findings will be detailed in the follow- ing section. Farrar(®), in a short discussion of the development of the meninges of the chick, finds in early stages three lamin about the spinal cord, ‘‘the middle one of which alone still presents the primitive features of the mesoblastic-sheath.” The inner layer, close to the medullary tissue, is highly vascular; in the outer zone “the con- nective-tissue elements are assuming elongated forms and crowding together with long axes parallel, giving a very close mesh with long but extremely narrow spaces, in contradistinction to the loose irregular reticulum of the pia-arachnoid.” The outer lamina becomes dura mater, while the inner two zones are considered together as the embryonic pia-arachnoid. Farrar defines the pia-arachnoid as develop- mentally a single membrane consisting of a loose reticulum, at the outer and inner borders of which limiting membranes are formed. REVIEW OF LITERATURE. 13 LITERATURE ON THE DEVELOPMENT OF THE MAMMALIAN MENINGEAL SPACES. The development of the meningeal spaces in mammals has not been studied extensively, and the literature in regard to it is quite meager. Only a very few workers have touched upon the subject except casually. Reford“47), working in the Anatomical Laboratory of the Johns Hopkins University, studied the development of these spaces by the method of injection with india ink. His work unfortunately has never been published, but it has been rather extensively referred to by Sabin9) in 1912 and by Cushing®) in 1914. Their summaries of this work are here included. Miss Sabin thus speaks of it: “In a study of the arachnoid made by the injection method in the Anatomical Labora- tory of the Johns Hopkins University by L. L. Reford, and as yet unpublished, it has been shown that the thinning out of the mesenchyme around the central nervous system is not haphazard, but that injections of the same stage give the same pattern, and that the form of the arachnoid space changes as the brain develops. That is to say, the arachnoid space has as definite a form as the ccelom, and it never connects with the lymphaties.”’ Cushing®) gives the following summary: “Tt was thought that an investigation of the cerebro-spinal spaces in the embryo would most likely shed light on the subject, and some unpublished studies in this direction were undertaken in 1904 and 1905 by Lewis L. Reford in Mall’s laboratory in Baltimore. In living pig embryos of various stages low spinal india-ink injections were made either into the wide central canal or into the subarachnoid space, and the embryos were subsequently cleared. It appeared from the course taken by the injection mass that the full develop- ment of the spinal arachnoid preceded that of the intracranial spaces, the impression being gained that the separation of the primitive meninx into its layers occurred later over the cerebral vertex than in the basilar portion of the chamber. Still, I never felt quite con- vinced that the failure of injection of the meninges over the surface of the hemispheres in many of Reford’s specimens was not due to the floating up of the brain against its envelopes by the introduction of the injection mass from below. However this may be, it was never- theless apparent that a venous injection of the body of the embryo was often produced, and the impression was gained that a communication existed between the basal subarachnoid spaces and the precursors of the sinusoidal veins of the cranial chamber which empty into the jugulars. If due to an artifact from a vascular rupture, at all events the communication always occurred at the same point. Reford, moreover, in agreement with Cruveilhier, Reichert, and Kolliker, came to doubt the existence of the foraminal opening described by Magendie, believing that the opening was an artifact and that the fluid escaped by seepage through a persistent membrane.” It is regrettable that Reford’s study has not been published, as it represents the only attempt to solve the problems of the development of the cerebro-spinal space by the method of injection. As stated in subsequent sections of this com- munication, his apparent failure to control pressures of injection and to use only granular suspensions is unfortunate. In a study of the development of the blood-vessels of the human brain, Mall'® noted the ease with which an extravasation into the embryonic arachnoid spaces could be brought avout by increasing the pressure in a venous injection. In a specimen of 46 mm. an arterial injection with aqueous prussian-blue resulted in a complete subarachnoid spread, due to rupture of the vessels as they perforated the 14 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. nervous tissue. In general, it was found that this rule held: an arterial extravasa- tion always took place from the perforating capillaries, while a similar venous rup- ture occurred in the veins themselves. Mall made similar observations on living pig embryos from 30 to 80 mm. in length, with analogous results. But when, in these embryos, the arachnoid spaces were completely filled by an intraventricular injection of india ink, no passage of the granular injection into the veins or sinuses occurred. The ventricular injection flowed into the extraventricular spaces “through the medial opening of the fourth ventricle.”’ From the spinal cord the ink extended for a short distance along the main trunks of the spinal nerves. In the larger embryos (above 50 mm.) the ink usually gushed from the mouth, reaching it by way of the Eustachian tube. Using, in the pig embryo, the heart as the mechanism for injecting the ink, extravasation from the cerebral vessels in the arachnoid spaces occurred. In one human specimen of 90 mm., Mall found both the arachnoid spaces and the cerebral ventricles filled with india ink after an arterial injection of that sus- pension. He states: “‘The injection passes through the medial opening into the fourth ventricle (Magendie), and apparently the ventricles are injected through this opening from the arachnoid.” To His5) and to Kdlliker" belongs the credit of first having established on a firm basis the development of all the meninges in man from mesenchyme. This perimedullary layer of mesenchyme Salvi) called the “ primitive meninx’’—a term now used extensively in comparative anatomy. The primitive meninx divides into two layers, the outer forming the dura and the inner the pia-arachnoid. Sterzi®3), working on the development of the human spinal meninges, advanced a view similar to that of Kolliker. The perimedullary mesenchyme (the ‘primitive meninx’’) divides into two portions, one hugging the inner surfaces of the vertebra and the other adhering to the cord. This inner layer of the perimedullary mesenchyme, according to Sterzi, should properly be termed the “ primitive meninx,”’ as it divides subsequently into dura and the “secondary meninx,” which in turn forms both arachnoid and pia. The denticulate ligaments develop in the “primitive meninx.” The dura and arachnoid in human embryos are modeled up to a certain point on the cord; then, with the augmentation of the subarachnoid space, they follow the outline of the vertebral canal. His'25) has given information regarding the development of the meninges, with particular reference to the formation of the subarachnoid space. He affirms the mesenchymal origin of all of the cerebro-spinal membranes. His describes the first differentiation of mesenchyme to form the meninges as consisting of two zones of condensation, the outer being closely associated with the developing perichondrium of the vertebral column and the inner facing upon the cord. Between these two zones of condensation the subarachnoid space develops, posterior and anterior spaces first appearing, with later fusion laterally. These appearances were met with in chicks of 10 to 12 days’ incubation. Quite soon after this process of space- development a separation occurs which gives rise to a complete subarachnoid space. Later the splitting-off of dura from the vertebral periosteum takes place. METHODS OF INVESTIGATION. 15 III. METHODS OF INVESTIGATION. In the study of any problem dealing with the development of fluid-spaces within the body, the method of investigation must of necessity be such as to offer excep- tional opportunities for control. In the present work several well-known and generally accepted anatomical procedures were naturally suggested, such as injec- tion of the spaces about the central nervous system, reconstruction from serial sections, or merely study of the various stages by means of serial sections. It was ascertained early in the investigation that by injection and serial see- tions without reconstruction the necessary stages in the process of meningeal differ- entiation could be established. In regard actually to the physiological aspects of the problem more reliance was placed on the results of injection than on any histo- logical differentiation, for, as explained above, considerations of the pathway and of the flow of the cerebro-spinal fluid were deemed most important. No method of injection, however, holds out much promise in such a problem unless it can be applied, under conditions approximating the normal, within the spaces about the nervous system. The greatest objection to reliance upon injections in this problem is in relation to pressures. From the very nature of the case it will be realized that any ordinary injection into the embryonic central canal or perispinal space must result in an extraordinary increase in the normal tension of the fluid. This objec- tion applies to any method employed, whether that of a simple syringe and needle, the glass tube and bulb devised by Knower'*), or a glass capillary-tube contrivance. The erroneous conclusions drawn by investigators from the employment of excessive pressures of injection are nowhere more strikingly illustrated than in studies of the circulation of the cerebro-spinal fluid. Many such examples were recently brought forward in a critical review) published in connection with a study of the fluid. In the embryo, with structures and membranes still of very little tensile strength, the consequences of a disregard for the pressures of injection are even more disastrous. A second criterion for the study of fluid-pathways in the body is necessarily the type of injection mass. Not only should attention be paid to the pressures involved, but the peculiarities of the particular body-fluid concerned must be con- sidered. Adopting for this work on the embryo the same standards followed in the previous investigation on the adult, true solutions were used in place of the customary granular suspensions. Emulsions and viscous solutions were not employed because of their obvious disadvantages in studying the passage through membranes. India ink and process black (in which carbon granules are the particulate matter) were also used, but only for comparison with the standard true solution, as the likelihood of the insoluble granules being phagocyted within the period of experimentation or of being caught mechanically in tissue meshes appeared a priori to be too great. In any study of fluid-pathways in the body, not only must the injection fluid be a true solution, but it must also be one which is not attracted to particular cells (as with many stains). Likewise, colloid stains (such as the benzidene group) could not be employed, because of the fact that certain cells (macrophages, as described 16 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. by Evans\!4)) phagocyte the small colloidal particles. In addition, the true solution must be readily precipitated as an insoluble salt, capable of remaining unchanged in histological technique. After trying many salts in long-continued injections into the adult cerebro-spinal spaces, it was found that solutions of potassium ferro- eyanide and iron-ammonium citrate in equal parts were admirably adapted to the purposes of the experiment. By the addition of a mineral acid (preferably hydro- chlorie) ferric ferrocyanide could be precipitated. This prussian-blue is insoluble in the routine technique and is readily identified in sections. After mounting in damar or balsam the blue granules can be observed unchanged for several months, but after a year there is some deterioration in the specimen, due to a conversion of the blue into indefinite greens. Trxt-FIGURE 1.—Schematic sketch of mechanism used for replac- ing ventricular and spinal fluid of an embryo with a foreign solution or suspension. The system is here shown in balance, the difference in fluid-level in reservoirs and needles repre- senting the hydrostatic pressure necessary to overcome the capillary resistance of tubes and needles. The stands hold- ing the injecting needles may be moved about without altering the balance of the system. As one reservoir is raised, the other is lowered in a corresponding degree. In regard to these two major factors in the employment of injections (pressure and true solution) it was found necessary to devise a method of experimentation which would satisfy the requirements of the problem. Solutions of the ferrocyanide and of the citrate were non-toxic within the central nervous system and afforded an excellent histological means for following the fluid-pathways. It was hoped at first that a simple “replacement’”’ type of injection might be employed, as in the adult animals. In this procedure a given amount of fluid was withdrawn from the subarachnoid spaces and immediately replaced by an equal quantity of the injection fluid. The method was successfully tried on fetal cats of considerable size, but METHODS OF INVESTIGATION. 17 was impracticable on small embryos. After such a replacement the animals were allowed to live for varying periods of time (up to 3 hours) and then killed. It was soon ascertained that the essential circulation of the cerebro-spinal fluid was established in pig embryos of less than 30 mm. in crown-rump measurement. Hence the ordinary method of replacement had to be discarded for some more delicate system. With the realization that a simultaneous withdrawal and intro- duction in a living embryo would be far preferable to a two-stage procedure, the extremely simple apparatus pictured in text-figures 1 and 2 was employed. This device consists of two glass tubes of uniform and like bores, suspended from above by a string running over a pulley. To the tapering lower ends of these reservoirs are attached rubber tubes which connect the reservoirs to two needles. These needles are held at the same level by two metal brackets which can be moved at will on a level glass plate. TEXT-FIGURE 2.—Diagrammatie representation of the method of replacing the cerebro-spinal fluid inalivingembryo. Thespinal needleis inserted into the central canal of the spinal cord, while the cerebral needle is introduced into one of the cerebral ventricles. The canal of the spinal cord and the cerebral ventricles are represented by the interrupted lines. The foreign fluids are introduced by the spinal needle and with- drawn by the cranial. The apparatus is employed as follows: Both tubular reservoirs are filled up to the point where the fluid is just ready to fall from the needle in a drop. This point is easily obtained by filling the reservoirs slightly in excess and allowing this excess fluid to run off from the needle. With the system thus in balance the needles lie in the same horizontal plane and can be moved without altering the balance of the solutions. The injection is made by inserting one needle into the central canal of the spinal cord and the other into one of the lateral ventricles; then as the reservoir connecting with the spinal needle is raised the other is lowered, so that an amount of fluid equal to that introduced into the spinal canal is withdrawn from the cerebral ventricles. In this way the whole contents of the cerebral ventricles and central canal of the spinal cord can be slowly withdrawn without increasing the pressure in the central nervous system. The initial pressure necessary to secure this flow is only that required to overcome the capillary resistance of the medullary-canal system. In practically all cases this can be accomplished by using a positive pres- sure of less than 60 mm. of water (associated with a negative pressure of the same degree). 18 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN, In the present study the above procedure was the routine method of injection employed. Pig embryos, brought from the abattoir, contained in the uterus, were found to be wholly satisfactory material. If not permitted to cool excessively in transit the embryos lived for at least two hours in a 38° incubator. On being received at the laboratory a section of the uterine wall containing the placenta was excised, with the embryo left connected by the umbilical structures. As soon as the technical preparations for injection were completed the amnion was opened and the embryo placed upon a padded block at the proper level. The first needle was then inserted into the easily discernible central canal of the spinal cord and the second into the left cerebral ventricle or into the mesencephalic ventricle. By elevation of the reservoir connected with the first needle the cerebro-spinal fluid was replaced by the injection solution. As soon as the replacement was complete the needles were withdrawn and the embryo and its uterine portion replaced in the incubator. The heart of the embryo could be easily observed in the smaller forms and served as the index of a continued circulation. The incubation of the embryos was continued for varying periods of time, but it was soon ascertained that a period of over 30 minutes generally resulted in a complete spread of the injection solution. For comparison the period of incuba- tion was lengthened and shortened, but the best results were usually obtained with a 45-minute incubation after the replacement. Injections of the necessary true solutions were made, in the routine experiment, with a 1 per cent concentration of potassium ferrocyanide and iron-ammonium citrate in distilled water. By a1 per cent solution is meant a salt concentration of this amount (potassium ferrocyanide, 0.5 gm.; iron-ammonium citrate, 0.5 gm.; water, 100 c.c.). The resultant true solution should be practically isotonic with the body-fluids. In this way any injurious consequences due to hypertonic or hypo- tonic solutions were apparently overcome. The factors of osmosis and diffusion also had to be considered in this connection. Other concentrations of the so-called “‘ferrocyanide mixture”’ were used, but only for the sake of comparison or for the purpose of investigating some particular phase of the problem. The results obtained by the use of these concentrations were not relied upon as affording standards for the normal pathway of the cerebro-spinal fluid. In addition to the replacement type of injection, many observations were carried out on pig embryos, with a simple syringe-injection of the ferrocyanide solu- tion into the central canal of the spinal cord or into the cerebral ventricles. It proved to be a very simple matter to regulate the pressures by this method, and three arbitrary standards (mild, moderate, and strong) were found to be of value in a comparison of the extent of the spread obtained by replacement and by injection. The prussian-blue reaction (formation of ferric ferrocyanide) was obtained in these experiments by fixing the whole embryo in an agent containing hydrochloric acid. For histological study the best results were obtained by immersing the specimen from 1 to 10 minutes in a 10 per cent formaldehyde solution containing METHODS OF INVESTIGATION. 19 1 per cent hydrochloric acid. After this primary procedure, during which the ferrocyanide was precipitated, the embryo was transferred to Bouin’s fluid (satu- rated aqueous picric acid, 75; formaldehyde 40 per cent, 20; glacial acetic acid, 5). The specimens were allowed to fix over night and were then dehydrated in graded alcohols. From 30 per cent alcohol, use was made of 4 per cent changes up to 60 per cent; and from this point to absolute the changes were by 2 per cent gradations. In addition to the technique outlined above, Carnoy’s solution and 10 per cent formol were employed. The Carnoy fluid, containing acid (absolute alcohol, 60; chloroform, 30; glacial acetic acid, 10; hydrochloric acid, 1) proved to be of particular service in the study of specimens cleared by the Spalteholz method; histologically, however, it has not been as valuable as Bouin’s fluid. Besides the ferrocyanide solution, two other injection masses were constantly employed. Solutions of silver nitrate in concentrations of 0.5 per cent were injected into the central canal of the spinal cord and into the cerebral ventricles. This method, with reduction of the silver salt in the sunlight, gives very pleasing prepara- tions. It is, however, subject to obvious limitations. The intraspinous toxicity of the silver, together with its action as a precipitant of albuminous substances, renders its use unsatisfactory in replacement experiments. Furthermore, it reacts apparently with any protein tissue, irrespective of the true function of that tissue (as, for example, its coagulation of the lining ependyma of the ventricles). India ink, the other substance employed, is of extreme value in anatomical studies. Because of the suspension of carbon granules it possesses the disadvan- tages already commented upon for the study of any true pathway of fluid. It has been of service, however, in the present work in showing marked differences in spread from that of true solutions and in furnishing information in regard to fluid passage through a membrane. This investigation has been carried out on the basic idea of correlating the physiological spread of the embryonic cerebro-spinal fluid with the gradual trans- formation of the perimedullary mesenchyme into the three fully formed meninges. This has necessitated a histological study of the embryo. Pigs for the most part were the animals used, but the findings have all been verified by a study of the same regions in the human embryos in possession of the Carnegie Institution of Washing- ton. In addition, certain structural characters have likewise been identified in sections of chick, rabbit, and cat embryos. It was early apparent that the material to be of value must be free from any great shrinkage about the central nervous system. Comparative freedom from this artifact was obtained by fixing the embryo alive in Bouin’s fluid and dehydrat- ing by 2 and 4 per cent gradations of alcohol. The material was chiefly cut in paraffin after being embedded by means of xylol. The methods of investigation outlined in the foregoing paragraphs have been followed throughout the major portion of the work. In many minor instances other procedures not commented upon have been employed; these will be detailed in appropriate subdivisions of this paper. 20 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. IV. INJECTIONS AND REPLACEMENTS IN THE CEREBRO-SPINAL SYSTEM. RESULTS OF REPLACEMENTS IN THE VENTRICULAR SYSTEM OF TRUE SOLUTIONS. The results of experiments carried out on embryo pigs by the technical pro- cedures outlined in the previous section will be detailed here. The study was made on this animal because of the facility with which it could be obtained living and in good condition and also because it exhibits the characteristic meningeal anatomy of all mammals. The chick could not be used in this investigation on account of the dissimilarity between the avian and the mammalian meninges. The chief problem concerned here was the actual physiological extent of the cerebro-spinal spaces. This apparently could be ascertained by the replacement of cerebro-spinal fluid by the ferrocyanide mass. But there was also to be considered the passage of fluid from the ventricles out into the periaxial* spaces, corresponding exactly to a similar passage in the adult. If into the central canal of the spinal cord of a living pig embryo of 9 mm., crown to rump measurement, an injection of the ferrocyanide solution be made under very mild syringe-pressure, the ventricles can be fairly well filled without rupture of any element. Incubation of this experimental embryo with its circula- tion continuing almost unabated for an hour should cause a further spread of the fluid throughout the normal canals. If at the end of this time the whole embryo is fixed in an acid medium the ferrocyanide will be precipitated in situ. Such a specimen, subsequently cleared by the Spalteholz method, is represented in figure 1., In this drawing the spread of the injection solution is clearly shown. Running upward from the point of introduction, wholly within the central canal of the spinal cord, it reaches the bulbar region and extends outward into the large fourth ventricle, appearing as a dense collection of the prussian-blue. Cephalad from this region it spreads in diminishing intensity until it is finally lacking in the diencephalon. The injected solution, then, in spite of the unavoidable increase in the normal intramedullary pressure, is contained only within the medullary-canal system (cen- tral canal of spinal cord and cerebral ventricles). There is no evidence of any spread outwards, either from the third or fourth ventricle. In the next stage of meningeal development the replacement method can be used, as the embryo is no longer too small for its employment. In figure 2 is repre- sented an embryo of 13 mm., in which the circulation continued for 90 minutes after the replacement. The same general picture shown in figure 1 results. The whole medullary-canal system is filled with the precipitated prussian-blue, which is densest in the region of the fourth ventricle. The roof of the ventricle, however, shows a striking difference from that of the ventricle in the embryo of 9mm. Just posterior to the cerebellar lip is a regular oval, which is covered from within by a dense collection of prussian-blue granules, causing it to stand out in clear contrast to the thinner and more evenly distributed blue lining of the remainder of the Fe ale gating act} the term *‘periaxial '’ has been used in the sense of ‘‘around the central nervous system ’’ or arounc 1@ cerebro-spinal axis. tThroughout this work the reference “figure ’’ 1, etc., refers to plate illustrations; the word “ text-figure'’ refers to the illustrations inserted in the text. INJECTIONS AND REPLACEMEN'S IN THE CEREBRO-SPINAL SYSTEM. 21 roof. This oval area is comparatively large and comprises a portion of the superior or anterior half of the ventricular roof. This area, differentiated from the remainder of the rhombencephalic roof, is clearly shown in figure 2, a drawing of a cleared speci- men of this stage. With the exception of this strikingly dense area in the rhombic roof, the injec- tion spread in an embryo of 13 mm., subjected to replacement of the cerebro-spinal fluid by the ferrocyanide, differs in no way from that in the embryo of 9mm. Care- ful inspection of figure 2 is convincing that the spread still remains within the medullary canals, with no extension of the fluid into the spaces outside of the cerebro- spinal axis. It seems justifiable, then, to speak of the cerebro-spinal spaces at this stage of development as being only intramedullary in type, with no indication as yet of a meningeal fluid cushion (corresponding to the adult subarachnoid space). With the use of larger embryos, however, for the medullary replacement with ferrocyanide and citrate, the picture gradually changes. The first indication of a more advanced stage of development is obtained in embryos whose length exceeds 14mm. Figure 3, of a pig embryo of 14.5 mm., is included here as representing this further extension of the injection fluid. The cerebro-spinal fluid of this speci- men was replaced, by the compersating mechanism, by a solution of potassium ferrocyanide and iron-ammonium citrate. The embryo was then kept alive (as judged by the heart-beat) for a period of one hour. At the end of this time it was fixed in an acid medium and subsequently cleared in oil of wintergreen after careful dehydration. The essential differences between an embryo of this stage and one of the stage represented in figure 2 concerns the spread of the injection fluid from the roof of the fourth ventricle. Both specimens show a complete filling of the intramedullary system (cerebral ventricles and central canal of the spinal cord) with the precipitated prussian-blue granules. The specimen of 13 mm. (fig. 2) is characterized by a dense oval collection of the prussian-blue on the upper and inner surface of the rhombic roof. In the specimen shown in figure 3, in contradistinction to this localized aggregation of granular matter, there is a delicate extension of the injec- tion fluid caudalwards from the roof of the fourth ventricle. This fusiform projee- tion is here readily made out, lying beneath the skin over the ventricular roof and separated quite distinctly from the easily discernible line of the roof. This outward extension of the fluid has a fairly wide and deep origin from the upper portion of the roof, but tapers caudally to a sharp point with considerable rapidity. At the stage of 14 mm. the roof of the fourth ventricle shows the small depres- sion which marks the formation of the chorioid plexuses. With this depression occurring transversely the relation of the external surface of the embryo to the ventricular roof necessarily alters somewhat in this region. The chorioidal depres- sion of the roof gradually becomes separated from the skin; and it is into this area between the skin and the ventricular ependyma that the first spread from the cerebral ventricles occurs. At this stage, illustrated in figure 3, the injection is intramedullary in type, with but slight extension into the pericerebral tissues. 22 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. The pericerebral spread may be made out in nearly all replacements in embryos of 14 mm., but in a few cases the injection has remained intramedullary in type. In embryos of 16 mm. the spread into the pericerebral tissues is invariably found. Often, with this extension of the replacement solution outside the ventricles, the oval area noted in the stage of 13 mm. persists. (This phenomenon is especially well shown in a simple injection of silver nitrate, illustrated in figure 11.) The next stage of importance in the development of the cerebro-spinal spaces is represented in figure 4, a drawing of a pig embryo of 18 mm. in which a typical intramedullary replacement of the cerebro-spinal fluid with a solution of potassium ferrocyanide and iron-ammonium citrate had been made. Here, with the exception of the region of the roof of the fourth ventricle, the replaced fluid is contained solely within the central canal of the spinal cord and within the cerebral ventricles. The roof region, however, exhibits a new phenomenon, which distinguishes it from the stage shown in figure 3. The chorioid plexus invagination has become strongly developed, dividing the roof into two parts. These roof divisions have been termed superior and inferior, the former lying anteriorly and orally from the chorioid fold. The general surface outline is but little changed, due to the mesenchyme filling up the area between roof and skin. From two areas in the entire roof of the fourth ventricle the foreign fluid has escaped into the pericerebral tissue. These points of fluid passage le in the two divisions of the ventricular roof. The superior area of escape corresponds to the oval outlined by the prussian-blue in figure 2 and to the point of emergence of fluid shown in figure 3. The lower area of fluid escape is in the inferior half of the ventricular roof, where the ependymal lining and its support- ing tissue are developing into a well-marked dorsal distension. This area corre- sponds to Blake’s'%) caudal protrusion, though, as Heuser‘2*) has pointed out, the shape of the structure in the pig in no way resembles the “finger of a glove.” The extraventricular spread of the injection fluid in this specimen is consider- ably greater than in the pig embryo of 14 mm. (fig. 3). On the whole, however, the distribution of the replaced fluid is not extensive as compared with the adult rela- tionship, where the central nervous axis is entirely surrounded by its subarachnoid cushion of cerebro-spinal fluid. From the superior area of fluid passage the replaced solution (as shown by the resultant precipitation of the prussian-blue) has passed both superiorly and inferiorly. In the median line, and extending laterally but slightly, a projection of the blue may be seen occupying a large portion of the extraventricular area formed from the chorioidal invagination. This area of fluid passage occupies at this stage about one-third of the total transverse diameter of the ventricular roof. From it the blue tapers caudally, diminishing in all directions. Above, the precipitate may be made out extending superiorly over the cerebellar lip. Its extension into the pericerebellar tissue is not marked; here again it tapers from the area of fluid passage, its midline prolongation stretching farthest anteriorly. This relationship is easily made out in figure 4, a frank lateral view of such an experi- mental replacement. The granules which result from the introduced ferrocyanide solution are found only in the central canal of the spinal cord and not in any peri- spinal arrangement. INJECTIONS AND REPLACEMENTS IN THE CEREBRO-SPINAL SYSTEM. 23 In the pig embryo of 18 mm., shown in figure 4, the replaced solution has been carried somewhat farther than in the embryo of 14 mm. (fig. 3). The chief point of differentiation lies in the fact that in the latter stages two areas have apparently become permeable to the intraventricular fluid, so that a larger periaxial spread has resulted. Then, too, the extension of the ferrocyanide solution from the superior area is considerably greater, overlapping the cerebellar lip and filling in some degree the pericerebral tissue in the chorioidal invagination. With a definite periaxial spread established for the cerebro-spinal fluid in pig embryos of 14 to 18 mm., it seemed not unreasonable to expect a gradual increase in the extent of the future subarachnoid distribution in more advanced stages. The earliest extension of the fluid into the peribulbar tissues occurred with the inception of the infolding of the ventricular roof to form the chorioid plexuses of the fourth ventricle. Its further extension, particularly its passages through a second area, occurred with the greater development of the chorioidal invagination (7. e., 18 mm. stage). A still more extensive pericerebral flow of the ferrocyanide and citrate is illustrated in figure 5. Here the cerebro-spinal fluid in a living pig embryo of 19 mm. was replaced by the ferrocyanide solution. The embryo was kept alive for about an hour after the replacement and was then fixed in toto in an acid fixing medium, which caused the precipitation of the prussian-blue. On clearing subse- quently by the Spalteholz method the spread of the solution was found to be some- what more extensive than in the stage of 18 mm. (ef. figs. 4 and 5). In figure 5 the whole periaxial area over the roof of the fourth ventricle is shown to be completely filled by a dense aggregation of the prussian-blue granules. The separation of the two areas of fluid passage can not be made out in such a specimen. This dense periaxial extension almost completely covers the cerebellar lip, not only in the medial region but laterally to the limit of the ventricular roof. The injection precipitate lies directly beneath the skin in this area, but more posteriorly its separation from the skin becomes more marked. Tracing this dense periaxial injection posteriorly, it is seen (fig. 5) to end somewhat abruptly in the region of the cephalic flexure. The line of termination of the denser mass, to the ventral surface of the medulla, tapers somewhat anteriorly. This extraventricular spread is medial to the otic vesicle, but extends peripherally along the caudal cerebral nerves, reaching outward as far as the peripheral ganglia. The periaxial spread also closely covers the ventral surface of the medulla and extends in this plane around the pontine flexure for a short distance upwards along the basilar surface of the mid-brain. Examined from its dorsal aspects, the superior portion of the spinal cord is found to be covered (in a perispinal relation) by a fine deposit of the prussian-blue. This is shown in figure 5. Caudally from the higher cervical region there is no evi- dence indicating a further spread in the perispinal tissues. Such a spread from above downward is wholly at variance with Reford’s47) conception of a development of the spinal meningeal spaces before the cerebral. The complete filling here of the central canal of the spinal cord and of the cerebral ventricles with the replaced fluid, 24 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. with no evidence of a periaxial spread except in the region of the fourth ventricle, indicates that in the pig embryo the adult human relationship between the cerebral ventricles and the subarachnoid spaces endures. There is apparently in this embryo no evidence of the foramina of Bichat and of Mierzejewsky, a finding in accord with the observations of Dandy and Blackfan®), In the slightly larger embryos the further extension of the embryonic extraven- tricular spaces progresses rapidly. Figure 6 represents such an extension in a pig embryo of 21 mm., in which the normal cerebro-spinal fluid was replaced by a dilute solution of potassium ferrocyanide and iron-ammonium citrate. In this specimen the central canal of the spinal cord and the cerebral ventricles are completely filled with the precipitated prussian-blue. But in addition there is almost a total filling of the periaxial spaces. Viewed laterally the densest aggregation of the blue granules is again in the region of the roof of the fourth ventricle. Asin the embryo of 19 mm. (fig. 5), the whole extraventricular tissue posterior to this ventricular roof is filled with the granules precipitated from the foreign solution. The spread from this region is similar to that in the previous specimen, except in its far greater extent. The granules may be traced caudalwards in the perispinal spaces to the point of injection. The arrangement of the precipitated material, both within the central canal of the spinal cord and surrounding it in the perispinal relationship, is well shown in figure 7, a frank dorsal view of the same specimen represented in figure 6. The greater density of the perispinal granules in the upper region of the cord, as contrasted with the granules in the thoracic region, is probably of importance in indicating the direction of the flow from above downwards. The increased amount of the injection fluid in the region about the point of insertion of the spinal needlé.is in all likelihood due to a local spread from the needle, such as frequently occurs in a very limited area. The phenomenon may, however, be due to an actual increase in the size of the potential perispinal space, though observations upon other embryos of the same stage of development argue against this view. The segmental outlining of the caudal portion of the perispinal space is to be noted in this figure. The cephalic regions in the specimen of 21 mm. show a quite extensive spread (fig. 6), and there is the same general distribution of the granules about the medulla, as in the specimen shown in figure 5. The rhombencephalon is completely sur- rounded by the blue, the ventral sheet inclosing it tightly. Laterally the prussian- blue is shown in a dense mass, in intimate relation to the cranial nerves as they join the brain-stem. The cerebellum is practically completely covered by the precipi- tate; from the ventral portion of the pericerebellar granules the replaced solution (as evidenced by the granules of prussian-blue) spreads forward and surrounds a portion of the mid-brain. Only the ventral surface of the posterior half of the mid-brain is circumscribed by the granules; anteriorly it is wholly surrounded by the periaxial injection; more anteriorly the extension is limited to the mesial strue- tures, leaving unsurrounded the cerebral hemispheres, although creeping between the hemispheres and the mid-brain. The peculiar avoidance by the replacement fluid of the extreme dorsal half of the mid-brain is also to be made out in the dorsal view of the specimen (fig. 7). INJECTIONS AND REPLACEMENTS IN THE CEREBRO-SPINAL SYSTEM. 25 The two lateral extensions from the ventral sheet of the injection granules approach on either side this mesencephalic eminence. The peculiar appearance of the injec- tion spread caused by the chorioidal invagination of the roof of the fourth ventricle is also here illustrated. In this specimen, then, of a pig embryo 21 mm. the periaxial spread is almost complete, the only areas not entirely surrounded being the anterior mesencephalon and the cerebral hemispheres. In an embryo but a few millimeters larger this periaxial extension of the solution is complete. The mesencephalon first becomes entirely covered by the prussian-blue precipitate, with later extension over the hemispheres. This complete periaxial injection occurs usually in replacements in embryos varying in length from 24 to 28 mm. A specimen exhibiting a complete extension of the replaced solution around the central nervous system is shown in figure 8. This specimen was prepared by replac- ing the cerebro-spinal fluid in a living embryo of 26 mm. and then keeping the embryo alive for an hour. After fixation in an acid medium, dehydration, and clear- ing, the injection was found to occupy the whole medullary-canal system and also to surround completely the cerebro-spinal axis, as shown in the lateral view. The striking features of this stage are similar to those observed in the younger specimens— the dense accumulation of granular material in the region of the roof of the fourth ventricle, the surrounding of the central portion of the caudal cranial nerves, and the thin pericerebral covering by the replacement mass. In addition the specimen exhibits in the thoracic region an extension of the granular material laterally along each spinal nerve. An observation of this peculiarity reveals the prussian-blue extending outwards only as far as the ganglia on the posterior roots. The relationships, then, observed in an embryo pig of 26 mm. are those which exist in the adult; the cerebro-spinal axis contains cerebro-spinal fluid within its cerebral ventricles and within the central canal of the spinal cord, while in turn it is completely surrounded by cerebro-spinal fluid within the subarachnoid space. Communication between the ventricles or intra-medullary system and the peri- spinal spaces occurs only in the region of the fourth ventricle. Here again the adult human relationship holds. The evidence, therefore, from a study of the fluid spread in a replacement experiment with the use of true solutions, indicates that in pig embryos of about 26 mm. an adult distribution of cerebro-spinal fluid occurs. THE RESULTS OF INJECTIONS OF TRUE SOLUTIONS. In the preceding section there have been detailed the results of experiments on living pig embryos in which the cerebro-spinal fluid of both the central canal of the spinal cord and the cerebral ventricles has been replaced by a dilute solution of potassium ferrocyanide and iron-ammonium citrate. After the replacement, car- ried out so as to avoid any increase in the normal tension, the embryos were incu- bated for varying periods of time so that the normal current of the fluid might cause an extension of the ioreign solution. In the experiments which will be recorded in this section the same true solution was injected from an ordinary syringe and the 26 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. salts immediately precipitated as prussian-blue. The purpose of these observations was solely to ascertain the effect of injections at pressures above the normal tension, so that the conclusions drawn from the replacement method might be more fully substantiated. It was soon ascertained that the pressures caused by injections with a simple syringe could be fairly well controlled and that several degrees of tension might be employed. Thus it was found to be simple and serviceable to designate the injec- tions as those made with mild, moderate, or strong syringe-pressure. Most of these injections were made into the central canal of the spinal cord, but occasionally into the perispinal spaces or cerebral ventricles. Injections under equivalent pressures in the central canal of the spinal cord or into the cerebral ventricles always gave corresponding results. It is necessary to record that the injections, even under strong pressure, were not carried to the point of macroscopic rupture. The so-called mild syringe-pressure, making use of solutions of potassium ferro- cyanide and iron-ammonium citrate, resulted in extensions of the prussian-blue wholly similar to those obtained in the replacement experiments which were carried on for 30 minutes and over. This similarity indicates a complete filling of the avail- able cerebro-spinal system in the replacement method, for certainly (even in the mildest syringe injections) the intraventricular pressure must be excessively increased. Figure 1 shows a specimen under such conditions, with a marked thin- ning of the injection mass in the region of the fore-brain. This finding is customarily present in the injections under mild pressure, due to the pushing upwards of an existent ventricular fluid. When moderate pressures are employed with the syringe the picture gradually changes. The essential difference in the results obtained by moderate syringe injection and by the replacement method lies in the greater extension of the foreign solution in the smaller embryos. Thus in figure 9 the spread of the injection precipitate in a pig embryo 16 mm. is shown to be about as extensive as that obtained by the replacement method in an embryo of 19 mm. (fig. 5). The extraventricular distribution of the injected solution around the medulla, the extension (even more marked here) along the central roots of the caudal cranial nerves, and the localized perispinal spread are easily made out in this specimen of 16 mm. This general rule applies to all of the results obtained with the use of syringe- pressures above the mildest. Dependent upon the degree of syringe-tension, the spread extends in simple ratio. Thus, by the use of moderate pressures of injection into the central canal of the spinal cord, a complete intramedullary and periaxial spread was secured in a pig embryo of 22 mm. somewhat earlier than the equivalent stage was obtained by the use of the replacement method. With the highest syringe-pressures (insufficient, however, to cause macroscopic rupture) the same general type of injection spread was obtained, bringing the more complete stages down into smaller and smaller embryos. Most of these embryos, however, on microscopic section showed obvious rupture of some part of the central nervous system. INJECTIONS AND REPLACEMENTS IN THE CEREBRO-SPINAL SYSTEM. 27 The most important feature of these findings in the embryo pig injected with true solutions under moderate pressures from a syringe concerns the fact that the extension of the injection coincides, except as to the size of the embryo, in every instance with that obtained by the replacement method. Thus similar and analo- gous spaces are filled by injections under syringe-pressures in small embryos and by the solution under normal tension in larger embryos. It must be assumed, then, that the pressure of injection is sufficient to dilate potential cerebro-spinal spaces which normally would not be concerned in the pathway taken by the cerebro-spinal fluid. No evidence of new or abnormal pathways for the fluid is afforded by the observations made with the increased pressure; these phenomena indicate great potential strength in the tissues which limit the immature cerebro-spinal spaces. Injections with a simple syringe may be made with such a degree of pressure that gross rupture of the tissues becomes apparent. In such an injection into the central canal of the spinal cord the infundibular region ordinarily ruptures in the smaller embryos (under 15 mm.), while in larger embryos rupture usually occurs into the subcutaneous tissues of the back of the neck over the fourth ventricle. In discussing the effects of the introduction of solutions of ferrocyanide under pressures higher than normal into the central canal of the spinal cord, it may be appropriate to record observations made in the attempt to inject the cerebro-spinal spaces from the perispinal space. In embryos under 15 mm. in length it is quite difficult to make a perispinal injection. As the embryos exceed this measurement the injection becomes increasingly easy, but not until a length of 20 mm. is attained can it be made under the mild pressure advisable. These observations tend to substantiate the findings already recorded in both the intramedullary replacements and the injections under mild pressure. RESULTS OF INJECTIONS OF NITRATE OF SILVER. In a number of experiments a dilute solution (0.5 per cent) of nitrate of silver was injected into the central canal of the spinal cord and the salt then reduced in the sunlight. This solution, although a true one, is wholly unsuited for the replace- ment type of injection, on account of its great toxicity and its power to coagulate protein. It was employed here only for the simple type of injection. The results obtained by this intraspinous injection of solutions of nitrate of silver were of but little value in the determination of a pathway for the cerebro- spinal fluid, but they vividly present certain aspects of the problem. Thus, in figure 11, a drawing of a specimen (pig) of 16 mm., the area through which fluid passes in the superior portion of the roof of the fourth ventricle is clearly outlined by a denser deposition of the silver. This specimen was prepared by introducing the solution of nitrate of silver into the central canal of the spinal cord under the so-called moderate syringe-pressure. The drawing shows a slight, cone-shaped extraventricular spread of the injection fluid. This spread takes place solely from the superior area of fuid passage, a result in accord with the finding that the solu- tion of potassium ferrocyanide and iron-ammonium citrate passed first through the superior area. Of course it is realized that the precipitant action of the silver may 28 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. have exerted a more potent action on the structures constituting the lower area of fluid passage. Another interesting phenomenon of the injections of silver nitrate is shown in figure 12. The embryo of 13 mm. here represented was injected under strong syringe-pressure with a solution of silver nitrate into the central canal of the spinal cord. On subsequent reduction and clearing it was found that the excessive pressure had resulted in a complete intramedullary injection with a localized pedunculate spread into the tissues from the roof of the fourth ventricle. This bulbous extrava- sation into the extraventricular tissue has not been observed in any specimens except those into which the solution of silver nitrate was injected. Such a spread is prob- ably to be accounted for by an immediate coagulation of the surrounding tissue. The extensive use of solutions of silver nitrate as a means of demonstrating vascular channels naturally suggests a careful comparison of the results obtained from its use and those obtained from the employment of other available true solu- tions, in regard to the evidence afforded by the two methods of intraspinous injec- tions. The chief objection to the use of silver nitrate, as has already been mentioned, is its power to coagulate protein. This is illustrated by many features of the specimen shown in figure 11—by the sharp outlining of the area of fluid passage, the markings on the caudal process of the fourth ventricle, and the delimitation of the cerebellar lip. But much more marked are the evidences of this coagulative power as shown in figure 12, the pedunculated extraventricular spread, the transverse corrugation of the cerebellar lip (amounting to circumscribed indentations), and the peculiar outlining of the roof attachment to the bulb. These phenomena obtained by the intraspinous injection of solutions of silver nitrate must be classed as arti- facts. The different degrees of this corrosive action of the silver probably result from the varying rates of reduction of the salt to the metal, a factor which is not easily regulated. The findings, therefore, with this method are worthless unless controlled. Many embryos of varying sizes were injected with the silver nitrate. In the main these observations followed the course of development of the cerebro-spinal spaces as evidenced by the replacement experiments with the ferrocyanide. The injections required moderate pressure in the syringe in order to secure more than a local extension from the roof of the fourth ventricle, and to secure the same extent of spread it was generally necessary to use embryos a few millimeters larger than those required in the replacement experiments; but this is to be expected, in view of the probability of a constant precipitation of the albuminous tissues by the injection fluid. Specimens prepared by the intraspinous injection of silver nitrate, then, afford but little reliable evidence in this problem except of a substantiative sort. The findings by this method indicate that the perispinal and pericerebral spaces, in pig embryos of 25 mm. and upward, could be filled by an injection of silver nitrate under moderate pressures into the central canal of the spinal cord. The point of pas- sage of the fluid from the intramedullary to the periaxial system was in the region of the roof of the fourth ventricle. INJECTIONS AND REPLACEMENTS IN THE CEREBRO-SPINAL SYSTEM. 29 THE INJECTION OF INDIA INK. The objections to the use of any fluid of insoluble particles in suspension have already been discussed in considering the methods of injection which were possible for use in this study; but for comparison with results obtained by more promising methods and to ascertain to what extent injections with india ink are reliable they will be further considered here. No granular substance other than india ink (ear- bon granules) was employed in this investigation. In every way this suspension possesses advantages over other possible masses—in its ease of preparation, in the small size of the granules, and the insolubility in the reagents used for microscopic technique. Suspensions of india ink (diluted from 4 to 10 times) were introduced first into the medullary-canal system of living pig embryos by the replacement method. In no case, however, even though the circulation of the embryo may have continued for 90 minutes, was there any evidence of an extension of the replaced mass outward into the periaxial spaces. The carbon granules remained wholly within the ven- tricles, a striking difference from the results obtained by the ferrocyanide replace- ments. It would appear, then, without the further evidence afforded by micro- scopic section of the specimens, that there is an existing mechanism which prevents the passage of the carbon granules from the fourth ventricle into the periaxial spaces. This finding was found to be constant in all the living embryos subjected to the cerebro-spinal replacement. Quite similar to these resuits by the replacement method are those from the injection of a suspension of india ink under mild syringe-pressure. In no instance, provided the pressure was maintained at a low enough degree, was there any passage of the granular material into the periaxial tissue. In embryos of over 30 mm., however, even with the lowest pressure, it becomes increasingly difficult to prevent a sudden spread into the periaxial spaces. The type of spread indicates a sudden release of some restraining agent and suggests a rupture of a membrane. This spread is usually local and takes place from the roof of the fourth ventricle. With moderate and strong syringe-pressures, however, it is possible to secure a periaxial spread, but this is quite different from the distribution of the injections by the use of ferrocyanide solutions. Figure 10 illustrates a specimen of a pig embryo of 21 mm. into whose central spinal canal india ink was injected under strong syringe-pressure. The resultant spread of the injection is easily discerned; the cerebral ventricles are quite filled with the carbon, while from the superior portion of the roof of the fourth ventricle a dense but localized periaxial spread is made out. This extraventricular extension of the ink is well defined; it stretches caudalwards for a slight distance, curving about the bulbous caudal portion of the ventricle and extending lateralwards but a short distance. The median portion of the cerebellar lip is covered by the granules. Evidences of the excessive pressure at which the injection was made are shown by the lines of invasion of the spinal cord and mid-brain. A comparison of the spread of this injection mass with the exten- sion of a ferrocyanide replacement in an embryo of the same size (21 mm.) is afforded by figures 10 and 6. With such a divergence in the results obtained by the two 30 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. methods of approach it is not surprising that observations such as Reford’s‘?) fail to coincide with these findings. The unsuitability of suspensions of granular material in the investigation of the cerebro-spinal spaces has been many times verified in this work. In the further study of the course of the spread with injections of india ink it was found that, in pig embryos of approximately 22 mm. and over, a partial periaxial injection could be secured by plunging the syringe-needle into the perispinal spaces. The carbon granules could subsequently be seen filling the perispinal spaces and also mounting upwards in partial pericerebral relationships, particularly around the medulla. This result was obtained by the use of strong syringe-pressures. Appar- ently the resistance to the spread of the ink in injections or replacements in the medullary-canal system occurs in the passage of the fluid from the roof of the fourth ventricle into the periaxial spaces. So far as is known, Reford” did not control his injection pressures. These results with the injection of india ink under strong pressures coincide with the idea of his observations afforded by the abstracts given by Sabin) and Cushing’). Suspensions of india ink, then, injected under mild syringe-pressure or by the replacement method, offer no evidence, in the pig embryo, of a passage of the cerebro-spinal fluid into the periaxial spaces. Only by employing pressures much above the normal tension can such evidence be obtained. V. UNDESCRIBED STRUCTURES IN ROOF OF THE FOURTH VENTRICLE. The results of the replacement of the existing cerebro-spinal fluid by a true solu- tion of potassium ferrocyanide and iron-ammonium citrate in a living pig embryo indicated, as detailed in the foregoing section, that the fluid passed from the ven- tricular system into the periaxial tissues in the region of the roof of the fourth ventricle. This important transit of the fluid, agreeing with the established con- ception of the relationship in the adult, was first observed in an embryo pig of 14 mm. (fig. 3). At this stage the exudation of the replaced fluid occurred in one defined area, seemingly corresponding to the dense oval in a smaller embryo shown in figure 2. Such a passage of fluid from ventricle to periaxial tissue is necessarily a physio- logical phenomenon, and it was in the hope of finding an anatomic basis for this phenomenon that the roof of the fourth ventricle was studied histologically. It was realized that failure to demonstrate anatomically differentiated structures would not vitiate the physiological observations, but that a correspondence between function and structure was most desirable. Hence observations were undertaken to deter- mine, if possible, an area of histological differentiation in the roof of the fourth ventricle which might be concerned in the primary passage of fluid from the cerebral ventricles into the periaxial tissues. The investigation concerned first the exami- nation of this region in pig embryos of 14 to 15 mm., at which stages the fluid passes from a single area. Subsequently, similar studies were undertaken in regard to the second, more inferior area (shown in figure 4). The results of these studies will be given here. UNDESCRIBED STRUCTURES IN ROOF OF THE FOURTH VENTRICLE. 31 AN UNDESCRIBED AREA IN THE SUPERIOR PORTION OF THE ROOF OF THE FOURTH VENTRICLE. THE AREA MEMBRANACEA SUPERIOR IN THE PIG EMBRYO. Examination of the roof of the fourth ventricle in a pig embryo of 14 mm. revealed a peculiarly differentiated area in the superior portion. The general topog- raphy of this area is shown in the rectangular area marked off in figure 32—a median sagittal section from a pig embryo of this critical stage. In figure 33 this rectangular area is enlarged to show the morphology in greater detail. In this figure the densely staining ependyma lining the fourth ventricle ap- proaches from both sides. The superior portion of the ependyma ends abruptly, while the inferior line of the layer tapers more slowly. Between these two points is an area having none of the characteristics of the ventricular lining at all other points. The comparatively smooth contour of the ependymal cells is replaced by an irregular cell-border. The pyknotic nuclei of the cells have been replaced by less densely staining, elongated, spindle-like nuclear bodies. The cell-layer lining the ventricle is here really only of a single cell in thickness, although blood-capillaries closely applied to it suggest a greater thickness. The mesenchyme between this layer and the peripheral epidermis is quite thin, but resembles in every way the mesenchyme in the immediate neighborhood. There is, therefore, as pictured in figures 32 and 33, an area in the roof of the fourth ventricle which is morphologically dissimilar to the characteristic ependyma lining the cavity. Is this the result of some distortion in fixation or in the routine histological technique? Is it a constant finding and, if so, what is its history? Does it arise at a definite period and persist throughout intra-uterine life only or through adult life also? The question of the actual existence of this area, or of its being caused by technical manipulations, is one which must be answered. That this differentiated portion of the roof of the fourth ventricle is not an artifact is verified by the general history of its formation, by its invariable occurrence (not only in the pig but in other animals), and by its general histological appearance. Moreover, the physio- logical importance of this area undoubtedly inclines one completely from the possible explanation that it is due to an artifact. No single finding wholly excludes such a possibility; rather is one convinced, by many features, of its actual occurrence. Considering the fact, then, that this differentiated structure in the roof of the fourth ventricle may be found in all embryo pigs at the stage of 14 mm., it becomes necessary to ascertain at what time in the development of the embryo it first appears and how it is formed. Obviously the most satisfactory method is to trace the area through the lower stages and also through the older embryos. For the sake of greater clearness, however, a description of the area will be given from its first differentiation through its maximum transformation to its final disappearance—for the structure is only temporary. In pig embryos ot 8 mm. and less in crown-rump measurement, the roof of the fourth ventricle is formed of cells morphologically and tinctorially different from 32 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. those lining other parts of the ventricular cavities. These cells are quite unlike the deeply staining ependymal cells, which can be so readily identified as the lining cells in older embryos. In this younger stage of 8 mm., the entire ventricular roof is composed of several layers of cells with round or somewhat oval nuclei and fairly abundant cytoplasm. The cell-boundaries are not well defined. The nuclei are not deeply tinged with hematoxylin. The chromatin material is sparse and irregu- larly distributed. Nucleoli are prominent. The cytoplasmic border lining the ven- tricular cavity is rough and ragged at times, often blending with the coagulated albumen of the cerebro-spinal fluid. Altogether, these lining cells bear a much greater resemblance to the epithelial cells than to the ependymal. These characteristics of the lining cells of the roof of the fourth ventricle are shown in figures 24 and 25, from a pig embryo of 8 mm. The close association of the roof cells to the surface epithelium is easily made out in figure 25, as well as the general character of the lining cells. At the stages of 8 mm. and under, in the pig embryo, the roof of the fourth ventricle is relatively quite large. In its whole extent it is formed of the peculiar lining cells described above. With the growth of the embryonic nervous system, the roof of the fourth ventricle is subjected to alterations in form and position; to some extent these changes influence the cells which line the cavity in the early stages. In pig embryos between 8 and 12 mm. in length the roof of the fourth ventricle undergoes a change. The ependyma, which from comparison with later stages is regarded as typical, begins to encroach upon the epithelial-like cells which are so numerous in the 8 mm. stage (fig. 25). The area occupied by these cells diminishes, not only relatively but absolutely. It becomes smaller and the cells gradually change their character. These changes are shown in figures 26 and 27, from a pig embryo of 1l mm. Figure 26 gives the location, in a sagittal section near the mid- line of the area in figure 27, taken at a higher magnification. In figure 27 the densely staining lips of ependymal and nerve cells are seen approaching each other. For a considerable space in the central portion of the photograph there is an area similar to that shown in figure 33. But considered in connection with figure 25 this area represents the epithelial-like cells of the roof of the fourth ventricle. This relationship is more clearly shown in figures 28 and 29, taken in a more lateral plane from the same embryo (11 mm.). Examination, how- ever, of the area in figure 29 shows the epithelial-like cells again apparent in the roof of the fourth ventricle. The process of transformation, then, as shown in these photographs from an embryo pig of 11 mm., concerns a gradual encroachment upon the area of epithelial- like cells by the more densely staining and more closely packed ependymal cells. Gradually the epithelial-like cells in the central portion of the area lose their former character (fig. 27), while around the periphery, especially on the lateral sides, the epithelial-like appearance persists (fig. 29). On the lateral side of this area, just as the typical ependymal lining is about to become isolated (fig. 29), the epithelial-like lining cells form a several-celled layer. UNDESCRIBED STRUCTURES IN ROOF OF THE FOURTH VENTRICLE. 33 The nuclei are poor in chromatin material and the cytoplasm somewhat small in amount. The inner cytoplasmic border lining the ventricle is in contrast, by its ragged outline, with adjacent smoother ependyma on both sides. At this stage of the pig embryo the characteristics of the epithelial-like cells are still to be made out, but a gradual transformation is becoming evident. The metamorphosis becomes much more marked in the central portion of the area, as shown in figures 26 and 27. In these figures the whole central area seems to have lost some of its former character as an intact cell-layer. Closer examina- tion, however, under higher power demonstrates that it still possesses an intact surface as a lining for the ventricle. Delicate cytoplasmic strands stretch in a continuous line across the whole area between the lips of denser typical ependyma. The nuclei in this differentiated area are seemingly altered from their rounded form and have elongated almost into spindles. The inner cytoplasmic border is charac- teristically rough, with small amounts of coagulated albumen adhering to the pro- cesses. The area, then, in its central portion, at the stage of 11 mm., has assumed the character of the stage of 14 mm. (fig. 32). On the periphery, however, the cells still resemble those of smaller stages (8 mm.). From the pictures presented by the intermediate stages (figs. 27, 28, and 29) the differentiation goes on very rapidly, so that in the pig embryo of i3 mm. there is rarely any evidence of the epithelial-like cells. Figures 30 and 31 are photo- micrographs of a sagittal section of an embryo pig of 13 mm.; here there are no evidences of the epithelial-like cells. The whole area, pictured in figure 31 as sharply delimited from the tongues of typical ependyma above and below, has become well differentiated. The cell-character observed in figures 27 and 33 (elon- gated nuclei and scanty strands of protoplasm) has become very obvious. The ragged and roughened intraventricular border, the coagulated albumen, and the abrupt transition from the neighboring typical ependyma are well shown in the photomicrographs of this specimen. The differentiation of this area in the roof of the fourth ventricle of the pig embryo proceeds at a very rapid rate, so that within the growth of a few millimeters (from § to 13 or 14) a great histological change occurs. Figures 32 and 33, already described, show the extent of this metamorphosis in a pig embryo of 14mm. The process, however, continues, modified possibly by the changing of the roof of the fourth ventricle. For this roof structure is subjected to marked alteration in stages of 14 mm. and upwards, both by the lateral development of the chorioid plexuses and by the readjustment of the cervical and pontine flexures. Its maximal differ- entiation may be said to appear at a stage of 18 mm.; this is maintained through several millimeters, until undergoing final retrogression. This maximal change in the roof of the fourth ventricle is shown in figures 34, 35, 36, and 37. Several points of interest are brought out in these photomicro- graphs. Figure 35 represents an enlargement of the rectangular area in figure 34, taken from transverse sections of an embryo pig of 8 mm. The area is particularly well shown in this figure, in which, from the right, the typical ependyma, in a fairly 34 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. smooth single-cell layer, approaches the differentiated cells in the central portion. On the left, too, similar typical ependyma is shown. In the central area, which has been repeatedly described, the elongated nuclei, the strands of protoplasm, and the ragged, irregular intraventricular surface are well presented. The photomicro- graph has been reproduced to show the relation of this differentiated area to the various blood-channels in the supporting mesenchyme. Apparently the whole ven- tricular roof is, at this stage, a site for an extensive capillary plexus; from both sides, as shown in figure 35, vessels (one of great caliber) approach the central area of differentiation. Directly beneath this area smaller capillary channels can be made out, from which, apparently, a slight extravasation of red blood-cells has occurred. Here, as in the greater part of the basilar pericerebral region, extravasa- tion of the blood-cells is very frequent. This phenomenon has already been pointed out by Mall. The large extent and the great differentiation of this peculiar area in the roof of the fourth ventricle are well shown in figures 36 and 37, taken from a transverse section of a pig embryo of 18 mm. In the photomicrograph of higher magnifica- tion the two sharp tongues of typical ependyma are quite striking. Their abrupt termination in the wide, differentiated area has nowhere been more convincingly shown. ‘The resemblance of these lining cells in the central area to the mesenchymal elements adjoining is here also seen. The most interesting of all the phenomena exhibited in this reproduction, however, is the attachment, apparently by precipi- tation, of the coagulated albumen of the cerebro-spinal fluid. This coagulation, in this specimen, delimits the differentiated area in the roof of the fourth ventricle. The phenomenon is seemingly only an amplification of a similar attachment of small fragments of the albuminous precipitate shown in other figures. Beyond the stage of 18 mm., which may be termed the maximal stage, the differentiated area in the roof of the fourth ventricle undergoes a regression. This is apparently due to the morphological alterations in this rhombic roof. The cho- rioid plexuses in embryos over 18 mm. long deeply invaginate the fourth ven- tricle, possibly drawing some of the true roof with them, but surely encroaching upon the mid-line with their lateral tuftings. This growth tends to decrease the available extent of the differentiated area, but an even more potent factor is the rapid development of the cerebellum. The caudal growth of the cerebellar lip soon largely occupies or replaces the superior half of the roof. These two factors, the cerebellar growth and the enlargement of the chorioid plexuses, render the per- sistence of the differentiated area impossible, so that a regression or disappearance is to be expected. With these considerations before us, the study of sectioned pig embryos of a greater length than 18 mm. becomes important. The process of disappearance, however, does not occur at once. Thus, in an embryo pig of 19 mm. (figs. 42 and 43) the differentiated area is as large and as characteristic as in the stage of 18 mm. This same appearance and maintenance of size may be observed through the next several millimeters’ growth, but in pig embryos of 23 mm. the chorioid plexus has UNDESCRIBED STRUCTURES IN ROOF OF THE FOURTH VENTRICLE. 35 usually developed to such an extent that a continuation of the former size becomes impossible. This is shown in figures 44 and 45. Figure 45, the enlarged squared area from figure 44, is a photomicrograph from a pig embryo of 23 mm. The dif- ferentiated area, due to the factors favoring its regression, now appears in close proximity to the chorioid plexus. It has more the appearance of a degenerating area at this stage than in any of the younger embryos, but it still shows a character- istic delimitation of both edges—on the one from the typical ventricular ependyma, and on the other from the differentiated ependyma of the chorioid plexus. The cytoplasmic strands of the area which forms the ventricular border do not show to advantage in the photomicrograph, but the same ragged character with the covering of coagulum may be made out. The process of regression, mechanical as it perhaps is, has begun at this stage in the pig, and in the course of the next few millimeters’ growth will become even more active. With the encroachment of the chorioid plexuses and the downward growth of the cerebellar lip, the superior portion of the ventricular roof soon disappears, and is practically non-existent in embryos of 30 mm. and more in length. The differ- entiated area thus encroached upon from the sides and above becomes a mere vestige of its former size. Thus in a pig embryo of 32 mm. (figs. 46 and 47) it appears as a very small break in the lining continuity of the ventricular ependyma. Without the intermediate stages such a picture would undoubtedly be considered as an artificial erosion of the ependymal lining of the ventricle, but when studied in connection with figure 45 the true vestigial character of the area becomes estab- lished. The final fate of this differentiated area in the roof of the fourth ventricle is a complete disappearance, with the occupation of the region by chorioidal epithelium and cerebellum. In this study it was impossible to find traces of the differentiated areas in pig embryos of over 33 mm. in length; vestiges may persist, but so small as to present difficulties of decision. The persistence of such a differentiated vestige in rare instances would not be surprising; the transitory character of the area and the method of disappearance make this seem not unlikely. This transitory area of differentiation in the roof of the fourth ventricle of the pig has not, so far as can be determined, been noted or described by any previous author. His‘25), in a retouched photomicrograph of a sagittal section of a human embryo of 17 mm., reproduced the area as differentiated from the roof, but he has made no comment upon it. I have called this differentiated area in the superior portion of the rhombic roof ventricle the “area membranacea superior ventriculi quarti.”’ This terminology is based on the anatomical character of the area as a continuous membrane, but chiefly on its physiological significance. For, as will be shown in the succeeding section of this paper, the transit of embryonic cerebro- spinal fluid from ventricle to periaxial tissue occurs in this area, which functions apparently as a physiological membrane. With such a physiological conception of the area, the term ‘‘area membranacea”’ seems most suitable, inasmuch as it also meets the anatomical requirements. 36 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. THE AREA MEMBRANACEA SUPERIOR IN THE HUMAN EMBRYO. The finding of the differentiated area in the superior portion of the roof of the fourth ventricle in the embryo pig suggested the value of a study of the same region in the human embryo in the further solution of the problems underlying its occur- rence. Hence this region in the roof of the fourth ventricle has been examined in the sectioned human embryos of the Department of Embryology of the Carnegie Institution of Washington. It was found that a similar area occurred in the human embryo of approximately the same age. The study of the roof of the fourth ventricle is usually more difficult in the human embryo than in the pig. This is due to the fact that the roof of the fourth ventricle quickly suffers from poor fixation and dehydration—collapse or inversion of the whole structure being commonly met with. It is rarely possible, in the younger embryos, to secure the most satisfactory fixation, whereas in the pig these factors may be controlled as desired. Furthermore, the undue pressures to which the human ovum is frequently subjected in abortion may cause crushing of the more delicate parts of the nervous system. It is probably best, in the human embryo as in the pig, to trace the formation of the area membranacea superior ventriculi quarti from its beginning, through the various differentiations. In a human embryo of 4 mm. (No. 836 of the Collection of the Carnegie Insti- tution of Washington) the entire roof of the fourth ventricle is composed of cells with round or slightly oval nuclei and palely staining cytoplasm. The nuclei of the cells are poor in chromatin material as contrasted with the pyknotic character of the typical ependymal cells. The lining tissue is of the thickness of several cells. The ventricular cytoplasmic border is fairly smooth at this stage. This characteristic ventricular lining is shown in figures 40 and 41, both taken from embryo No. 836. The whole picture is similar to that exhibited by the pig embryo of 8 mm. (figs. 24 and 25). A similar accumulation of epithelial-like cells is found in a human embryo of 7 mm. (No. 617 of the Carnegie collection). This is pictured in figures 48 and 49. The photomicrograph of higher magnification shows these poorly staining cells heaped up in a rather localized part of the ventricle, fairly sharply delimited from the adjoining ventricular lining. This accumulation of cells in the roof of the ven- tricle invariably occurs, and it must not be considered as being due to the distortion of the ventricular roof. The reason for the asymmetry of the rhombic roof shown in these figures lies in the fact that in this embryo, as in practically all the embryos of similar stages in this collection, some degree of distortion of the roof of the fourth ventricle is present. Photomicrographs (figs. 50 and 51) taken more posteriorly (from embryo No. 617) give strong evidence of this distortion. They are repro- duced not only to show the possible distortion, but also to give a further picture of the lining of the ventricle, with its epithelial-like cells in several layers (fig. 51). Similar accumulations of these epithelial-like cells are to be found in human embryos of 9 mm. Reproductions of a much fragmented specimen of this size UNDESCRIBED STRUCTURES IN ROOF OF THE FOURTH VENTRICLE. 37 (No. 721) are given in figures 52 and 53. In the latter figure the complete occupa- tion of the ventricular roof by these cells is well illustrated. Moreover, the speci- men shows the many-layered stage to a degree but seldom found. It is unfortunate that such a degree of fragmentation and distortion is found throughout this specimen. Thus far, in human embryos up to and including 9 mm. in length, the roof of the fourth ventricle has shown the same architecture as appears in the pig. As will be recalled, the first evidence of a further differentiation of these cells in the pig embryo was found at a stage of 11 mm. (figs. 26 and 27). In one human embryo of this stage (No. 544) a distinct break in the roof of the fourth ventricle can be made out. This is shown in two photomicrographs (figs. 54 and 55). The picture in this case is somewhat obscured by the shrinkage and distortion of the ventricular roof, but a distinct differentiation of the lining epithelium can be made out. On the caudal side of figure 55 considerable nervous tissue is seen. Just superior to this (toward the left) the lining tissue is almost lacking, a few nuclei, only, preserving the contour of the ventricle. Above this area appears again the ventricular lining of many layers of cells. It has been quite difficult to interpret these findings. The area under discussion shows a rather typical adherence to the coagulated albumen; there is evidence of its extension also into the adjacent mesenchyme, a finding observed in no other similar stage. The caudal position of the opening, the char- acter of the tissue approximating the ventricular cavity, and the presence of the albumen in large amount in the adjacent mesenchyme—all indicate that in great measure the pictures presented in this specimen are largely artifacts. It seems most likely, though, that some differentiation of the tissue in this area has occurred. In a human embryo of 14 mm.,* as in the pig of the same stage, the area membranacea superior has attained a great degree of differentiation. This is particularly well shown in figures 56 and 57, the latter being an enlargement of the squared area in the former. These photomicrographs are from embryo No. 144 of the collection of the Carnegie Institution of Washington. Figure 57 shows a charac- teristic which distinguishes the area membranacea from that of the pig, although in the later stages of the pig embryo (figs. 45 and 47) this feature is present. This concerns the marked decrease of cellular tissue in the membranous area. In figure 57 the deeply staining typical ependyma is shown approaching from below. These cells end abruptly at the border of the area membranacea; the ventricle in this area is lined by cells possessing small elongated nuclei and long cytoplasmic processes, which unite to form a ventricular lining. The oval nuclei along the ventricular border become more closely massed together in the superior portion of the area, but nowhere is there the same architecture as in the equivalent stage in the pig (fig. 33). A feature of the histological appearance of the membranous area in the pig embryo is also shown in figure 57; this is the marked adherence of the coagulated albumen of the cerebro-spinal fluid to the area membranacea superior. The roof of the fourth ventricle in the human embryo is subjected to the same factors causing changes in the form and relationships which were commented upon *Measured on the slide after mounting. 38 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. in the pig; but these play little part until the chorioid plexuses become of sufficient size to divide the ventricle into a superior and inferior portion. In the human embryo, as in the pig, the superior half of the ventricular roof is sacrificed to the greater growth of the cerebellum. In human embryos of 17 mm., however, these factors have not begun to influ- ence the membranous area. This is shown in figures 58 and 59, photomicrographs from embryo No. 576. The section is somewhat to the side of the midline, but in the superior portion of the roof of the fourth ventricle the differentiated membra- nous area can be made out. The sharp delimitation of this area from the denser typical ependyma on both sides is quite apparent. The ragged character of the ventricular border, with its few elongated spindles, seems wholly in keeping with the transverse view of this area afforded by figure 37. Embryo No. 576 exhibits one characteristic of the area membranacea superior very frequently seen in human embryos, but almost invariably absent in these stages in the pig. Along the lateral margins of the superior membranous area are dense borders of the many-layered epithelial-like cells which lined the ventricular roof in younger stages. This feature is well shown in figures 60 and 61, the latter figure being a higher magnification of the former. The cellular border of the superior area reaches transversely only through a few 15-micron sections, but it extends throughout the whole cephalo-caudal diameter of the area. It seems likely that this represents purely a survival of the epithelial-like cells in the younger embryos. In rarer instances the whole area membranacea superior may be surrounded by such a border of many-layered cells, but even in these cases the superior and inferior margins are quite thin. No apparent agencies favoring the disappearance of the superior membranous area in the roof of the fourth ventricle of the human embryo are apparent in stages up to the fetus. Thus, in human embryos of 18 mm. this differentiated area in the roof has reached its maximal differentiation. A section from an embryo of. this size (embryo No. 409) is reproduced to show the distortion and its influence upon the topography of the area membranacea. The two photomicrographs (figs. 62 and 63) show the extreme collapse and distortion of the roof of the fourth ventricle. In the figure of higher power (No. 63) the membranous area appears facing poste- riorly, due to the shrinkage; the proper leader runs to this area. It shows the differ- entiation from the adjoining typical ependyma which is characteristic of the fully developed area membranacea superior. In a beautifully preserved and sectioned human embryo of 21 mm. (No. 460) in the collection of the Carnegie Institution of Washington the area membranacea superior appears as a sharply delimited area (figs. 64 and 65). These figures give a very good idea of the definiteness of the area when the fixation and dehydration approach the perfect. The tissue of this membranous area lining the ventricle here appears to be wholly lacking in an epithelial covering; the mesenchyme seems to serve as the ependymal lining. Study of this area, however, through different stages argues most strongly against such a view. UNDESCRIBED STRUCTURES IN ROOF OF THE FOURTH VENTRICLE. 39 The process of regression of the area membranacea superior in the human embryo differs somewhat from that described in the pig. This alteration in the mode of disappearance is largely due to the fact that in the period of growth from 20 to 35 mm. the superior portion of the roof of the fourth ventricle in the human embryo is not sacrificed to the cerebellar lips; for in the human the cere- bellum grows largely into the fourth ventricle, enlarging beneath the superior part of its roof. Thus, the attachment of this part of the roof is not greatly interfered with by the rapid development of the cerebellum. The total extent, then, of the superior portion of the roof is hardly altered in these stages in the human, while in the pig embryo the roof is shortened by its attachment to the inferior portion of the cerebellar lip, which retains its earlier characters. These differences in the relationship of the superior portion of the ventricular roof in human and pig embryos may be seen by comparison of figures 74 and 89. Another factor which renders the mode of disappearance different in the two embryos concerns the greater tufting and development of the chorioid plexuses of the fourth ventricle in the pig. This greater size and complexity of the plexus causes an encroachment upon the roof structures which, in the pig embryo, seems of considerable importance in the final closure. In the human embryo, however, it has been found very difficult to explain the final disappearance of the superior membranous area on the same mechanical factors which seemed so well to account for its transitory characters in the pig; but at approximately the same stage of growth the process of regression occurs in the human fetus. The area maintains a fair size in stages up to a length of 23 mm. Thus, in figures 89 and 90 (No. 453 of the Carnegie collection) a sagittal section from a human fetus of this size is illustrated. In the higher power (fig. 90) the superior membranous area is shown, rather sharply delimited on its superior border by the typical, dense ventricular ependyma. Below, its edge is irregularly formed by the deeply staining ependyma over the invagination of the chorioid plexus. The cell-character of this area resembles that shown in the photomicrographs from the specimen of 21 mm. (figs. 64 and 65). There is left in the area no indication of the cellular architecture which characterized the original ventricular ependyma; the cells with their elongated cytoplasmic processes here have the oval nuclei which are found almost invariably in this membranous area. In the human fetus of 26 mm. (No. 1008 of the collection of the Carnegie Institution of Washington) there is but slight evidence of a superior membranous area in the upper portion of the roof of the fourth ventricle. The evidence present in this specimen consists in a localized thickening of the lining cells of the ventricle in the situation of the area in other stages. This thickening is illustrated in figures 91 and 92; it consists of several layers of epithelial-like cells, similar in all respects to the many-layered border shown in figure 83. The picture is somewhat obscured by the vascular plexus directly beneath the ventricular lining. There is difficulty in determining exactly when the last evidences of the supe- rior membranous area in the roof of the fourth ventricle may be found. This is 40 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. due to the likelihood of artifacts disturbing the character of the ventricular lining in human material, where the freshness and fixation of the specimen may not be ideal. In the larger specimens in the collection of the Carnegie Institution, which are well fixed and sectioned, the existence of the area membranacea superior could not be wholly verified. Thus, in specimen 405 (26 mm.) the presence of the area seemed probable though not definite. In another embryo of this same size (No. 782) the existence of this area was still more questionable. In a larger embryo (30 mm., No. 75) the presence or absence of the area could not be assured; many indications suggested its existence, but the resemblance to an artificially separated ependyma was strong. In all specimens of human embryos of over 30 mm. examined, no evi- dence of the area membranacea superior could be found. It appears likely, then, that the final disappearance of this differentiated area in the roof of the fourth ven- tricle occurs at a slightly earlier stage in the human embryo than in the pig. The final disappearance of the area membranacea superior in the human embryo is not accompanied by the same ingrowth of typical ependyma that characterizes the process in the pig. There is a great tendency, in the human, as indicated in figure 92, for a replacement of the area by the same type of epithelial-lke cell which comprised the whole ventricular roof in the earlier stages (fig. 41) and later formed lateral borders for the superior membranous area (fig. 83). Thus, in a human embryo of 24 mm. (No. 632 of the Carnegie collection) there is evidence of a very- small membranous area surrounded by a border of epithelial-like cells. In a slightly larger specimen (No. 840, 24.8 mm.) the whole membranous area is occupied by the epithelial-like cells. The frequent association of these cells with the area indicates that in disappearing the area membranacea is probably replaced first by these cells, which in turn disappear, so that the whole roof is finally composed of the typical, densely staining ependyma. THE AREA MEMBRANACEA SUPERIOR IN OTHER ANIMALS. In order to ascertain whether the area membranacea superior existed in other animals examinations of serial sections of the rabbit, cat, sheep, and chick of suitable stages were made. All of these animals were found to possess a differentiated area in the roof of the fourth ventricle. Opportunity was afforded for the study of serial sections of the head of a chick* of 121 hours’ incubation. The head was carefully dehydrated and embedded by Dr. E. R. Clark, and was subsequently sectioned by Dr. C. R. Essick. The material was beautifully fixed and dehydrated, showing practically no evidence of shrinkage. Typical portions of the superior membranous area are reproduced in figures 66, 67, 68, and 69. Figure 67, taken near the crown of the embryo and representing the squared area in figure 66, shows the two dense masses of ependyma separated by the more lightly staining area membranacea. The cellular character of this differen- tiated zone resembles more the histological features of the similar area in the pig than those of the human embryo. This resemblance is also to be seen in figure 69, taken *This chick measured 14 mm. in 40 per cent alcohol. UNDESCRIBED STRUCTURES IN ROOF OF THE FOURTH VENTRICLE. 41 more posteriorly than the two preceding figures. The dense ependyma approaching on both sides is sharply delimited at the edge of the broad membranous area. This is composed of cells having elongated, chromatin-poor nuclei, and long cytoplas- mic processes, which form the ventricular roof. The adherence of the albuminous coagulum occurs here also. In the rabbit the occurrence of the superior membranous area was verified as in the other species studied. In a rabbit embryo of 13 mm. (series x in the embryo- logical collection of this laboratory) the area was well differentiated from the sur- rounding typical ependyma. The cells of the area resembled those of the adjacent mesenchyme. The ventricular surface was roughened by the projection of num- erous protoplasmic processes. An albuminous coagulum was attached to the cells of the membranous zone. One sheep embryo from the collection of this laboratory was also studied. The sections, although labeled as an embryo of 10 mm., resembled in every way a pig embryo of 18 mm. The area membranacea was easily identified in the roof of the fourth ventricle; it is similar in every respect to the same area in the pig and the human embryo. In a cat embryo of 10 mm. asmall but highly differentiated area membranacea superior was made out. The most striking feature in this specimen is the great adherence of the coagulated albumen to the cells of the area and the resemblance of these cells to the mesenchymal elements adjacent. The edges of this differentiated area are sharp and clear-cut. No attempt was made to identify the area membranacea superior in other animals—as further suitable material was not immediately available. The chief study has been made on pig embryos and on human embryos. The occurrence of the area in the cat, sheep, and rabbit probably indicates its existence in all mammals. The finding of such an area in the chick is also suggestive. GENERAL CONSIDERATION OF THE AREA MEMBRANACEA SUPERIOR. The occurrence of a definite area of differentiation in the superior portion of the roof of the fourth ventricle has been pointed out in preceding subdivisions of this paper. It has been described in detail in the pig embryo and in the human embryo; it has been identified also in cat, sheep, rabbit, and chick embryos. It remains here to discuss the general characteristics of this area. No description of such an area of differentiation in the ventricular roof has been found in the literature. It may be that the distortion of this structure in the course of the usual embryological technique has rendered its discovery less likely. His’5), in his description of the ventricular roof, has not commented upon the occurrence of this membranous area, even though in a retouched photomicro- graph of his fetus C—1 (a human specimen, of the beginning of the third month) the area membranacea superior can be made out. Likewise in his description of the plica chorioidea he fails to mention any differentiated areas in the roof, although plate 1, in his ‘‘ Die Entwickelung des menschlichen Rautenhirns, von Ende des ersten 42 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. bis zum Beginn des dritten Monats,’’ shows a slight irregularity in the roof. Prac- tically all of the contributions to the anatomy of the roof of the fourth ventricle deal with the lower half of the structure, with particular reference to the occurrence of the foramen of Magendie. The general biological process involved in the formation of the area membra- nacea superior concerns a differentiation of the epidermal elements which line the ventricular cavity. This differentiation, both in human and in pig embryos, first begins with the occurrence in the ventricular roof of an area of epithelial-like cells. These, in the course of enlargement of the roof, become more or less isolated in the superior portion of the structure, and then undergo a metamorphosis into the typical cells of the membranous area. They are characterized by oval or elongated nuclei (rather poor in chromatin as compared with the nuclei of the typical ependymal elements) and by cytoplasmic strands (in which the cell-boundaries are very poorly marked) which compose the ventricular border. The ventricular surface in the area membranacea is more ragged and irregular than where lined by typical epen- dyma. In many instances, as in figure 57, from a human embryo of 14 mm., this transformation has proceeded to such an extent that the epithelial character of the lining cells is almost wholly lost, and the ventricle seems, in this area, to be lined by mesenchyme. Study of the membranous area in many stages convinces one that such an hypothesis is untenable; in every case the ventricle must be considered as being lined by epidermal elements, no matter to what extent the process of differ- entiation has proceeded. There is no real evidence to support the view that the ependymal lining of the ventricle has been replaced by mesenchymal elements to form the area membranacea superior. In general the area membranacea superior is a rounded oval. Its measurement is quite difficult except when fixation and dehydration have been excellent, because of the highly abnormal distortion of the ventricular roof which frequently occurs in the technically poor specimens. Measurements have been made in a considerable number of favorable specimens, both of human and pigembryos. With the history of this area in mind, it will be realized that the size of the structure necessarily varies with the length of the embryo, attaining its greatest dimensions at about the length of 18 or 20 mm. Herewith is a short table of the measurements taken. Dimensions of area membranacea superior. | secies, | No.of | Length of} Width | Length|| . No. of | Length of Width Rpecies. | specimen.| embryo. | of area. | of area. cae specimen.) embryo. | of area. mm. mm. mm, mm, mm, mm, Rigs is teste } 98 12 0.37 0.5 Pig ver. totes 121 16 0.6 0.48 Rabbit....... | 107 13 0.95 | 0.4 Human..... 576 17 1.5 0.9 IMIAN Fain ye 144 14* 1.25 ted Sheep: |. «5. 108 | 18 (?) 0.8 0.8 Pig eisai 5 ate ec 119 14 0.45 0.6 Pig fe. dee é | 0.9 0.4 Chidhy occas 2 106 14 0.65 0.85 Pigiaiack shee 0.8 0.7 In a rough way, then, we may consider the area membranacea as an oval; in some cases the longitudinal diameter exceeds the lateral, and in others the reverse *Measured on slide after mounting. UNDESCRIBED STRUCTURES IN ROOF OF THE FOURTH VENTRICLE. 43 holds. The measurements given above were taken from mounted sections and are probably somewhat disturbed by the histological technique which was followed. The borders of this oval area membranacea are usually fairly regular and smooth, but in some instances they are irregular, due to the fact that small exten- sions of the area run into the bordering ependyma. These extensions are more commonly met with at the stage when the area has reached its maximum size, as in figures 38 and 39, photomicrographs from an embryo pig of 19 mm. The higher power of these two photographs shows two areas in the smoother ependymal wall. These are extensions of the area membranacea, and within a section or two directly connect with the differentiated area. Both of these small spots on the circumference resemble technical errors; their ragged appearance, the relative exca- vation of their surface, and the intact ependymal borders would seem to encourage such a view; but when considered in connection with the character of the whole area membranacea they assume a definite relationship in this regard. Other similar areas, rather rare in occurrence, are found separated entirely from the main area membranacea. These isolated areas are of the same size as those shown in figure 39. In significance and character they are probably identical with the larger area mem- branacea superior. Most of the general features of the area membranacea superior have been com- mented upon in descriptions of the various stages of differentiation in both pig and human embryos. The characteristics most commonly observed concern the dif- ferentiated character of the cells of the area, the sharp borders of the typical epen- dyma, the ragged ventricular surface throughout the whole extent, and the peculiar adhesion of the albuminous coagulum from the embryonic cerebro-spinal fluid to the lining cells. ‘The area membranacea superior should be considered, then, as a transi- tory focus of differentiation of the typical ependymal lining of the roof of the fourth ventricle. AN UNDESCRIBED AREA IN THE INFERIOR PORTION OF THE ROOF OF THE FOURTH VENTRICLE. With success attending the effort to find in the superior portion of the rhombic roof an anatomically differentiated area which would furnish a morphological basis for the physiological phenomenon of the extraventricular passage of the cerebro- spinal fluid, attention was necessarily directed to the inferior portion of this roof (considering the whole roof structure to be divided by the chorioid plexuses). The spread of the replaced injection fluid (fig. 4) into the periaxial tissues through two points in the roof of the ventricle suggested a study of this stage (pig embryo of 18 mm.) as the basis of the investigation. As a histologically differentiated area in this inferior portion of the roof is easily made out, the complete history of the area will be given chronologically. It has been termed the “area membranacea inferior ventriculi quarti,’’ the terminology being based on the same physiological and anatomical features which led to its adoption in the case of the analogous area in the upper portion of the roof. 44 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. THE AREA MEMBRANACEA INFERIOR IN THE PIG EMBRYO. The inferior portion of the fourth ventricle shows no evidence of a differentiation from the typical lining ependyma until the length of 15 mm. is reached. In this development consideration must be given to the factors concerned in the process. It will be recalled that in the younger embryos, both pig and human, up to and including a length of 9 mm. the whole roof of the ventricle is occupied by the epi- thelial-like cells. With rapid growth of the medulla and corresponding enlargement - of the fourth ventricle the roof becomes elongated and widened. This process results in the isolation of the area composed originally of the epithelial-like cells and the subsequent formation of the superior membranous area. The epithelial-like cells remain in the superior portion of the enlarged ventricular roof, while the whole inferior half is composed of the densely staining, typical ependyma. The division of the roof by the laterally developing chorioid plexuses becomes evident in pig embryos of 14 mm. At this stage the whole inferior portion shows a ventricular lining composed of the typical ependyma. The first indication of a differentiation in this inferior half of the roof was found in a pig embryo of 15mm. This is illustrated in figures 70 and 71. The sagittal section from which these photomicrographs were taken is near the mid-line of the embryo, as is indicated by the partial section of the central canal of the spinal cord (fig. 70). The division of the ventricular roof into two parts is also indicated in figure 70 by the invagination of the chorioid plexus. The squared area in the lower half is reproduced in figure 71 under higher magnification; here the first evidence of an ependymal differentiation is observed. The dense line of the typical ependyma appears from both sides, but in the center of this ventricular lining a small area of differentiation is seen. This area, isolated by the abruptly terminating pyknotic ependymal elements, is composed of two or three layers of less deeply staining cells. The nuclei are round, rather larger than those of the adjacent mesenchyme, and contain little chromatin. The cytoplasm stains fairly well with eosin and is not scanty in amount. The cells resemble those epithelial-like elements which so largely make up the ventricular roof in the earlier stages. No albumen is found near this point of differentiation, although the whole ventricular cavity is filled with the normal amount. In figure 70 the marked zone of the area membranacea superior may easily be seen. After this initial indication of a differentiation in pig embryos, the further dif- ferentiation of the tissue proceeds but slowly until the length of 18 mm. is attained. Thus, in a similar specimen from an embryo pig of 18 mm. the area of differentiation is not greatly increased in size. This is shown in figures 72 and 73. In the higher- power figure (fig. 73) both the superior and inferior membranous areas can be made out by the attachment to these areas of the protein coagulum of the ventricular cerebro-spinal fluid. In the higher-power figure (fig. 73) of the squared area from figure 72, the area membranacea inferior shows the same character as exhibited by the specimen of 15 mm. (fig. 71). The opening maintains the same approximation to the lateral lip UNDESCRIBED STRUCTURES IN ROOF OF THE FOURTH VENTRICLE. 45 of the medulla, but the area is larger and the histological character more nearly approaches the permanent feature of the tissue. The nuclei in this zone are paler than those of the adjoining ependymal elements and contain less chromatin. The cytoplasm is not scanty, nor is it very abundant in amount. The area is also characterized by the occurrence of the cells in a layer, two or three cells in thickness. In view of the very slow differentiation of the area membranacea inferior in the growth of the embryo from 15 to 18 mm., the enormous enlargement of the region within the next few millimeters’ growth is very astonishing. This period, as has been pointed out, is a critical one in the extension of the embryonic cerebro-spinal fluid from a ventricular to a periaxial relationship. Apparently, in the course of the embryo’s growth during these next few millimeters the whole inferior roof of the ventricle undergoes a transformation and enlargement, so that the differentiated area membranacea comes to occupy practically the whole inferior half of the roof. This portion of the roof, persisting, enlarging, and suffering no extension of nervous tissue upon it, becomes the tela chorioidea inferior. The rapid differentiation of the whole inferior half of the roof of the fourth ventricle is a very interesting process. Apparently the typical ependymal ele- ments, visible on both sides of the membranous area in figure 73, undergo a very rapid alteration, so that in the course of a few millimeters’ growth the cubical lining of the ventricle is replaced by a low-type cell, with round or oval nuclei, staining much less densely than do the ependymal elements. The whole area membranacea rapidly becomes a membrane in the true sense of the word; it is a continuous, intact layer of cells, generally only one cell in thickness, closing in the fourth ventricle from the chorioid plexus above and the bulbar lips on the sides. The general characteristics of this transformation are seen in figures 74 and 75. These photomicrographs are taken from a sagittal section of a pig embryo of 23 mm. On one side of the sharply delimited membrane shown in figure 75 is a tongue of nervous tissue of the medulla; on the other is the differentiated ependyma of the chorioid plexus; between these two structures stretches uninterruptedly the area membranacea inferior. The flattened cells of the membrane, with their oval nuclei and almost continuous cytoplasm, effectually close the whole ventricle. The pho- tomicrograph also shows an interesting characteristic of this membranous area which is universally present in the larger forms; this is the relatively unsupported character of the membrane. The highly vascular mesenchyme posterior to the area has gradually developed, during growth, larger and larger interstices between the cytoplasmic processes. The phenomenon is not due to shrinkage, but is intimately connected with the formation of the future cisterna cerebello-medullaris. This phase of the mesenchymal differentiation will be more fully considered in an appro- priate section of this paper. It will suffice here merely to record the lack of support of the membrane. Another phenomenon of importance in the cerebro-spinal fluid relationships of this stage is shown in figure 75. In the mesenchymal spaces directly beneath the membranous area there is a large amount of albuminous coagulum. This phenome- 46 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. non does not occur to any appreciable extent in earlier stages or in other parts of the mesenchyme, except about the nervous system. The close association of the coagulum from the ventricular cerebro-spinal fluid with the inner border of the area membranacea (shown in figure 75 as a slight roughening of the border) is of very great significance in thisconnection. In one point in the membranous area (fig. 75) the albumen can be traced almost without interruption from the ventricle into the wide spaces of the mesenchyme (cf. fig.8). This observation strongly suggests that the embryonic cerebro-spinal fluid, which is rich in protein material, is passing, in this stage of embryonic growth, from the ventricle into the periaxial mesenchyme; and such an interpretation becomes established by the comparative findings in the embryo of the same stage in which a replacement of the cerebro-spinal fluid by the ferrocyanide solution had been effected. These comparable findings are surely of the utmost importance for the final solution of the problems centering about the embryonic cerebro-spinal fluid. In the later stages of development of the area membranacea inferior in the pig embryo the same structural relationships persist that are shown in figure 75. Figures 76 and 77 are photomicrographs taken from a sagittal section of a specimen of 32mm. In the enlargement of the squared area, from the first of these figures, the continuity and completeness of the membrane are well established. The photo- graph shows well the flattened character of the cells comprising the membrane and its sharp differentiation from the nervous tissue and ependyma below and from the ependyma and chorioid plexus above. Most important in this case is the distribu- tion of the albuminous coagulum. Within the ventricular cavity this appears in considerable amount, and in several places it is in close adhesion to the lining area membranacea. This albuminous precipitate may likewise be traced in some places apparently through the cellular membrane into the periaxial spaces. For here, as indicated in figure 75, the clotted albumen from the cerebro-spinal fluid apparently exists in large amounts in the space just posterior to the membrane—the future cisterna cerebello-medullaris. Delicate strands of mesenchyme are still observed running through the wide space, but in general the whole tissue has returned to the line of the future arachnoid. The relative lack of substantial support of the mem- brane is well brought out in figure 77. A characteristic feature of this membrane, which Blake) has championed, and which is indicated in figures 76 and 77, is the posterior bulging of the roof—‘‘the caudal process like the finger of a glove.” Another section from the same pig embryo, taken more laterally, is represented in figures 78 and 79. In the photomicrograph of higher power the flattened char- acter of the lining cells, the intactness of the membrane in isolating the ventricular cavity, the unsupported freedom of the membrane, and the relation to the albumen coagulum on both sides are of particular interest. The ultimate fate of the area membranacea inferior will not be more fully entered into until the early history of the similar area in the human embryo has been detailed. For in this connection the occurrence of the foramen of Magendie requires discussion, and it seems best to delay the further consideration of the present topic until the whole question can be reviewed. UNDESCRIBED STRUCTURES IN ROOF OF THE FOURTH VENTRICLE. 47 THE AREA MEMBRANACEA INFERIOR IN THE HUMAN EMBRYO. The same process in the formation of an area of differentiation in the inferior portion of the roof of the fourth ventricle may also be followed in the human embryo. Unfortunately, however, human embryological material can rarely be subjected to the immediate fixation and preservation which yield excellent histo- logical results in the more plentiful specimens. It does not seem strange, therefore, that the determination of the exact stage at which an area of differentiation can be made out in the ventricular roof should be practically impossible; for, in poor tech- nical procedures, the roof of the fourth ventricle suffers almost more than does any other portion of the specimen. In a human embryo of 13 mm. (No. 695 in the collection of the Carnegie Insti- tution of Washington) there is slight evidence of a differentiation in the lower por- tion of the rhombic roof. The changing character of cells in this specimen is not marked, but as the central portion of this inferior roof is reached the ependymal cells seem to assume gradually a more cubical morphology. Associated with this change in shape, there is also a slight loss of the deeply staining character of their nuclei. The whole differentiation, however, is slight and would be commented upon only from the conception of this area in the pig embryo. The first definite evidence of differentiation in the inferior portion of the ven- tricular roof was found (specimen 390 in the Carnegie collection) in a human embryo of 15.5mm. This initial differentiation occurs, then, in the human embryo of approximately the same length as in the pig. The specimen showed the same change in character of the lining ependyma as was found in the pig. The deeply staining ependymal elements are replaced in a limited central area in the inferior portion of the roof by cells with more elongated nuclei, poorer in chromatin, and resembling somewhat the epithelial-like cells which early filled the ventricular roof. These cells tend to compose a layer of more than one cell in thickness—a feature particularly noticeable in the peripheral portions. The size of the area membranacea inferior observed in specimen 390 suggested that the earliest evidence was probably to be observed in somewhat smaller speci- mens. This could not, with the material at my disposal, be verified, but it is prob- ably safe to assume that the first signs of an ependymal differentiation will be found in human embryos of about 15 mm. This time of appearance of the area in the human would coincide with its time of primary differentiation in the pig embryo. In this limitation of the first appearance of the area membranacea inferior, the standard has been an unmistakable differentiation of ependyma and not an isolated change of a lining-cell or two which might have been the result of the technical procedure. Such a criterion was necessitated by the very marked changes in the ventricular borders observed in specimens in which distortion of the chorioidal roof had occurred. The area membranacea inferior very rapidly increases in extent after the onset of the process of epencymal differentiation. This was likewise observed in the pig embryo, although perhaps more stages could be made out. In a human embryo of 48 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. 16 mm. (No. 406 of the collection of the Carnegie Institution) the area membranacea inferior is quite extensive, as is shown in figures 80 and 81. In the photomicro- graph under higher power (fig. 81) the densely stained ependyma approaches the membranous area (amz) as tongue-like processes from above and below. These tips gradually lose their dense character and are prolonged as a delicate membrane, lining, in this localized area, the ventricular cavity. The nuclei of the cells here are not heavily laden with chromatin; they are oval and somewhat larger than the more densely packed nuclei of the typical ependymal element. Unfortunately, the middle portions of the membranous area in this specimen are surrounded by extrav- asated red blood-cells obscuring somewhat the structure (fig. 81). The process, though, of the differentiation of these ependymal elements into paler and larger epithelial-like cells is quite apparent. As in the pig, the tendency of the differentiated ependymal cells forming the area membranacea inferior to lose in some degree their distinctive appearance and to approach in character the undifferentiated mesenchymal element is apparent in the human embryo very shortly after the original steps in the process of differen- tiation have occurred. Photomicrographs from two human embryos of 17 mm. have been included to show this phenomenon. Thus, in figure 88, an enlargement of the blocked area from figure 58, the area membranacea inferior (ami) is well defined. The sagittal section from which this photomicrograph was taken is from embryo No. 576, in the Carnegie collection. Above and below the dense line of ependyma may be made out; this tapers quite abruptly, to be succeeded by the cells of the area membranacea inferior. These cells, products of ependymal differentiation, have lost much of their epithelial-like appearance; they now show rather small, oval or rounded nuclei, poor in chromatin. The cytoplasm of the cells is small in amount, but not disproportionate for the size of the nucleus. The ventricular border of these cells (fig. 88) exhibits a rather characteristic phenomenon, the adherence of a slight albuminous coagulum. The fine processes of this coagulum fuse with the eyto- plasmic borders of the cells and render these borders vague and indefinite. Beneath the cells of this inferior area small vascular channels may be made out. These tend to make the membrane appear denser than its cellular character warrants. In another section from this same embryo (No. 576) the inferior membranous area is shown in relation to the tufted chorioid plexuses (figs. 82 and 83). In the reproduction under higher magnification (fig.83) the ependymal lining may be traced caudalwards to a gradual fusion into the area membranacea inferior. From the rather high cubical cells in the immediate proximity to the plexuses the ependymal elements become reduced in size and in height, and then rather abruptly the pyknotic character of the ventricular lining is lost. This loss of the deeply staining character coincides with the superior border of the area membranacea inferior (ami). The membrane of this area shows the same cell-character as already described for this embryo. On the superior side of the plexuses (fig. 83) the lateral border of the area membranacea superior (ams) is shown composed of epithelial-like cells. UNDESCRIBED STRUCTURES IN ROOF OF THE FOURTH VENTRICLE. 49 The apparent tendency of the cells composing the inferior membranous area to lose the epithelial-like character, as shown in the figures from embryo No. 576, is not an invariable phenomenon. Rather is an aggregation of epithelial-like cells met with in human embryos very commonly in this area, not only in embryos of small size, but also in small fetuses. This phenomenon is illustrated in figures 84 and 85, reproductions of photomicrographs from a human embryo of 18 mm. (No. 409 in the collection of the Carnegie Institution). In figure 85 the total transverse extent of the area membranacea inferior (am?) is illustrated, with the villous chorioid plexuses appearing to the left. Although this membranous portion of the embryo has been distorted somewhat by the technical procedures to which the specimen was sub- jected, the cellular character of the membranous area is well indicated. The most striking feature, apart from the characteristic tinctorial differentiation from the typical ependymal elements, consists in the marked clumping of the cells in certain parts of the membrane. On one lateral extent the membrane is thickened into a bulbous swelling several cells in thickness. These cells have palely staining nuclei, poor in chromatin, with an oval or round form. In other places in the membrane smaller but no less characteristic clumps of similar cells may be made out. Between these cellular aggregations the membrane stretches in a continuous line with but few nuclei. Analogous clumps of cells, with pale, rounded or oval nuclei, may be made out in figures 86 and 87, taken from a human embryo of 19 mm., No. 431 in the collec- tion of the Carnegie Institution. Only a small portion of the membrane is repro- duced in the figure under higher magnification, but a characteristic clump of epithe- lial-like cells (epc) is shown. These cells of the differentiated ependyma here again have oval and rounded nuclei, poor in chromatin, similar to those which have been pointed out many times in the foregoing pages. A second broadened area in the inferior membrane is also shown in figure 87. The further development of the area membranacea inferior proceeds in the human embryo in a manner very similar to that described for the pig. In the stages but slightly above those already described the differentiation goes on slowly, but within a few millimeters the cellular pictures resemble those given for the embryo of 17 mm. (figs. 82,83, and 88). The cellular clumps which appeared quite frequently in the embryos under 20 mm. have not been found in the larger forms. Thus, in an embryo of 23 mm. (No. 453 in the collection of the Carnegie Institution) the inferior membranous area (ami) appears as an extensive membrane comprising almost wholly the inferior portion of the chorioidal roof. The membrane is here of a single cell in thickness; these cells are rather small, with oval nuclei, simulating in some measure those of the surrounding mesenchyme. The most interesting phase of the membranous area at this stage of 23 mm. concerns its completed cellular differen- tiation and its rather slow increase in size. Wholly similar pictures of the inferior membranous area of the roof of the fourth ventricle are afforded by a human fetus of 26 mm. (figs. 91 and 92). These photomicrographs were taken from embryo No. 1008 in the collection of the Car- 50 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. negie Institution. In this specimen (fig. 92) the fourth ventricle seems almost to lack a lining of ependymal (epidermal) elements in the area membranacea inferior (ami). ‘The cells of this area are small, inconspicuous in their distinctions from the underlying mesenchyme. The whole character resembles that of the superior area membranacea shown in figure 57. The appearances exhibited by the inferior membranous area in the stages above 26 mm. are modified in great part by the development of the great cisterna cerebello- medullaris. As in the pig, the breaking-down of mesenchyme to form this cistern results finally in the almost total isolation of the inferior membranous area. The cistern is fairly rapidly formed when once the process begins, and so in an embryo of 35 mm. (No. 199 in the Carnegie collection) the isolated character of the area mem- branacea inferior (amt) may be easily made out. This is shown in figure 94, an enlargement of the blocked area in figure 93. The general architecture of the mem- brane, particularly its intact character, appears in this photomicrograph, but its finer structure is obscured by the albuminous coagula which adhere on both sur- faces. The cell structure of the area membranacea resembles closely that described in the embryos already pictured. Discussion of the final disposition of the area membranacea inferior will be undertaken in the following subdivision of this paper, in order that the findings in the pig and in the human embryo may be correlated. GENERAL CONSIDERATION OF THE AREA MEMBRANACEA INFERIOR. The ependymal lining of the caudal portion of the roof of the fourth ventricle undergoes a process of differentiation which results in the formation of the area membranacea inferior. This transformation has been observed in pig and human embryos; in both, the first definite evidence of the cellular change has been observed in specimens of 15 mm. The essential phases of the process are identical in the two embryos. The tendency of the deeply staining typical ependymal elements is to lose their highly pyknotic character; the nuclei become poorer in chromatin and the cytoplasm somewhat more abundant. In the first stages of the metamor- phosis the lining cells come to assume epithelial-like appearances, but in the final change the nuclei become small oval bodies, poor in chromatin, resembling to some degree the nuclei of the adjoining undifferentiated mesenchyme. In the human embryo, a tendency for the epithelial-like characters to persist in isolated cellular aggregations is apparent. After the initial process of differentiation has begun, the area membranacea inferior increases rapidly in extent and the differentiated cells which characterize it come to occupy the greater portion of the caudal part of the chorioidal roof. In the somewhat later stages the area membranacea is almost wholly unsupported by other tissues, due to the development of the cisterna cerebello-medullaris. As soon as the cistern forms, the area membranacea serves as practically the sole dividing membrane between the ventricular system and the future subarachnoid spaces. UNDESCRIBED STRUCTURES IN ROOF OF THE FOURTH VENTRICLE. 51 The ultimate fate of this area membranacea inferior is necessarily involved in the distribution of the tela chorioidea inferior. Likewise it necessitates a discussion of the possible formation of the so-called foramen of Magendie and its mode of origin from the ‘‘caudal process” of Blake. It is proposed to discuss briefly some of these questions in the hope that some phases of the problem may be brought forth. It must be clearly understood that the questions of the ultimate fate of this area membranacea inferior probably differ considerably in the different species of mammals. In the horse and in the pig the absence of the medial foramen (Magendie) is fairly well established, but in man its existence seems to rest on equally firm grounds. While, primarily, this investigation has not been concerned with the possible existence of the foramen of Magendie, the question has been presented many times in regard to the pig and human embryos examined. As far as can be determined, no descriptive study of the development and differentiation of the inferior portion of the rhombic roof has been published. Heuser’s23) studies on the form of the cerebral ventricles of the pig have afforded a very good conception of the gradually changing relationships in this region. Hess?) has devoted attention to the histological appearances of the inferior roof in the embryo. One of his interesting observations concerns the caudal portion of the rhombic roof in a fetal cat of 10 em., where he noticed a very sudden interruption in the epithelial lining of the ventricle, with a complete closing by a fibrous net. This description by Hess is the only comment upon the histological appearance of the ventricular roof that has been found. His®5) pictures, without comment, in a retouched photomicrograph, a differentiated area in the proper situation in his fetus C-1 (beginning of the third month). The many writers in embryology have commented upon the roof of the fourth ventricle. Minot), in 1892, stated regarding it: “Several writers have thought that the membrane was broken through at several points, but it probably is really continuous throughout life. The fourth ventricle is to be regarded, then, as an expansion of the central canal permanently bounded by the original medullary walls.’ Kollman‘2), on the other hand, advances the view that during the third month the rhombic roof is broken down to form the foramen of Magendie and the two foramina of Luschka. Streeter 4), in his chapter on the development of the nervous system in the Keibel-Mall Handbook of Embryology, advances a similar view. The majority of investigators to-day incline to the belief that the roof of the fourth ven- tricle in man is perforated to form the median foramen of Magendie. Hess2) has advanced a conception of the foramen of Magendie that is sup- ported by numerous observations. To test Kolliker’s statement that the fourth ventricle remained closed during human embryonic life, Hess sectioned the region in human fetuses, new-born infants, and in adults. The lengths of the fetuses cut were as follows: 7, 12.5, 15, 16, and 17 em. In the 47 cases the roof showed a medial opening (Magendie), except in one case, in which it was closed by a “thin pial membrane.” Hess’s conception of the process of formation of this membrane 52 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. was that in early embryological life the rhombic roof was bordered by a regular, meshed tissue. Later the small meshes in this tissue fused to form the larger fora- men of Magendie. Blake’s® hypothesis of the formation of the medial foramen has been quite extensively quoted in the more recent publications on this subject. In a study of the chorioidal roof Blake found a caudal bulging of the inferior velum; this out- pouching became more and more extensive in the older embryos.. In man this pouch became sheared off at its neck, leaving the foramen of Magendie. In addition to the few studies referred to above, there have been in the past 25 years a great number of articles (notably those of Wilder®® and Cannieu™)) offering evidence that this median foramen of the fourth ventricle is an existent, functional opening. Into this literature it is not proposed to go in the present communication; it may be stated that in the larger part the views presented have been in favor of the consideration of the true occurrence of the foramen of Magendie. The material on which this study is based has been purely embryological in type, so that no reliable data regarding the foramen of Magendie could be obtained. But even in the largest fetuses examined, there was no evidence which indicated a breaking-down or a shearing-off of the inferior roof of the fourth ventricle. In the largest human fetus at my disposal, in which the histological material was good enough to permit an accurate examination of the chorioidal roof (embryo No. 448, 52 mm. in the Carnegie collection) the area membranacea inferior appeared as an intact membrane supported only be a few pial cells. In the pig the material at hand has been such that accurate study of the roof could be made in specimens up to 20 em.; in all of these later fetal pigs the roof has been wholly without foramina. If, however, in these larger stages the histological procedures have not been of the best, ruptures and other artificial separations are very frequently found. The area membranacea inferior, then, may be regarded as a region of ependymal differentiation. Whether it persists as an intact membrane or undergoes, in certain animals, a perforation to form a foramen of Magendie can not be here answered; this study has been concerned solely with the embryology of the cerebro-spinal spaces, and it affords no evidence in favor of or against the existence of such a fora- men. Nor has any study been made of the two foramina of Luschka, the two open- ings from the lateral recesses of the fourth ventricle into the subarachnoid spaces. It can be stated, however, that these foramina are not in existence at the time of establishment of the circulation of the cerebro-spinal fluid. This phenomenon, as recorded in the previous section, occurs in pig embryos of 26 mm.; at this time the lateral recesses are anatomically and physiologically closed. PASSAGE OF FLUID THROUGH ROOF OF THE FOURTH VENTRICLE. 53 VI. PASSAGE OF FLUID THROUGH ROOF OF THE FOURTH VENTRICLE. On pages 20 to 30 is a description of the passage of a true solution, substituted without increase in pressure for the embryonic cerebro-spinal fluid, through the roof of the fourth ventricle into the extraventricular or periaxial spaces. This extension of fluid occurred in two localized areas, one in the superior half and the other in the inferior half of the rhombic roof. Histological study of these regions revealed a localized differentiation of the ependyma, both in the upper and lower halves of the ventricular roof. It becomes necessary, then, to correlate, if possible, the areas of this fluid-passage to the anatomical differentiations pointed out. THE ACCUMULATION OF INJECTION-MASSES IN THE SUPERIOR MEMBRANOUS AREA. It has already been recorded that the first evidence of a change in the reaction to a replacement injection occurred in an embryo about 13 mm. long (fig. 2). This stage was characterized by a dense collection of the precipitated granules in a definite area in the roof of the fourth ventricle. At this stage also the area mem- branacea superior is well differentiated (fig. 31). That the site of the granular accumulation is this membranous area is easily proved by an inspection of figure 117, which represents an enlargement of the squared area in figure 116. In the low- power photomicrograph the prussian-blue granules are not represented, but are found scattered through the ventricles, with a definite collection in the posterior region of the fourth ventricle. Under a higher magnification (fig. 117) the blue can be traced in but small quantity along the normal ependymal lining (shown to the left in the figure), but as soon as the differentiated area (area membranacea superior) is reached the granular material is heaped up in a dense mass, which extends as a thickened pad into the ventricle. The same phenomenon of the accumulation of the injection fluid in the superior membranous area is shown in figures 112 and 113, the second photomicrograph representing the area outlined in the first, but reproduced under much higher magnification. In this specimen (an embryo pig) a dilute solution of silver nitrate was injected into the central canal of the spinal cord. On histological examination the accumulation of the silver also shown in figure 11 was found. Thus, in figure 113 the ventricular epithelium can be made out in the upper right-hand corner, while below (in the area membranacea superior) the silver is densely accumulated. The explanation of this phenomenon of accumulation in the superior membra- nous area is not wholly clear. It occurs only in stages in which the histological differentiation of the ventricular roof has proceeded to some degree and in stages where the fluid-passage into the periaxial tissues is not wholly unobstructed. This aggregation of the precipitated granules of prussian-blue and of the reduced silver in a localized area certainly suggests a physical explanation, as in these cases the physical laws of precipitation and reduction must hold. The many figures of the superior membranous area of the ventricular roof show that in the stage under con- sideration the cell-outlines projecting into the ventricles are rough and ragged as contrasted with the smoother and more regular surface of the adjoining ependyma. 54 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. Could not these roughened, irregular cell-surfaces become the site of the first and most extreme precipitation of the prussian-blue and of the reduction of the silver? Certainly they would serve much more efficiently as the foreign substances about which precipitation would occur in greatest amount. This physical explanation finds many arguments for its support in these studies. Another explanation of the phenomenon concerns the normal flow of the fluid and the relation of the direction of this flow to the roof of the fourth ventricle. As has already been emphasized, it is difficult to assume that there is any marked pro- duction of cerebro-spinal fluid before the periaxial spread occurs. Such an assump- tion would argue against the development of any special current toward the roof of the fourth ventricle in any stage smaller than that represented in figure 3, and would vitiate the explanation of the occurrence of the granular accumulation shown in figure 2 (a pig embryo of 13 mm.). In the later stages (16 mm., cf. fig. 11) this explanation would probably suffice for the phenomenon exhibited. THE SITES OF FLUID PASSAGE THROUGH THE ROOF OF THE FOURTH VENTRICLE. With consideration of the evidence presented as to the accumulation of the precipitates of the injected fluid about the area membranacea superior during certain stages in the development of the cerebro-spinal spaces, it would seem that the same area must be concerned in the passage of fluid from the ventricular cavities into the periaxial tissues. This view receives support from the reproduction of a cleared specimen (fig. 11) in which an injection of silver nitrate had been made into the central canal of the spinal cord. The pressure employed was great enough to force the fluid into the periaxial spaces, but the resultant picture clearly showed the oval outline of the area membranacea superior. The study of the passage of fluid from the ventricular to the extraventricular spaces can best be made by simple histological serial sections. In these observations pig embryos in which the cerebro-spinal fluid had been replaced by the compensating device, supplying a true solution of potassium ferrocyanide and iron-ammonium citrate, were sectioned and examined with reference to the sites of fluid passage. The results of these studies are given here in order that the whole question of the connection of the cerebral ventricles with the subarachnoid spaces may be discussed. In the stage represented by figure 3 (in which fluid passes from one area in the roof of the fourth ventricle into the extraventricular tissues) histological sections show that the point of fluid passage is localized and concerns solely the area mem- branacea superior. ‘The replaced fluid (as demonstrated by the subsequent precipi- tation of the prussian-blue) passes through this entire membranous area into the adjoining mesenchyme. The process is wholly confined to this area; the adjoining \/ ependyma is entirely impervious to the ferrocyanide. This phenomenon of passage of the replaced fluid through the superior membranous area is well shown in figures 14, 18, and 23. The distribution of the minute granules of prussian-blue in the cells of the superior membranous area is of importance in any discussion of the passage of fluid through a membrane; for this area (in the superior portion of the roof of the PASSAGE OF FLUID THROUGH ROOF OF THE FOURTH VENTRICLE. 55 embryonic fourth ventricle) must be considered as a membrane permeable in certain degrees to the fluids bathing it. That the area membranacea is intact and does not contain stomata or other minute foramina has been demonstrated histologically. Further evidence of the entire lack of intercellular stomata is afforded by the distri- bution of the prussian-blue granules precipitated in situ after the replacement of the cerebro-spinal fluid by the ferrocyanide solution. Figure 14 is a reproduction of the superior area from a transverse section of a pig embryo in which the routine replacement had been made. The position of the area is shown by the squared outline in figure 13. On both sides the impermeable ependyma is seen, with granules of the blue adhering to the ventricular border of the cells, but not penetrating them at all. To the left of the drawing the few epen- dymal cells possess, beneath their central border, a chain of the granules which have entered from the abrupt edge of the area membranacea. In the cellular border between the two lips of the ependyma, the area membranacea superior, the passage of the replaced fluid is easily made out by the resultant blue granules. The area is roughly delimited by a ventricular collection of the blue granules. Examination of these cells shows that the prussian-blue is present within the cytoplasm, avoiding the nuclei with perfect precision. Some of the cells are rounded and almost free from the granules; others, particularly those whose cytoplasm is elongated, are completely filled with the granules, the nuclei standing out in a blue granular cytoplasm. The question of the passage of the fluid between the cells must also be answered by the histological evidence. In the same drawing (fig. 14) in one or two places there are indications of a slight stream of granules between the cells of the area membranacea superior. This apparent transit of the fluid through intercellular passages is particularly clear in the small areas where the cellular cytoplasm is relatively free from the granular deposits. But upon careful examination of these areas under oil immersion it is always apparent that the adjoining cytoplasm is also involved in the granular precipitation, indicating that the cells, although almost free from the deposit, are also engaged in the process of the fluid passage. Com- pared to the whole area of fluid transit, the points indicative of a passage through possible intercellular stigmata are almost negligible. It seems not unlikely that the outlining of canals between cells may be a physical phenomenon, as in most cases no cellular borders (as demonstrated by the precipitated granules) can be made out. These peculiarities of fluid passage may be seen in figures 14, 18, and 23. Consideration of all the evidence afforded by histological examinations of the essential character of the area membranacea superior and of the passage of fluid through it inclines one inevitably to the belief that this area functionates as a cellular membrane. The fluid passes through it as through any permeable living membrane. Histologically the passage is for the most part through the cytoplasm of the cells, but occasionally an intercellular course is suggested. Both processes are wholly compatible with the accepted view of a cellular membrane devised for the passage of fluid through it. 56 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. The same phenomenon of the passage of fluid from the fourth ventricle into the periaxial spaces is beautifully illustrated in figure 23. This drawing is from a trans- verse section of a pig embryo (23 mm. in length) in a stage when the superior mem- branous area is rapidly being encroached upon by the developing cerebellum and by the caudal chorioid plexuses. Between the deeply staining ependymal cells on either side the membranous area is densely outlined by the deposition of the granules of prussian-blue in the cytoplasm of the cells of the area membranacea superior. The avoidance of the nuclei of these cells by the ferrocyanide is well demonstrated in this reproduction, as is also the impenetrability of the ependymal cells. In a specimen of this nature the question of the passage of the injection fluid through possible intercellular foramina loses its significance; for the drawing shows clearly the importance of considering the entire area membranacea as a functioning whole— a permeable, living, cellular membrane. It has been shown in a foregoing section of this memoir that histologically the area membranacea superior decreases to an almost negligible remains in speci- mens of embryo pigs over 30 mm. long. This same rule apparently holds for its functional importance, as determined by the relative and absolute amount of prussian-blue granules deposited in the cells of the superior membrane. This decrease in the functional importance may be inferred from figure 47, a photomicro- graph from a pig embryo of 32 mm. Apparently the size of the membrane deter- mines in large measure the amount of the replaced fluid which passes through it. Thus far we have been concerned solely with the passage of fluid through the area membranacea superior. In the earlier stages of from 14 to 23 mm. the importance of the superior membrane functionally is great, but in the later stages the inferior membrane assumes far greater significance. This is demonstrated not only by the structural history of the two areas, but by the functional index afforded by the replacement of the cerebro-spinal fluid by a foreign solution. In the foregoing section the first evidence of any histological differentiation in the inferior portion of the roof of the fourth ventricle was shown to occur in pig embryos of 15 mm. in length. From this stage upwards (figs. 4, 5, etc.) a portion of the inferior roof allows fluid to pass through it. The exact point of fluid passage is the localized ependymal differentiation forming the area membranacea inferior. This relationship is easily verified by reference to figure 18. In this drawing of a median sagittal section of a pig embryo the two localized points of fluid passage into the periaxial tissue are readily identified; they are quite limited in comparison to the extent of the periaxial spread. Figure 16 represents the inferior membranous area of the roof of the fourth ventricle from a pig embryo of similar size (18 mm.). The histological character of the inferior area is well shown in this drawing. It will be seen that, except in small areas, the histological differentiation of the ependyma has not proceeded to any great extent; the fluid from the ventricular cavity (as traced by the precipitated granules) closely follows the points of greatest cellular differentiation. There is no possibility of an interpretation of the findings concerned with the existence of PASSAGE OF FLUID THROUGH ROOF OF THE FOURTH VENTRICLE. 57 intercellular stomata; the passage of fluid is here again to be looked upon as a transit through a cellular membrane. The same general phenomena of the passage of fluid through a localized area (the area membranacea inferior, in the caudal portion of the roof of the fourth ventricle) that have been observed in the superior portion of the roof are shown in figure 18. Chief among these phenomena is the careful avoidance by the precipitated granules of the ependymal lining of the ventricles and the adherence of the granules to the lining walls at the points of fluid passage. The ependymal lining, except in the two areas of differentiation, is everywhere impenetrable to the solution of the ferrocyanide. As the size of the embryo increases the functional importance of this more caudal area becomes much greater (cf. figs. 3, 4,5, and 76). The whole caudal half of the fourth ventricle becomes an area of ependymal differentiation and of fluid passage. It serves everywhere as a complete diffusing membrane, unbroken by the occurrence of stomata. Through this whole membrane the replaced solutions of potassium ferrocyanide and iron-ammonium citrate pass with apparent ease, as demonstrated by the precipitated granules of prussian-blue (fig. 18). From stages of 24 mm. and over, the lower membranous area is the only one of significance in the total fluid passage. The areas, therefore, through which the replaced solution of potassium ferro- cyanide and iron-ammonium citrate passed, in the experimental pig embryos, are the two areas of histological differentiation in the roof of the fourth ventricle—the are membranacee superior et inferior. There is no evidence whatsoever of any other point of escape of the fluid from the ventricular system into the periaxial spaces. The precipitated prussian-blue does not penetrate any of the lining cells of the ven- tricle except in the two areas under consideration. Nor is any evidence afforded by histological study of the escape of ventricular fluid through the described fora- mina of Bichat and of Mierzejewsky. FACTORS CONCERNED IN THE EXPERIMENTAL FLUID PASSAGE. It becomes necessary to discuss the question of the passage of the replaced fluid through the two cellular membranes in order to ascertain to what extent the results obtained by the method may be relied upon. Naturally in such questions the factors concerned in the normal transit of body-fluid through such structures must be considered. Probably the most essential element in obtaining reliable results in any injec- tion is the control of the pressure at which the foreign fluid or mass is introduced. This matter has been fully discussed in the résumé of the methods employed; it is sufficient to reaffirm here that, in these observations, the normal cerebro-spinal tension has not been disturbed because of the use of a compensatory replacement. Other experiments, carried out under increasing pressures of injection, have been made, in order to compare the results with those furnished by the replacement- method. DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. fa | CO Consideration must next be given to the factors of diffusion, filtration, and osmosis in the passage of fluid through the roof of the fourth ventricle. The third factor, however, may be largely excluded, owing to the fact that the solutions of potassium ferrocyanide and iron-ammonium citrate employed were for the most part practically isotonic with the body-fluids. Furthermore, the use of hypertonic solutions apparently gave no different results (except in the increased density of the resultant precipitate) from those obtained by the employment of the isotonic solu- tions. Finally, it was found to be of service to use hypotonic replacement solutions in order to obtain very slight precipitates; in these experiments also the spread of the replaced ferrocyanide solution was similar to the standard result afforded by the isotonic solution. These observations with varying concentrations of the foreign solutions replacing the cerebro-spinal fluid serve to indicate that osmosis plays but little part in the passage of fluid through the roof structures of the fourth ventricle. Undoubtedly the factor of osmosis can not be ignored in any consideration of the passage of fluid through a cellular membrane, but it seems unlikely that with solu- tions of practically the same salt-content it should be of great importance. The influence of diffusion in this passage of the solution of the ferrocyanide and citrate from the cerebral ventricles into the extraventricular space is probably great. The whole plan of the experiment concerns the introduction of salts foreign to the body-fluids, even though in analogous concentrations. It seems not unlikely that as soon as the replacement of the existent cerebro-spinal fluid is effected the ferro- cyanide and citrate must immediately begin to diffuse out into the periaxial tissues and the normal salts return to the ventricles. Probably, however, this same phenomenon plays a normal réle in the human body. Jacobson’s@?) extensive and important studies on the chemistry of cerebro-spinal fluid have shown that the ven- tricular cerebro-spinal fluid is not identical with the subarachnoid fluid. The differences in the two fluids are probably to be accounted for by the fact that the ventricular fluid represents the pure elaboration of the chorioid plexuses, whereas the lumbar subarachnoid fluid is composed not only of the products of the chorioid plex- uses but also of the fluids from the perivascular system. In this transference of the ventricular fluid to the subarachnoid space diffusion may play some part, the rela- tive importance of which can hardly be estimated. But will diffusion alone account for the passage of the experimental fluid in the ventricle through two well-defined areas into the periaxial tissues? Will diffusion account for the varying extent of the injection in different stages of embryonic development? There are several arguments against according diffusion a maximal importance in the process. In the first place, an injection of the solution of the ferro- cyanide under mild syringe-pressure will give a spread similar in every respect to those obtained by the replacement method. This indicates that the course taken by the two solutions is not necessarily the result of diffusion, but rather of the capa- bilities of the tissues for fluid-spread; and similarly the passage of this true solution through the roof areas need not be solely a diffusion process, but may be accounted for by the true flow of the fluid in this direction. Again, in the stages represented PASSAGE OF FLUID THROUGH ROOF OF THE FOURTH VENTRICLE. 59 in figure 2 one would expect as extensive a spread of the replacing solution into the periaxial tissue were diffusion the active force in the movement of the fluid. Instead of such a periaxial spread the injection fluid remains wholly within the ventricular system, indicating that other forces than that of diffusion play an active réle, in the more advanced stages, in the movement of the fluid. Finally, if diffusion is to be considered the sole agent in the distribution of the replacing fluid, why does not the ferrocyanide penetrate all the cellular structures lining the ventricular cavity? Surely it would be expected that diffusion between the body-fluids and the ferro- cyanide solution would occur in each ependymal cell—a phenomenon observed only in the cells comprising the ventricular surfaces of the membranous areas of the rhombic roof. While acknowledging that diffusion and osmosis may play important parts in the process of the passage of fluid from the fourth ventricle into the periaxial tissues, it seems apparent that some other factor or factors must be the determining agent or agents. It is not unlikely that the formation of cerebro-spinal fluid by the cells of the chorioid plexus may cause, in the replacement experiments, further passage of fluid into the extraventricular regions. Such an elaboration of fluid, with the ventricles filled with the experimental solution, would result in an increase in the normal ventricular tension. If this be the real explanation, the passage of the fluid into the extraventricular spaces would result in part from the increase in pressure on one (the ventricular) side of the membrane. The process, then, would be one of filtration through the membrane from the point of higher to that of lower pressure. This explanation best seems to cover the results obtained by the replacement method, and is supported by the histological examination of the developing chorioid plexuses and by many other features which are dealt with in other sections of the paper. This view is also strongly supported by the results of injections under mild syringe-pressure. On the basis that the passage of fluid from the fourth ventricle into the periaxial tissues is in large measure a process of membrane filtration, the phenomenon of the fluid transit of the replaced solutions may be taken as a real index of the circulation and distribution of the cerebro-spinal fluid. It may be assumed, therefore, that the resulting distribution of the prussian-blue granules represents the course and extent of the fluid channels of the embryonic cerebro-spinal fluid. The discussion of the fluid passage outward from the cerebral ventricles into the subarachnoid spaces has thus far been concerned with the processes involved for the transit of the true solutions of the salts. There is, however, an undoubted passage outward, as has already been indicated in a foregoing section, of the pro- tein content of the normal cerebro-spinal fluid. This occurs in specimens in which a truly definitive membrane, intact throughout, can be seen inclosing the chorioidal roof. The explanations which suffice for the passage outward of the true solutions will not serve for this phenomenon. The cells of the body probably are equipped to handle colloidal solutions in several ways, but two methods seem possible as explanatory of the problem at hand. 60 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. In the first place, it is conceivable that the cells in the differentiated arese mem- branacez could phagocyte the colloidal albuminous particles of the ventricular fluid and excrete them into the subarachnoid spaces on the other side of the membrane; but it does not seem probable that this explanation is correct. Much more likely is it that the colloidal masses may follow the same laws of fluid-passage as the true solutions. But in such a passage through a cellular membrane the rate of passage will be much slower with the colloid. These two theories regarding the passage of the albumen of the ventricular cerebro-spinal fluid into the subarachnoid spaces are not based on any findings presented in this article, but are ventured as being in keeping with current physio- logical explanations of such phenomena. On the basis of the second hypothesis, the failure of granular material to pass through the cellular membrane of the chori- oidal roof must be explained as being due to the inability of the cells to handle the foreign material except in sizes which could be absorbed. The fact that the origi- nal unit was not phagocyted or passed through the membrane probably depended on the size of the molecule and the specific character of the lining-cells. THE PASSAGE OF SILVER NITRATE AND INDIA INK THROUGH THE MEMBRANOUS AREAS IN THE ROOF OF THE FOURTH VENTRICLE. Thus far in the discussion of the passage of the experimental fluids through the ventricular roof, true solutions of potassium ferrocyanide and iron-ammonium citrate only have been considered. This solution, as has been pointed out in this and in a previous article), is non-toxic and is not taken up by the cells. With the dilute solutions (0.25 to 0.5 per cent) of silver nitrate, a far different problem is presented. Replacement experiments with this salt are rendered impossible by its intraspinous toxicity and by its precipitating action upon protein; but beautiful preparations may be made by this method by the simple injection with a syringe into the central canal of the spinal cord. With mild syringe-pressure the result of such an injection with silver nitrate is in all cases a simple ventricular spread, with no extension into the periaxial tissues. This general rule holds in all stages in which the central canal can be definitely entered without causing a spread into the perispinal tissues. This failure of the spread to extend into the periaxial tissues under mild pressure is undoubtedly due to the coagulating effect of the silver, which renders further passage of the fluid impossible. The reduced silver collects about the superior membranous area in the roof of the fourth ventricle, outlining it distinctly. This phenomenon is illustrated in figure 115 (a transverse section of a pig embryo of 19 mm.). At this stage the replacement of cerebro-spinal fluid by a ferrocyanide solution results in a quite extensive spread (cf. fig. 5). With increased pressures of injection the silver may be pushed into the periaxial tissue through the roof structures of the fourth ventricle. The transit of the injec- tion-mass occurs in the area membranacea superior in practically all cases (ef. fig. 12). The inferior membranous area, in the earlier stages, is almost invariably impermeable PASSAGE OF FLUID THROUGH ROOF OF THE FOURTH VENTRICLE. 61 to the silver (unless the injection-pressure is extreme). When the superior area is examined after such an injection under high pressure the silver is found deposited throughout the cells of the area, extending only a short distance into the adjacent tissue. This feature of the injection is pictured in figure 113. In these injections the high pressure undoubtedly suffices to force the silver through the coagulated area membranacea. Its coagulating effect on the ependyma is almost equally marked, but the point of least resistance is apparently in the membranous area, allow- ing the fluid to pass through it. Replacements of the cerebro-spinal fluid with diluted solutions of india ink within the medullary-canal system of small pig embryos never result in any exten- sion of the granules into the periaxial tissues, for under the normal tension in the ventricles of the pig the aree membranacee are impermeable to the passage of granular material. After such a replacement the carbon masses may be found everywhere throughout the ventricles, but not in the periaxial tissues. However, india ink may be forced into the periaxial tissues by the use of high pressures of injection, as shown in figure 10. In this specimen of a pig embryo (21 mm. in length) the periaxial spread occurred solely from the superior membranous area. This is analogous to the results obtained with silver nitrate, shown in figure 12. Without doubt in the earlier stages the superior area is much more permeable than the inferior. Histological examination of these specimens after an injection of india ink under high pressure reveals that the carbon granules gain the extraventricular space only through the area membranacea superior; some cells in this area are crowded with the granules, but for the most part extensive intercellular stomata have been made. The whole process must be viewed asa result of the excessive pressure of injection. In the more advanced stages of the pig embryo (80 mm. and upwards) the pressure necessary to occasion an extraventricular spread of the india ink after intraspinous injection decreases somewhat, so that with mild syringe-pressure a local periaxial spread from the fourth ventricle may be obtained from an injection into the central canal of the spinal cord. This is in accordance with the observation of Mall®), who found that the injection flowed ‘‘through the medial opening of the fourth ventricle.” The opening in these cases is in the area membranacea inferior, and in many instances subsequent examination showed rupture of the membrane with escape of the ink, even though the injection-pressure was moderate. Taken as a whole, then, the findings are against the passage of solutions of silver nitrate or suspensions of india ink from the ventricles into the periaxial tissues, except when injected under pressures far above the normal intraventricular tension. RELATION OF THE EPENDYMAL DIFFERENTIATION TO THE PASSAGE OF FLUID. Under this heading it is proposed to discuss the relationship, if any, existing between the stages of differentiation of the ependyma of the roof of the fourth ven- tricle and the passage of fluid through the two membranous areas. The discussion must necessarily be of a temporal character, with an attempt to consider possible factors in the process. 62 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. The most important question in this connection is whether the ependymal differentiation is necessary for the passage of fluid through it. In the pig embryo of 13 mm. the area membranacea superior has reached a stage of marked differ- entiation (fig. 31), but at this same stage (fig. 2) there is no evidence of any passage of fluid through the roof of the fourth ventricle into the periaxial tissue, only an outlining of the oval membranous area. Here, then, the histological differentiation has definitely preceded the assumption of function on the part of the area mem- branacea superior. The passage of fluid through the lower area occurs at a rela- tively earlier stage than it does through the superior opening. The first evidence of differentiation of the inferior roof of the fourth ventricle was observed in pig embryos of 15 mm. in length. At 18 mm., even though the process of differen- tiation was far from complete, some of the replaced fluid was able to pass through the lower area (figs. 4, 16, and 18). A consideration of these observations leads to the assumption that some histo- logical differentiation of the ependyma is necessary for the extraventricular passage of the replaced fluid. In the case of the superior area the differentiation occurs at a considerable developmental interval before fluid passes through it; in regard to the inferior area the assumption of function occurs at a somewhat earlier period in its differentiation. This slight difference between the two areas may possibly be explained on the basis that as soon as the stage of 14 mm. is attained (by the pig embryo) a greater amount of cerebro-spinal fluid is produced than can be cared for by the more slowly enlarging ventricular cavities. As soon as this disproportion occurs the excess of fluid is poured into the periaxial tissues through the already differentiated area membranacea superior; therefore, when the inferior area first shows evidence of formation there is still this excess of fluid in the ventricles. The fluid apparently avails itself almost at once of the new opening and its functional existence becomes immediate. It is apparent, moreover, that the capacity of the membranous areas for the passage of fluid is considerably in excess of the demands made upon them, and furthermore, that the provision for the passage of increasing amounts of fluid is completed before the demand arises. In the passage of fluid from the ventricles into the mesenchyme, there is one other factor which has not as yet been considered. This concerns the potentiality of the adjacent mesenchyme to afford channels for the fluid poured into it. Were resistance offered to the flow of solutions through the mesenchymal tissue spaces, fluid could escape from the ventricles in only very small amounts, if at all; as soon, however, as easily traversed fluid channels became established, the cerebro-spinal fluid could readily escape through the two membranous areas. The question as to what part the embryonic cerebro-spinal fluid plays in the further development of the meningeal spaces also arises in this connection. It is at present impossible to assign to any one of these factors a specifie réle in the passage of fluid from the fourth ventricle into the periaxial spaces, but it is important to consider them as possible determining agents. The evidence all indicates that the rate of production of the embryonic cerebro-spinal fluid is the most important factor, by far, in the extra- ventricular escape of the fluid. GENERAL HISTOLOGICAL DIFFERENTIATION OF CEREBRO-SPINAL SPACES. 63 VII. GENERAL HISTOLOGICAL DIFFERENTIATION OF THE CEREBRO-SPINAL SPACES. The general problems concerned in the formation of the meninges and of the spaces inclosed within them deal with the gradual adaptation of a primitive undiffer- entiated mesenchyme to the anatomical and physiological requirements of the adult. Originally the meninges were held to be derived from the same epidermal infolding which gave origin to the central nervous system; then, with increasing knowledge of the structure, the dura alone was said to be a product of the middle germ-layer; and finally, by the researches of His>) and of Kolliker,?) the mesenchymal origin of the three meninges was established. The general process of the differentiation and the stages in this transformation have not been reported in great detail; here, too, the investigations must have an outlook for physiological anatomy as well as for pure morphology. It may be well to comment briefly on the relationships of the three meninges found in adult mammals. The dura is well established as the fibrous-tissue envelope of the leptomeninges and the central nervous system. But there is a tendency to regard the arachnoid and pia mater as constituting one structure—the lepto- meninges or “‘pia-arachnoid,”’ in the terminology of Middlemass and Robertson®). This difference of opinion in regard to the two inner meninges is due to their struc- tural and intimate relationships. The arachnoid may well be assumed to be a single membrane, worthy of being regarded as a single structure if one considers only its outer continuous membrane as the essential structure. But the inner surface of this membrane sends processes inward to fuse with the pia mater, which is so closely applied to the nervous tissue. These processes divide the subarachnoid space (included between arachnoid and pia) into the well-known meshes in which the cerebro-spinal fluid circulates. From the standpoint of these channels (the subarachnoid spaces) the arachnoid constitutes the parietal and the pia the visceral layer. Thus the intimate structural unity of the two membranes seems, in the opinion of many investigators, to warrant their designation as a single membrane. This view, however, has been strongly opposed by Poirier and Charpy‘), who considered the distinction of three meninges very essential. Hence, in considering the trans- formation of tissues in the embryo, regard must be had for the dura as a well-differ- entiated structure, and for the leptomeninges as units, but certainly to be regarded from the standpoint of the subarachnoid spaces. In this connection Sterzi’s(3) observations on the comparative anatomy of the meninges are of interest. It will be recalled that the dura in lower forms becomes well established before the lepto- meninges emerge from a primitive mesenchyme. THE PERIAXIAL MESENCHYME. Surrounding the central nervous system in young embryos is a rather thick cushion of undifferentiated mesenchyme, similar in all respects to the undifferen- tiated tissue in other parts of the embryo. But very soon in the course of develop- ment the nuclei in this mesenchyme increase along the clear marginal zone of the 64 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. spinal cord and basilar structures, forming the initial indication of the pia mater. This phenomenon is indicated somewhat in figure 40, a photomicrograph taken from a human embryo (No. 836) of 4 mm., the earliest stage here illustrated. The next essential change in the great differentiation of the meninges concerns a blastemal condensation of this same mesenchymal tissue to form ultimately the bony covering of the central nervous system and a portion of the dura; but between these two zones of differentiation the mesenchyme remains for a time almost unal- tered. A portion of this tissue will go to form the arachnoid membrane and the trabeculz which mark off the subarachnoid spaces. This process in the formation of the arachnoid will be discussed here; the formation of the pia mater and dura will be detailed in succeeding divisions of the paper. The differentiation will be discussed as a general process, in regard to both human and pig embryos, for in no respect has any essential difference between the two been observed. The general character of the periaxial mesenchyme may be commented upon here. The tissue is of a very loose and typical structure, forming a syncytial net- work of rather small mesh, but fragile. The nuclei of the cells are oval, with a definite chromatin content; the cytoplasm is largely devoted to the maintenance of long processes which connect with adjacent cells. Adhering to the cytoplasmic processes are very tiny albuminous coagula, of such small amount as to be hardly noticeable; also in the meshes of the mesenchyme very small quantities of this albumen may be identified. These albuminous coagula undoubtedly represent the protein of the tissue fluids in the undifferentiated stages. THE FORMATION OF THE ARACHNOIDEA. A general consideration of the problems here involved will surely shed light on some of the various factors concerned. It must be noted that in its develop- ment this membrane proceeds from an undifferentiated but small-meshed mesen- chyme into the adult structure which contains the relatively large cerebro-spinal channels. Then, too, the enlargement of the tissue meshes in certain places—as the future cisternee—must be enormous. Besides this necessary dilatation of the spaces in the periaxial mesenchyme, the outer portion of the tissue must separate from the future dura and form the outer surface of the arachnoid membrane. Here the process must be one of tissue condensation and proliferation. A similar ageney is involved in the growth of the mesothelial cells which cover the outer surface of the arachnoid and also the inner subarachnoid spaces. The general process, then, in the formation of the arachnoid membrane con- cerns a thinning and readjustment of the primitive mesenchyme in certain areas, while in others the process is reversed, the membrane reaching the adult form through proliferative and condensing phenomena. Such alterative processes must naturally result from the application of certain mechanical or vital agents in the growth of the embryo. Is the mere growth of the central nervous system sufficient to furnish these alterative agents, or must we likewise trace the corresponding devel- opment of the bony coverings of the brain and spinal cord? Neither factor seems GENERAL HISTOLOGICAL DIFFERENTIATION OF CEREBRO-SPINAL SPACES. 65 relatively of great importance when compared to the possible influence of the pres- ence and circulation of cerebro-spinal fluid on this periaxial tissue. This seems to be the most important factor, an internally-modifying influence to which the peri- axial mesenchyme is subjected in the formation of an arachnoid and its subarach- noid spaces. It will therefore be from this standpoint that the development of the spaces will be discussed; for, as has already been pointed out, the periaxial mes- enchyme becomes a functionally active tissue for the circulation of the cerebro- spinal fluid at a stage when differentiation has not begun. On this basis, the lack of differentiation shown in the periaxial mesenchyme in the stages before the ventricular cerebro-spinal fluid is poured into the mesenchyme in the neighbor- hood of the roof of the fourth ventricle is not surprising. The character of the periaxial mesenchyme in the early stages is reproduced in numerous photomicro- graphs (figs. 25, 49, 51, and 53). The mesenchyme is here characterized by a rather dense meshwork of cytoplasmic processes, interspersed by a considerable number of oval nuclei. The content of the interstices in albumen, as judged by the persisting coagula, is very small. This picture of the periaxial mesenchyme persists until cerebro-spinal fluid is poured from the ventricle through the area membranacea superior. As will be seen in figure 3, the first indication of an extraventricular spread of the replaced fluid in the ventricles occurred in a pig embryo of 14 mm. At this stage the membranous area in the superior portion of the roof of the fourth ventricle has already become well differentiated. The fluid from the ventricles, however, does not reach any considerable spread until after a length of 18 mm. is attained; the periaxial spread during this period of growth is wholly confined to the peribulbar tissues. It is quite important in this connection that the first obvious differentia- tion of the mesenchyme for the formation cf the arachnoid should appear during this period and should involve the peribulbar tissues. The first change to be noted in the transformation of primitive mesenchyme into the future arachnoid is an obvious thinning of the structure with a decrease in the number of nuclei per unit-volume. This is made out in a photomicrograph (fig. 57) of a section from a human embryo 14 mm.* long, when contrasted with a similar mesenchymal area posterior to the ventricular roof (fig. 53). In the pig embryo this thinning of the mesenchyme is as obvious at this early stage. The process of dilatation of the mesenchymal spaces at this stage hardly seems to concern a direct disruption of the syncytial strands, but resembles more the spread- ing of the cell-bodies by the introduction of more fluid into the tissue spaces. This process would certainly result in an appearance similar in every way to that repre- sented by figures 35 and 57. It probably also concerns other factors, as, possibly, the growth of the whole embryo without a corresponding degree of mesenchymal proliferation. In a human embryo of 17 mm. (No. 576) evidences are apparent of such a thinning of the mesenchyme about the medulla. Thus, in figures 58 and 59, from *This embryo measured 14 mm. on the slide. 66 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. this specimen, the cellular decrease can be made out both in the region of the roof of the fourth ventricle and around the basilar surface of the medulla. It will be noted that the differentiation (7. e., the thinning) about the roof has proceeded more rapidly than along the anterior bulbar surface. This is perhaps to be expected in view of the initial pouring-out of the cerebro-spinal fluid into the mesenchyme just posterior to the roof. In this mesenchymal differentiation a slightly increased amount of albuminous coagulum may be noticed. The truth of this is made obvious by an examination of figure 61, a photomicrograph from a human embryo of 17 mm. The almost entire freedom of the mesenchyme from albuminous detritus is most noticeable at earlier stages. As was pointed out in the description of the results of replacing the cerebro- spinal fluid, a marked change in the rate of development of the cerebro-spinal spaces in the pig-embryo ensues just after attaining the length of 18 mm. Within the growth of 2 mm. the injection spreads completely down the spinal cord and about the basilar structures of the cerebral cavity. This rapid extension finds its analo- gous process in the equally rapid changes which may be traced in the periaxial mesenchyme. Thus, in figure 72, a photomicrograph from a sagittal section of a pig embryo of 18 mm., the whole nervous tissue appears surrounded by a very thin, lightly staining tissue; this is the periaxial mesenchyme, which is undergoing its rapid metamorphosis. It will be noticed in this figure that the posterior structures (rhombencephalon) are surrounded by a much less dense mesenchyme than are the anterior (mesencephalon). This relative differentiation between the bulbar tissue and that around the mid-brain is only of temporal character; the mesenchyme about the medulla, as has already been pointed out, begins to differentiate first, the differentiation of the mesenchyme about the other nervous structures following somewhat later. Figure 73 is a photomicrograph of higher power, taken from the squared area in figure 72. It shows to what a surprising degree the mesenchymal differentia- tion has proceeded during a few millimeters’ growth. Two striking features of the process are brought out in this reproduction. In the first place, many of the mesen- chymal trabeculae have apparently been broken down, sacrificed to a few larger remaining strands. The cells connected with the destroyed trabecule appear to recede until one of the heavier surviving strands is met with, when they adhere and apparently aid in the future development of a permanent arachnoid trabecula. The second feature of importance in figure 73 concerns the large amount of albumen seen in the periaxial space. There is here a much greater amount of albumen than is ever found in the periaxial mesenchyme before the differentiating process which results in the future subarachnoid space has become definite. The occurrence of this large amount of albuminous coagulum is apparently related directly to the outflow of the embryonic cerebro-spinal fluid, for the embryonic fluid is very rich in protein material, as can be readily seen by the partial filling of the embryonic cerebral ventricles with the clotted albumen. GENERAL HISTOLOGICAL DIFFERENTIATION OF CEREBRO-SPINAL SPACES. 67 This process of the breaking-down of the mesenchymal spaces to form fewer and larger spaces goes on very rapidly in pig embryos as they exceed the length of 18mm. Thus, figure 75 (from a pig embryo of 23 mm.) shows a marked decrease in the mesenchymal elements about the medulla; the strands are becoming fewer in number, and the albumen-filled spaces are increasing rapidly in size, but decreasing in number. About the mesencephalon, however, the process has only just begun (also shown by fig. 74). In this photomicrograph (fig. 75) the mesenchymal elements have broken down somewhat; the spaces are becoming enlarged, and a fine albumi- nous coagulum fills the interstices between the mesenchymal processes. The whole picture conveys an excellent idea of the forces which convert the many-spaced mesenchyme into the much fewer cerebro-spinal channels. This general plan of the formation of the larger subarachnoid canals reaches its maximum in the formation of the various cisterne for cerebro-spinal fluid. The process is probably best illustrated in the case of the cisterna magna, which persists in the posterior cerebello-bulbar angle. Figures 74 and 75, taken from an embryo pig 23 mm. long, give an idea of the initial formation of the cisterna cerebello- medullaris. The mesenchymal strands, as shown in figure 75, are already broken down in part, and are profusely covered with albuminous coagula. The process has not proceeded to any extent in this specimen of 23 mm., but in the course of the next 10 millimeters’ growth extensive changes occur, as are shown in figures 76 and 77, photomicrographs from an embryo of 32 mm. In the space outside the inferior membranous area the mesenchymal trabecule have almost disappeared; the space— or cistern, as it should now properly be called—is almost completely filled with the clotted albumen. The mesenchyme is seen running through this embryonic cistern as a few isolated strands, but most of the tissue appears now as a fairly definite membrane on the outer side of the space. This membrane will go to form the inner surface of the dura and the continuous outer layer of the arachnoidea, as it furnishes a visceral layer for the subdural space. More laterally in this same specimen the formation of the cistern has progressed to an even greater extent. In figures 78 and 79 the total freedom of the lower portion of the cistern from trabecular strands is seen; above, the mesenchyme still sweeps down as a supporting structure for the chorioid plexus. 3 28 Present...... Pe 33. | Present...... 14 36 Present...... 6 39 Present...... 9 40 Present 8 55 Present ..... 4 66 Present...... 1 80 Present...... |; 42 90 Present...... 17 100 Present... 15 105 Present. . 20 118 Present...... 10 132 Present...... 18 155 Present...... 27 155 Present...... 39 155 Present... ... 25 158 Absent....... 32 160 Absent... ..... 40 163 Absent....... 41 170 Present...... 24 173 Absent....... 19 185 Absent....... 23 195 | Absent....... 11 209°, | Absent:....22. x: 21 23 J oS a 22 223 Absent....... 26 244 Absent....... 5 260 Absent:...... chorioid plexus in the form of globules of larger or smaller size. globules may be seen even in the surrounding cerebro-spinal fluid. ea Plaques of | glycogen. glycogen. Present...... Present): 2 245 Present...... Present...... Present....... IPTeSeNG 2s Absent....... Present...... Present...... Present...... Present...... Present...... Present... ... Absent.in ease Present Present...... Present...... Present...... Present ..... Absent: <4. 2. ¢ Present...... Present Present... ... Absents..5 6 Present... ... Absent.....:,.. Present... ..- Absent....... Present...... Absent: 20.0... Present. Absent osu Present Absent... 1. or Amount of glycogen. Intracellular palaces distribution plexus. of glycogen. General. ..... Basilar. Localized... . . Basilar. General. ..... Basilar. General. ..... Basilar General. Basilar General...... Basilar General. ..... General General. ..... General General. ..... General Genearl...... General. General...... Basilar. Localized... .. General. General...... General. Localized.....| General. Localized... .. General. Localized... .. General. aaa Distribution Goldmann) pictures the glycogen as occurring throughout the cells of the Some of these This general intracellular disposition was observed in this series in specimens measuring 66 mm. and over (fig. 95). Below this measurement the glycogen occurred practically PERIVASCULAR SPACES IN THE EMBRYO. 95 entirely in the basilar portion of the cell, central to the nucleus. Furthermore, in the stages between 30 and 60 mm. the glycogen globules were present in but small numbers and the glycogen was found in crescentic plaques (fig. 96). This formation of definite plaques is apparently to be ascribed to the fusion of the globules when the amount of glycogen becomes extreme. As far as is known this plaque formation with glycogen has not previously been noted; in one of Goldmann’s figures the fusion of some of the globules has apparently taken place. The table on page 94 records the findings in these observations. The occurrence of glycogen in the cells of the chorioid plexus only during a certain portion of embryonic life is, as shown by the foregoing table, a fairly definite phenomenon, but there is surely no indication that this temporary presence of the animal starch bears any relation to the assumption of function on the part of the chorioid plexuses. The evidence afforded by the extraventricular flow of the replaced fluid, with the apparent relationship of the developing chorioid plexuses to the periaxial extension of the fluid, argues strongly against such an assumption. XII. PERIVASCULAR SPACES IN THE EMBRYO. In 1865 His), using a puncture injection, found that each nerve-cell existed in aso-called space. These pericellular spaces connected, as demonstrated by the flow of the injection mass, with an extensive perivascular network, more complex in its gray matter than in the white. In all of His’s cases continuation of the injection led to a peripheral spread toward the pia, both in the spinal medulla and in the brain. Mott“), working on the brains of animals in which an experimental cerebral anemia had been produced by ligation of the head arteries, found the perivascular spaces enormously dilated and the perineuronal spaces likewise very evident. Direct connections between the perivascular and perineuronal spaces are pictured in Mott’s communication. The deduction which Mott made from his findings, regarding the possible absorption of cerebro-spinal fluid by the cerebral capillary bed from this perivascular and perineuronal system, was discussed by the present author in a paper two years ago®5), It was there shown that, with the use of true solutions as the injection (potassium ferrocyanide and iron-ammonium citrate), the whole perivascular system could be filled. This injection of the spaces, however, occurred only when the pressure conditions within the cranial ‘cavity were such that the subarachnoid pressure exceeded the vascular tension. This reversion of the pressure relations was accomplished by maintaining at normal the subarachnoid pressure with the injection fluid, and occasioning a simultaneous and complete vascular anemia. Under the routine conditions of injection (with undisturbed pressure relations) no injection of the perivascular system from the subarachnoid space resulted. It was found impossible to inject the perivascular system, using granular suspensions as the injection mass, without employing pressures far above the normal. From these results here recorded briefly, the belief was expressed in this former paper that each nerve-cell was surrounded by a capillary space which drained along 96 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. the perivascular channels into the subarachnoid spaces. Probably this system represents a mechanism for accessory tissue drainage comparable physiologically to the lymphatic channels of the other parts of the body. In view of these findings in the adult mammal it seemed desirable to ascertain at what period of intra-uterine life such function was acquired. It also seemed not unlikely that information of interest might be acquired from the embryonic intra- medullary circulation which would amplify our knowledge of this system in the adult. It was thought that there might be a correlation between the production of the perivascular fluid and the enlargement of the subarachnoid channels, similar to the evident connection between the chorioidal invagination and the extraventricular spread of the fluid. Experiments to demonstrate possible perivascular and perineuronal spaces were first attempted on rather large fetuses (pig), as follows: The spinal meninges were exposed in a fetus in which the heart was still beating vigorously. Into the spinal subarachnoid space was introduced a needle connected with a small reservoir, con- taining the injection solution (potassium ferrocyanide, 0.5 gm.; iron-ammonium citrate, 0.5 gm.; water, 100¢.c.). The reservoir was then adjusted so that a pressure of 160 mm. of water was maintained in the subarachnoid space. The arteries and veins in the neck of the fetus were then severed, and the subarachnoid pressure maintained at its former level. At the end of 20 minutes the head was placed in a fixative containing 1 per cent hydrochloric acid. This procedure, as outlined above, in the adult laboratory mammal, usually resulted in a complete injection of the perivascular system. In the embryo, how- ever, the procedure was uniformly unsuccessful. The injection solution, as shown subsequently by the precipitated prussian-blue, rarely ascended over a centi- meter above the point of injection. This indicated that the existent cerebro-spinal fluid was not replaced by the injection solution, and that the failure to demonstrate the perivascular system was to be explained on this basis, if the system were func- tional at this stage. Attempts were then made to replace the subarachnoid fluid with the injection solution before the cerebral anemia occurred. These attempts likewise met with failure, because of the impossibility of keeping the heart beating for any length of time in the larger pig fetuses. Other attempts were also made to demonstrate these channels, in larger pig embryos, by means of a procedure which in the adult gave at times good injections of these intracortical canals. This method differed from the method first employed only in the maintenance of a high pressure (100 mm. Hg) in the spinal subarachnoid spaces. It likewise met with failure, due apparently to the same causes which occasioned its failure in the adult: the high subarachnoid pressure operated chiefly to compress the cerebral and spinal tissues, rendering the injection of the perivascular spaces impossible. The same procedures were attempted in smaller pig embryos (15 to 60 mm.). The method usually successful in demonstrating the spaces (subarachnoid pressure slightly above normal, with subsequent cerebral anemia) failed, apparently because the cranial cavity at these stages is in no sense a rigid closed box, as in the adult. THE PERINEURAL SPACES IN THE PIG EMBRYO. 97 Any method of service in the adult—which must have in consideration the physical character of the skull as a closed box—was here necessarily doomed to failure. Together with these technical failures to demonstrate a perivascular system, it must be borne in mind that these are merely failures to demonstrate the existence of the perivascular system in the pig embryo. The system will probably be demon- strated as soon as a suitable technique is devised. The spaces are very likely present soon after the capillary plexus invades the nervous system, but the observation in many histological preparations of the spaces around the cerebral vessels must not be considered as offering proof of their existence, because of the likelihood of shrinkage influencing the picture. It is interesting, however, to note that elasticity of the cerebral tissues seems greatest along the course of the blood-vessels, for here the phenomenon of shrinkage is most frequently observed. The existence of the perivascular and perineuronal spaces, probably of only capillary thickness, must remain—in the embryo as in the adult—a subject of physiological demonstration; histological evidence, except with proper physiological regard, is of no value. The early development and function of such a system as the perivascular and perineuronal canals afford seems most likely from the standpoint of pure speculation. It is not improbable that fluid is poured from this system into the embryonic sub- arachnoid space at a period soon after the capillary plexus invades the cerebrum. There is no evidence, however, from the observations recorded in foregoing para- graphs, that adequate subarachnoid channels are afforded until the pig embryo reaches a length of about 25 mm. The hypothesis of Essick %) regarding the damming of the perivascular fluid as the cause of the two cava corporis striati is of extreme interest in this connection. It remains, however, for future work to afford real evidence in regard to the embryonic perivascular system. XIII. THE PERINEURAL SPACES IN THE PIG EMBRYO. The question of the existence of potential or actively functional spaces around the peripheral nerves is of great interest, partly because of the possible relation of these spaces to the developing lymphatic system, and also on account of the anatom- ical evidence of the possible existence of such spaces. It is realized that before much dependence can be placed on any theory regarding these potential spaces around the cerebro-spinal nerves, the possibility of their being purely artifacts must be dealt with. The methods of demonstration, in the adult, in the hands of the earliest workers were such as to favor the production of artifacts. As far as can be ascertained, Cotugno ), dealing with the nervus ischiadicus, was the first to conceive of these possible spaces. His method of demonstration consisted in filling the spinal subarachnoid space with mercury (in a cadaver placed in the erect posture). Globules of the mercury were subsequently found about the sciatic nerve in what then became the perineural spaces. Modern anatomical interest in these spaces was aroused by the remarkable injections of Key and Retzius®9). These investigators, by means of gelatin injec- tions into the spinal subarachnoid space, were able to demonstrate perineural 98 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. spaces around the cranial nerves, especially around the optic pair. Their results, however, are open to criticism, because of the excessive pressures employed (“not over 60 millimeters of mercury’’) and because the injections were made in fresh cadavers kept warm for periods of 10 or more hours. Some of the difficulties concerned in the problems of the perineural spaces were cleared up in a study) of the cerebro-spinal circulation published in 1914. In this work injections of true solutions (similar to those used in the present study) were introduced into the spinal subarachnoid space in living cats and dogs, under pressures but slightly exceeding the normal intraspinal tension. These injections were con- tinued for several hours, and the course of the injection fluid was then established by precipitating the solution in situ. By means of this procedure, which it was believed approached the physiological, the perineural spaces around the cranial nerves could be demonstrated. In these adult laboratory mammals the cerebral nerves without exception showed prussian-blue granules in a perineural relation, extending outward along the nerves beyond the termination of the dural cuff. This extension of the injection mass outward was more striking around the first two cranial nerves than about any of the others. Thus, the olfactory nerves uniformly showed perineural deposits beyond the cribriform plate, extending downwards into the nasal epithelium, while the optic nerves were surrounded by the granules in the infravaginal sheath, which spreads out over the posterior surface of the eyeball. The caudal cranial nerves were likewise characterized by extensive perineural injections. These findings were interpreted as evidencing a true perineural space, probably of only capillary thickness, which could be injected by filling the cerebro-spinal spaces with a demonstrable true solution. As far as could be made out under the microscope, they had no appreciable existence except when filled with the precipi- tated true solution. These spaces were not filled in the early moments of the injec- tions under low pressures, and could be demonstrated only when the injection had been continued for several hours. The perineural spaces are quite different from the spaces surrounding the spinal ganglia and the ganglia of the cranial nerves. These ganglia lie in the true sub- arachnoid space, with the dura investing the arachnoid membrane. Distal to the ganglion the dura ends upon each nerve. In the injection under low pressure with the ferrocyanide the cranial and spinal ganglia were all surrounded by the precipitated salts; the cranial nerves showed extensive perineural injections, whereas the spinal nerves rarely showed a true perineural injection, and then only of limited extent. The existence of perineural spaces in the embryo, however, has been under dispute. The larger nerves in sectioned embryos almost invariably show spaces about them, either a complete separation of the surrounding mesenchyme or a partial dilatation of the mesenchymal interstices. Sabin), in 1902, noted that in perispinal injections with india ink the spinal nerves could be outlined by the carbon granules, but in no case did such an injection run into true lymphatic channels. No evidence was afforded by her work of any lymphatic channels arising from these apparent perineural channels. THE PERINEURAL SPACES IN THE PIG EMBRYO. 99 In the course of this investigation of the cerebro-spinal spaces interest naturally turned to the perineural spaces. In the typical experiments (a replacement of the embryonic cerebro-spinal fluid with a demonstrable true solution in the living embryo), there was evidence of a spread of the replaced solution around the cranial nerves. Because of the procedure used (merely a filling of the ventricles and central canal of the spinal cord) no evidence of a perineural spread occurred until the foreign solution passed into the periaxial tissues. Here the spread chiefly involved the caudal cranial nerves curving around the lateral surface of the medulla in fan- shaped processes (figs. 5, 6, 8, and 9). The spread, however, was not extensive. In figure 8 a similar slight spread along the spinal nerves is to be made out. Closer study of these cleared specimens, and examination of the same and of similarly injected embryos after serial sectioning, convinces one that the apparent perineural spread in these cases extends around the sensory ganglia and not further toward the periphery. In no case, either in the caudal portion of the cranial or in the spinal region, has the replaced injection fluid passed the blastemal condensation of mesen- chyme. This finding is well shown by the distribution of the injection fluid in figures 9, 16, and 18. The optic nerves, however, possessing ganglia in the retina, usually show, in the typical replacements in the living embryo, a partial or complete surrounding of the nerves by the precipitated prussian-blue. An incomplete example of this—more typical, according to these observations, than a total cireumvention—is given in figures 19 and 20. The higher-power reproduction of this field is very interesting. It shows in the central portion the fiber bundles comprising the optic nerve, sur- rounded by mesenchyme and the developing ocular muscles. In the region between the nerve and the muscles is an undifferentiated mesenchyme which is characterized by a crescent of the precipitated granules of prussian-blue. The non-penetration of the surrounding tissue by the ferrocyanide is very well brought out in this drawing. The prussian-blue has reached its position about the nerve by extension from the pericerebral spaces; actually it has still the same distribution as noted in figure 8 above. The adult dura will completely surround the optic nerve in its whole extent; the subarachnoid space will likewise extend unbroken to the posterior surface of the eyeball. Hence it must be assumed that in this case the perineural space does not extend beyond the peripheral ganglion. With regard to the olfactory nerves, no evidence of a perineural spread was obtained in specimens of pig embryos up to 45 mm. in length. It seems obvious, then, that in the embryo pig true solutions, when substituted for the cerebro-spinal fluid, do not extend peripherally along the nerves any further than does the dura in the adult. The replaced fluid (if, as appears most likely, it indicates the true circulation of the cerebro-spinal fluid) extends only through the future subarachnoid space. Such a conclusion is best supported by the observations. The only discrepancy between the findings in the pig embryo and those in the adult with the same method lies in the fact that in the adult the cranial nerves showed a much more extensive perineural injection. This seeming discrepancy may be 100 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. accounted for in two ways. In the first place, the experimental replacement in the embryo pigs lasted at most one hour (due to the fact that the embryo’s heart frequently ceased beating at the end of this time), while in the adult cat or dog they were continued for several hours; and it was only in the long-continued experiments in the adult that the extensive perineural injections were obtained. On this basis it seems more than likely that the communications between peripheral perineural spaces and the subarachnoid space are very small and that diffusion must account for the slow filling of the peripheral system. The second explanation seems un- doubtedly to concern the time of development of these perineural spaces in the embryo. It may be that the spaces are morphologically non-existent until late in fetal life; in that case, of course, it is not strange that they have not been filled with the injection fluid. From the observations recorded above it is quite apparent that in the typical experiment in which the normal cerebro-spinal tension is not increased no evidence of the perineural space, as injected by Miss Sabin, has been adduced. However, the possibility of injecting these spinal spaces as was done by Miss Sabin is easily demonstrated. The injections may be made with ease, either with. granular sus- pensions or with true solutions. Success invariably attends such an injection into the perispinal tissues. The injection solutions easily run out around each nerve, more readily, apparently, in the younger embryo than in the older. It is not clear whether this difference is due to the fact that in younger embryos the resistance is greater to the perispinal flow and less peripherally, or merely to the fact that a ereater amount of fluid must be introduced in order to attain the same result. Careful repetition of these observations has led to the conclusion that such a demon- stration of the spinal perineural spaces results from excessive pressures of injection. Whenever the pressure exerted by the injection is but slightly above the normal, or does not exceed the normal (as in replacements), the perineural spaces are not injected around the spinal nerves. Miss Sabin’s conclusions from her results, that no connection exists between the spaces and the lymphatic system, seem to be wholly substantiated by these observations. The apparent perineural spaces around the embryonic nerves must be looked upon as artifacts. In tissue carefully fixed, dehydrated, and embedded, there is no real evidence of these spaces. Their size apparently varies with the care observed in the histological technique. GENERAL SUMMARY. 101 XIV. GENERAL SUMMARY. In the foregoing sections of this communication some of the problems concerned with the embryology of the cerebro-spinal spaces have been discussed and observa- tions have been presented in the hope that a better conception of the processes might obtain. It is purposed to present here briefly the results of these observations and to attempt to correlate the findings so far as is possible; and in this, as in the detailed reports in the preceding pages, the relationship of the physiological processes con- cerned will be referred to the morphological changes in the developing embryo. As a means of studying the physiological extent of the embryonic cerebro-spinal spaces, a method of replacing the medullary fluid with a foreign solution was devised. The procedure consisted in substituting, in the living embryo, a solution of potas- sium ferrocyanide and iron-ammonium citrate for the cerebro-spinal fluid. The embryos were then kept alive, for periods of about an hour, by placing them with the attached placente in an incubator at 38°. At the end of this time, which varied in the many experiments, the whole embryo was fixed in a medium containing hydrochloric acid, thereby precipitating an insoluble prussian-blue. Specimens prepared in this manner were studied after sectioning or after clearing by the Spalteholz method. Pig embryos, subjected to such experimental replacements, exhibited only an intraventricular retention of the foreign solution until after a stage of 14 mm. was attained. In the earliest specimens, embryos of about 9 mm., there was no charac- teristic distribution of the foreign solution, except that it remained within the medul- lary-canal system. In stages of about 13 mm. the replaced fluid also was retained within the cerebral ventricles, but in these specimens a dense accumulation of the precipitated prussian-blue may be made out in a distinct oval in the superior portion of the rhombic roof. This granular aggregation occurs against a histological differ- entiated area in the roof of the fourth ventricle—an area which represents apparently the more epithelial-like elements of the earlier roof-plate. This area must be consid- ered solely as a differentiation of the epidermal lining of the medullary-canal system. In living pig embryos of 14 mm. and over, the result of the routine replacement of the ventricular cerebro-spinal fluid was a slight extraventricular spread into the tissues posterior to the rhombic roof. The passage of this foreign solution outward oecurrec through the same area of ependymal differentiation, outlined by the col- lection of granules against its inner surface in the previous stage. The extraven- tricular spread remains definitely localized to a very small conical area which does not rapidly increase in size. The factors which cause this initial flow into the pericerebral spaces are of interest. It follows that in the growth of the embryo the production of the intra- ventricular and intraspinal cerebro-spinal fluid must necessarily keep pace with the increasing size of the cerebral ventricles. It is also necessary for the occurrence of an extraventricular spread of the fluid that the production of the fluid within the ventricles must exceed the amount required to keep the medullary-canal system filled. From our knowledge of the elaboration of the adult cerebro-spinal fluid, it 102 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. is impossible to conceive of the production of a true cerebro-spinal fluid in the peri- medullary mesenchyme. Such a view would be a reversion to the old hypothesis of Haller, who regarded the leptomeninges as the elaborators of the fluid. Likewise, the passage of the replaced foreign solution into the extraventricular spaces would render such a hypothesis untenable. Hence, it becomes incumbent to regard such an extraventricular spread of the experimental solution as an indication that the production of the cerebro-spinal fluid within the cerebral ventricles exceeds the capacity of the ventricles to care for the fluid. This argues strongly that the process of elaboration of the fluid in these pig embryos of 14 mm. is no longer sluggish, but that an active production, sufficient to cause a slight extraventricular flow during the observation, is now taking place. This acceleration of the flow is not great, but it represents a marked change in the relationship of the process of fluid elaboration to the increasing volume of the ventricles. It seemed desirable to endeavor to correlate this extraventricular spread of the experimental fluid with the morphology of some intraventricular structure at this critical stage of 14 mm. in the pig embryo. The first evidences of villous tufting in the chorioid plexus of the fourth ventricles were found to occur at this stage in the pig. Other studies of this plexus, particularly those which concerned the occurrence of glycogen in these glandular cells, were found to offer no additional evidence of value in regard to the onset of function in these structures. The corre- spondence between the initial tufting of the ependyma to form the rhombic chorioid plexuses and the initial extraventricular spread must be regarded as of the utmost importance. It would appear most likely that as soon as the chorioid tufts occurred an increased production of cerebro-spinal fluid took place, necessitating an extra- ventricular expulsion of the excess of fluid. Such a view receives the utmost support from these recorded observations; it is in keeping with the best conceptions of the processes of production of cerebro-spinal fluid in adult mammals. With the initial pericerebral extension of the experimental fluid occurring in pig embryos of about 14 mm., the further extension of this spread did not occur until after a length of 18 mm. was attained. At this stage the replaced foreign solution passed from the fourth ventricle through two areas in the roof-plate. The chorioid plexuses now have divided the roof into two portions; from each, fluid escaped. The superior area of fluid passage is the same which was concerned in the initial outpouring of the ventricular fluid. The inferior area, like the superior, is an area of ependymal differentiation, of which the first evidence may be made out in pig and human embryos of 15 mm. This differentiation consists in the transformation of the densely staining ependymal elements into cells with larger nuclei, poor in chromatin, and with more abundant cytoplasm. After the functional employment of the two membranous areas is established at about 18 mm. in the pig, the further pericerebral spread of the replaced solution occurs very rapidly. The peribulbar tissues are filled with the fluid and from this region extensions occur downward into perispinal spaces and upward into the more GENERAL SUMMARY. 103 basilar pericerebral spaces. Thus, the spinal spaces must be considered as develop- ing physiologically from above, and not from below upward, as Reford found. The complete filling of these perispinal spaces is found in pig embryos of 21 mm. At this stage the pericerebral spaces are filled, with the exception of those around the superior portion of the midbrain and about the cerebral hemispheres. The final filling of all the periaxial spaces occurred in pig embryos of about 26mm. This phenomenon may be taken to indicate the establishment of the true cerebro-spinal relationships of the adult, for in this case there is an intraventricular production of the fluid and an extraventricular spread. Likewise, the fluid returns to the venous system in embryos of over 23 mm., and this escape of the fluid from its periaxial bed is, as in the adult, directly into the venous sinuses of the dura mater. The rapidity of the further extension of the replaced solution after the stage of 18 mm. is passed is apparently due to a second marked acceleration in the rate of production of the ventricular cerebro-spinal fluid. As in the first instance, this increased elaboration seems connected intimately with the formation of the chorioid plexuses of the third and lateral ventricles. As soon as these tufts develop, the cerebro-spinal fluid is produced in amounts which far exceed the quantities for which the more slowly enlarging ventricles can provide. The histories of the two areze membranacez of the fourth ventricle are dissimilar. Both are areas apparently differentiated from the normal lining ependyma for a specific functional purpose—the passage of fluid from the ventricles into the future subarachnoid spaces. The superior membranous area reaches its maximum fune- tional importance in the stages of 18 to 20 mm. in the pig and also in the human embryo and from these stages on it slowly regresses. The final obliteration of the area, if it do not persist as an occasional small remnant, is due to the increasing growth of the cerebellum and the enlargement of the chorioid plexuses of the fourth ventricle. On the other hand, the inferior membranous area continues to increase both in size and functional importance after its initial differentiation from the ependyma; it finally occupies the greater portion of the velum chorioidea inferior. These observations can not solve the interesting question of a perforation of the inferior velum to form the foramen of Magendie. Of the factors which influence the passage of fluid outward into the periaxial spaces, it must be realized that probably there is difference in this regard between the true solutions of the salts and the colloidal suspensions. For the true solutions (as in the experimental replacements) diffusion probably plays some rdle; but that this is not the sole factor is shown by the failure of the fluid to pass through the membrane in the stages underl4mm. The findings of the granules of prussian-blue within the cytoplasm of the cells of this membrane indicates that the fluid passage is similar in every way to that through a true membrane. There is also a possible site of fluid passage between the cells of this membrane. But, surely, the most important factor in this process is one of filtration of the fluid from the point of higher pressure to one of lower. This is indicated by all of the findings: that the increased production of the fluid or the increased intraventricular pressure (whether 104 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. due to normal or experimental agencies) causes a marked extraventricular spread seems firmly established. For the colloidal suspensions (particularly the protein of the normal ventricular fluid) a slower process of diffusion and filtration seems the probable agency for passing the ventricular colloids into the subarachnoid spaces. That the results obtained by the method of replacement were not solely due to diffusion, but represent a filling of the physiological extent of the cerebro-spinal spaces, has been shown in many ways, but probably the chief argument against such a view is that wholly similar extensions of the foreign solution may be obtained by injections under mild pressures from a syringe; with increasing pressures these injections show the same type of spread, but always in a smaller embryo than the replacement method demonstrates as the standard for a given stage of the extension. The results recorded in the foregoing pages indicate also that suspensions (india ink) and true solutions (when powerful precipitants) are valuable only for affording comparisons in problems concerning the normal processes of absorption. Of interest in any discussion of the results of injections into the perispinal spaces or into the spinal central canal are the findings in regard to the perineural spaces. It is possible to inject such spaces around each of the segmental nerves, but only when the pressures of injections are extreme. In no case, however, were such injections found to enter the lymphatie system—a finding in accord with the obser- vations of Reford and Sabin. The physiological importance of these spaces in the adult is probably great, but the same methods of demonstration (with carefully controlled pressures) which suffice in the adult are unavailing in the embryo. The origin of the three meninges from the perimedullary mesenchyme is well established. His, Kélliker, Sterzi, Farrar, and others have placed this conception on a very firm basis. Most of the investigators have been concerned with the differentiation of the spinal meninges, while the observations here reported have been concerned solely with the cranial portion of these membranes. In general, the same phenomena in the transformation of the primitive periaxial mesenchyme as recorded by these earlier workers may be found in the cranium. The division of the primitive mesenchyme by a secondary condensation, a view advanced chiefly by Salvi, seems well supported. The findings in the cranium are in accord with this conception; the outer portion of this primitive meninx becomes the dura mater, the inner forms both the pia and arachnoid. The processes in the formation of the arachnoid are, however, quite diversified and concern both the formation of the subarachnoid spaces and the outer membrane of the arachnoid. Out of the rather loose-meshed periaxial mesenchyme, the subarachnoid spaces develop. The process concerns the transformation of the small “tissue spaces” of this mesenchyme into the larger subarachnoid channels, which are interrupted by the well-known arachnoid trabecule. Well-marked stages in this metamorphosis, which begins in the basis cranii, can be made out. The first appearance of a differ- entiation is seen in the gradual increase in the size of the mesenchymal mesh. This is closely associated with an increased amount of an albuminous coagulum which in a measure fills the larger interstices. Following this initial dilatation of the spaces GENERAL SUMMARY. 105 occurs a breaking-down of some of the syncytialstrands; these ruptured mesenchymal processes then retract and adhere to the persisting trabeculae. The process con- tinues with the formation of larger channels in this mesodermal tissue, with also the formation of the permanent arachnoidal trabecule. Throughout these larger spaces, in the smaller fetuses, the coagula of protein material are everywhere found, the remains apparently of the albuminous portion of the cireumambient fluid. In the formation of the various cisterne, particularly the great cisterna cerebello- medullaris, the process of the dilatation and confluence of the original mesenchymal spaces reaches its maximum. Here the breaking-down of the original syncytial strands proceeds to such an extent that very few of the strands remain to persist through life. Such a process of the enlargement of mesenchymal spaces to form the larger subarachnoid spaces, as described in some measure by His for the spinal meninges, is apparently intimately connected with the circulation through these spaces of the embryonic cerebro-spinal fluid. The fluid flows everywhere through the spaces, as evidenced by the replacement experiments and by the increased content in albumen, before the process of enlargement of the mesenchymal spaces begins. It seems most likely that this circulation of the fluid acts as the causative agent in initiating and probably also in completing the enlargement of the “tissue spaces.” The great content of albumen in the embryonic cerebro-spinal fluid has greatly facilitated the investigation, as the presence of the coagula from this protein has permitted the absolute exclusion of artifacts in the process of the tissue-dilatation. This mechanism of enlargement of the tissue spaces finds its analogue in the formation of the anterior chamber of the eye and in the perilymphatic spaces of the ear (Streeter). In both these situations, as in the meningeal spaces, there are special body-fluids, more or less characteristic in their physical and chemical char- acters, obviously subserving specialized functions. In both the eye and cranium, the absorption of the fluids is by way of special organs, directly into venous sinuses; in both, the origin of the specialized fluid is from epidermal organs; this fluid is at first poured into epidermal spaces and then subsequently into mesodermal spaces (subarachnoid space and anterior chamber of the eye). Thus, in these situations, the characteristic fluids have certain definite channels through rather larger spaces, connected finally with the venous system, and only indirectly with the lymphatic system. In no sense must the cerebro-spinal circulation be taken as a portion of the lymphatic system. Increasing knowledge of the cerebro-spinal fluid, of its physi- ology and chemistry, and of its pathway, have separated it permanently from any connection with the lymph of the lymphatic system, variable though that be. No longer may the meningeal spaces be compared to serous cavities, except possibly in the ease of the subdural space, and this space is really a space apart from the true cerebro-spinal or subarachnoid spaces. Quite similarly, in place of the many varying channels in the dura and to a lesser extent in the leptomeninges, which older writers considered lymphatic in nature, our increasing knowledge has caused 106 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. the introduction of specialized arachnoidal cell-chains running throughout the pachymeninx. Unquestionably, the cerebro-spinal fluid possesses its own peculiar and characteristic pathway, analogous in no way to the lymphatic vessels of other tissues. The outer continuous membrane of the arachnoidea forms as a mesenchymal condensation, at first in common with the inner surface of the dura mater, but soon separated from it by the subdural space. The very low cubical mesothelium which covers the arachnoid membrane on both surfaces and also invests the arachnoid trabecule differentiates apparently from the original mesenchymal elements in the periaxial tissues. One of the most interesting features of this study has been the relation of the various mesenchymal condensations to the foreign true solution which was intro- duced into the medullary-canal system. This fluid circulated throughout the peri- axial spaces which enlarge to form the subarachnoid channels, but it never pene- trated the primary blastema which served as a primitive dura, nor did it ever invade the pial cells which so closely adhere to the nervous tissue; likewise, as soon as the secondary mesenchymal condensation dividing the dura from the arachnoid spaces appeared, this condensation was impervious to the true solution. No evidence of any penetration, as might be expected as due to diffusion, could be made out. This summary has been included in order that some correlation between the topics discussed separately in the foregoing sections might be made. No attempt has been made here to present the findings in abstract form; these have been sum- marized at the end of each division of this communication. CONCLUSIONS. 107 XV. CONCLUSIONS. Based on the observations recorded in the foregoing sections, the following conclusions seem warranted: (1) During the early part of the growth of the pig embryo there is no extra- ventricular spread of the cerebro-spinal fluid. The first extension of the ventricular fluid into the periaxial tissues occurs in pig embryos of 14 mm.; the adult relation- ship of the ventricular and meningeal cerebro-spinal fluid is established in pig embryos of about 26 mm. (2) The ventricular cerebro-spinal fluid escapes into the periaxial tissues through two areas of ependymal differentiation in the roof of the fourth ventricle. Both of these areas differentiate at a slightly earlier period than that at which they function actively. The area membranacea superior undergoes a gradual regression and obliteration due to the changing form of the rhombic roof; the area mem- branacea inferior gradually occupies the major portion of the velum chorioidea inferior. (3) The embryonic cerebro-spinal fluid, as evidenced by the replacement with a true solution, spreads from the ventricles into the mesenchymal tissue about the central nervous system. It does not penetrate the cranial or vertebral blastemal condensations, nor does it invade the pial cellular layer. (4) The subarachnoid spaces arise by a process of breaking-down of the peri- medullary mesenchymal syncytium and a dilatation of the existent mesenchymal spaces. This phenomenon of the enlargement of the mesenchymal spaces is asso- ciated with the presence in the spaces of an increased amount of albumen. The process occurs at a period slightly later than that at which the initial flow of the cerebro-spinal fluid into the spaces is recorded. (5) The dura mater, arachnoid, and pia mater develop out of the perimedullary mesenchyme. The arachnoid trabecule are left by the breaking-down of the original mesenchymal strands, while the outer arachnoid membrane is formed, together with the inner surface of the dura, by a separate mesenchymal condensation. The dura develops between this secondary line of condensation and the embryonic skull. (6) There is indicated a very close relationship between the tufting of the chorioid plexuses of the fourth ventricle and the first extraventricular spread of the cerebro-spinal fluid. (7) By means of the method of replacement it is possible to demonstrate peri- neural spaces as far out along the nerve trunks as the peripheral ganglia. The extensive injections of the perineural spaces along the segmental nerves are not obtained by the method of replacement. The work, of which this paper forms the report, was done in the Anatomical Laboratory of the Johns Hopkins Medical School. It was largely due to aid received from the Department of Embryology of the Carnegie Institution of Wash- ington that the completion and scope of this paper were possible. The writer gladly acknowledges his indebtedness to the Carnegie Institution. January, 1916. FR | sare a a rohit) PACT PP a 4 ‘ ~ 4 7, ’ | “7 ea aay 7 av : 4 We ai aur tiw nye a myriis 1? eee : ’ cf : Li wrt hf 7 fiji) "Leer (+ g PAE Aa “a ¢ . ‘% A | Te i. ive ae ne aah es the Frege i iy : z < {0-8 — ; } rg Frise | (FP ee bry, Sali mg arr, ul vase He A fis > a F ' , ‘ a | , ys ati ‘ Terrie ti oe O° als : j hoot Jae uss ere Flat ett ie ir uit h years PO TT i | vr yl ‘ e a mg oe . ea (ot by ivy s } i1> ORs ‘e ws > Ti tie; siete. ‘eaHj ie a bay ‘4 Aase « sce teil! dvnilited "9 t pa i Wet Hise w , ora « if Mi A 4, 18 Beonut WT sviehe i - io A f ’ NAGS 19 iat ¥ “oe ie bo See ean mal well mene Sh) Adel me: re soe © betnotianl of ire aie iter. trot ed¥ to wheat Birth tera wl a> cabo 44) We arte ware HT rite tat o@ 8 dea Pity, ~ noltigl ail ~ ead “A 10. ZL. 12. 13. 14. 15. 16. 17. . CAPPELLETTI, . CATHELIN, . CoruGno [Cotunnivs], DoMENIco. BIBLIOGRAPHY. . ADAMKIEWwIczZ, ALBERT. Die Blutgefiisse des menschlichen Riickenmarkes. I Theil: Die Gefiisse der Riick- enmarks-Substanz. 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Arch. f. Anat., Physiol. u. wissensch. Med. (Du Bois-Reymond), Leipzig, 1872, 153. 7. Rerorp, L. L. Unpublished. Work referred to by Sabin (49) and Cushing (9). Ueber die Abflusswege des Liquor cerebrospinalis. Centralblatt f. Physiol., Leipzig and Wien, 1894, vii, 684. . ZurLehre vom Hirndruck. Wiener klin. Wo- chenschr., 1895, vim, 371. . Sapry, F. R. Development of lymphatic system. In Keibel and Mall’s Manual Human Embryology, London & Philadelphia, 1912, 1, 709. Satyr, G. Histogenése et structure des méninges. Thése de Paris, 1898. Quoted in Poirier et Charpy. Traité d’Ana- tomie humaine. Paris, 1901, tome m1, 113. . ScHROEDER VAN DER Ko tk, J. L. C. Bau und Fune- tionen der Medulla spinalis und oblongata. Aus dem Hollindischen iibertragen von Dr. Fr. W. Theile. Braunschweig, Vieweg & Sohn, 1859. . Sprva, A. Experimenteller Beitrag zur Kenntniss der Hyperiimie des Gehirns. Wiener med. Blatter, 1898, xx1, 17; 247. Experimentelle Untersuchungen iiber die Bil- dung des Liquor cerebrospinalis. Arch. f. d. gesamte Physiol., Bonn, 1899, txxvi, 204. BIBLIOGRAPHY. 52. Sprna, A. Ueber den Einfluss des hohen Blutdrucks auf die Neubildung des Cerebrospinalflissigkeit. Arch. f. d. gesamte Physiol., Bonn, 1900, Lxxx, 370. Untersuchungen iiber die Resorption des Liquor bei normalem und erhéhtem intracraniellem Drucke. Arch. f. d. gesamte Physiol., Bonn, 1900-1901, Lxxxin, 120; 415. 53. Svperz, G. Recherches sur l’anatomie comparée et sur l’ontogenése des méninges. Arch. Ital. de Biol., 1902, xxxvu, 257. Ricerche intorno all’ anatomia comparata ed all’ ontogenesi delle meningi, e considerazioni sulla filogenesi. Attidel R. Instituto Veneto di scienze, lettere ed Arti, 1900-1901, Lx, parte m, 1101. 54. Srreprer, G. L. Development of the nervous system. In Keibel and Mall, Manual Human Embryology, London & Philadelphia, 1912, 1m, 1. . The development of the venous sinuses of the dura mater in the human embryo. Am. Jour. of Anat., Philadelphia, 1915, xv, 145. 55. Weep, L. H. 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, Boston, 1914, xxx1 (n. s. xxv), 21. . Studies on cerebro-spinal fluid, No. III: The pathways of escape from the subarachnoid spaces, with particular reference to the arachnoid villi. Jour. Med. Research, Boston, 1914, xxx (n. 8. xxvi), 51. . Studies on cerebro-spinal fluid, No. IV: The dual source of cerebro-spinal fluid. Jour. Med. Research, Boston, 1914, xxx (n. s. xxv), 93. 56. WiLprer, B.G. Notes on the Foramina of Magendie in man andthe cat. Jour. Nerv. and Ment. Dis., New York, 1886, x11, 206. . The metapore (foramen of Magendie) in man and an orang. Med. News, Philadelphia, 1893, LXIII 439. . Meninges. Ref. Handbuch Med. Sciences, New York, 1893, rx (suppl.), 606. 57. Zrecter, P. Ueber die Mechanik des normalen und pathologischen Hirndruckes. Arch. f. klin. Chirur- zie, Berlin, 1896, Lu, 75. EXPLANATION OF PLATES. KEY FOR FIGURE-LEGENDS. ami, area membranacea inferior. dmc, dura mater cerebri (inner surface, in pme, pia mater cerebri. ams, area membranacea superior. approximation with arachnoid). pp», precipitated prussian-blue. cbl, cranial blastema. epe, epithelial-like cells lining ventricle. psn, reduced silver nitrate. cem, cisterna cerebello-medullaris. epe, ependyma. sas, subarachnoid spaces. chp, plexus chorioideus. 4ve, ventriculus quartus. efr, sinus transversus. Puate I. Fic. 1. Drawing of a pig embryo of 9 mm., into the spinal central canal of which an injection of 0.5 per cent solution of potassium ferrocyanide and iron-ammonium citrate was made under very mild syringe-pressure. The embryo was fixed in Carnoy’s fluid to which 1 per cent hydrochloric acid had been added. The speci- men was carefully dehydrated and cleared by the Spalteholz method. The resultant precipitate of prussian-blue is found wholly within the central canal of the spinal cord and within the cerebral ventricles. Enlargement, 11 diameters. Fig. 2. Drawing of a pig embryo of 13 mm., in which the cerebro-spinal fluid was replaced by a 1 per cent solution of potassium ferrocyanide and iron-ammonium citrate. The embryo was kept alive for 90 minutes after this replacement and was then fixed in 10 per cent formol containing 1 per cent hydrochloric acid. After dehydration the specimen was cleared by the Spalteholz method. The occurrence of a definite oval, outlined by the denser mass of the granules, in the roof of the fourth ventricle, is characteristic of this stage. Enlargement, 9 diameters Fic. 3. Drawing of a pig embryo of 14.5 mm. ir which the cerebro-spinal fluid was likewise replaced by the ferrocya- nide solution. After the replacement, the embryo was kept alive for 60 minutes; it was fixed in Carnoy’s fluid (with 1 per cent hydrochloric acid added) and after dehydration it was cleared by the Spalteholz method. The earliest indications of a periaxial spread of the replaced fluid from the roof of the fourth ventricle is here shown. Enlargement, 8 diameters. Puate II. Fic. 4. Drawing of a pig embryo of 18 mm., in which a typical replacement of the spinal fluid had been made. The animal was kept alive for 45 minutes and was then fixed, dehydrated, and cleared in the usual manner. The extraventricular spread of the replaced fluid from two areas in the roof of the fourth ventricle is well illustrated. Enlargement, 9 diameters. Fie. 5. Drawing of a pig embryo of 19 mm., in which likewise a typical replacement of the cerebro-spinal fluid by the ferrocyanide solution had been made. After this procedure, the embryo was kept alive for 55 minutes and was then carried through the routine technique for the Spalteholz method. The further pericerebral spread of the replaced fluid is recorded. Enlargement, 8 diameters. Puate ITI. Fie. 6, A frank lateral drawing of a pigembryo of 21mm. The typical replacement of the embryonic cerebro-spinal fluid by the ferrocyanide solution was effected in this embryo and it was then kept alive for 45 minutes. At the end of this time the embryo was fixed in an acid fluid, dehydrated, and cleared. The almost complete periaxial spread of the replaced fluid is indicated by the precipitated granules. Enlargement, 7.6 diameters. Fie. 7. A dorsal view of the embryo illustrated in fig. 6. The perispinal spread of the replaced fluid is well shown. Enlargement, 7.8 diameters. Puate IV. Fic. 8. Drawing of a pig embryo of 26 mm. in which the typical replacement of the cerebro-spinal fluid has been made. After the introduction of the ferrocyanide solution the embryo was kept alive for one hour; at the end of this time it was fixed in an acid solution, subsequently dehydrated, and cleared in oil of wintergreen. The specimen shows a complete periaxial spread of the replaced fluid, as evidenced by the precipitated granules, in addition to a total filling of the intramedullary system. Enlargement, 6.5 diameters. Fie. 9. Drawing of a pig embryo of 16 mm., in which the central canal of the spinal cord was injected with the ferro- cyanide soiu*ion under moderate syringe-pressure. After fixation in an acid medium the embryo was dehydrated and cleared by the Spalteholz method. The extraventricular spread in the peribulbar region is easily made out. Enlargement, 9 diameters. 111 112 Fic. Fic. Fie. Fic. Fic. Via. Fic. Fic. Fia. Fia. Fio. ‘ Fic. ‘ Fic. Fis. 10. 11. 13. 14. 16. 17. 18. 19. 23. DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. PuaTe V. Drawing of a pig embryo of 21 mm., in which an injection of diluted india ink was made into the central canal of the spinal cord. The pressure employed was the highest obtainable from the syringe, yet below the tension causing rupture. The specimen, after injection, was fixed, dehydrated, and cleared. The slight extent of the periaxial spread of the carbon granules can be easily seen. Enlargement, 7 diameters. Drawing of a pig embryo of 16 mm., in which an injection (under moderate syringe-pressure) of 0.5 per cent solution of silver nitrate was made into the central canal of the spinal cord. The silver was reduced in the sunlight, the embryo then fixed. After dehydration, the embryo was cleared in benzol and oil of wintergreen. Enlargement, 7.5 diameters. 2. Drawing of a pig embryo of 13 mm.; into the central canal of the spinal cord a dilute solution of nitrate of silver was injected under strong syringe-pressure. Reduction of the silver was accomplished by exposure to sunlight; the embryo was then fixed, dehydrated, and cleared. Enlargement, 9 diameters. Puate VI. Photomicrograph of transverse section of a pig embryo of 18 mm. Specimen obtained from an embryo in which the cerebro-spinal fluid was replaced by a 1 per cent solution of potassium ferrocyanide and iron- ammonium citrate. After this replacement the embryo was kept alive for 65 minutes. The resultant prussian-blue precipitate is not included in this photomicrograph. Enlargement, 13 diameters. Drawing of blocked area in fig. 13, under higher magnification and including the resultant precipitate of prussian-blue. The typical ependymal cells (epe) lining the fourth ventricle are shown on either side; between them occurs the area membranacea superior (ams). The transit of the replacement fluid through the membranous area and the spread through the adjacent mesenchyme are illustrated. En- largement, 245 diameters. . Photomicrograph of transverse section from embryo pig illustrated in fig. 13. Section taken from more caudal plane than that given in the former figure. The prussian-blue spread is not illustrated. Enlargement, 10 diameters. Drawing, under higher magnification, of the rectangular area in fig. 15. The passage of the replaced solution, as shown by the resultant precipitate of prussian-blue, through the area membranacea inferior (amt) is here illustrated. The extension of the replaced fluid through the adjacent mesenchyme and the non- penetration of the solution into the condensed mesenchyme are shown. Enlargement, 140 diameters. Photomicrograph of sagittal section of a pig embryo of 18 mm. Specimen obtained from an embryo in which the cerebro-spinal fluid was replaced by a 1 per cent solution of potassium ferrocyanide and iron-ammo- nium citrate. After this replacement the animal was kept alive for 45 minutes. Fixed for 5 minutes in 10 per cent formol containing 1 per cent hydrochloric acid; then over night in modified Bouin’s solution (saturated aqueous solution of picric acid 75, formaldehyde 10, glacial acetic acid 10). Dehy- drated by 2 and 4 per cent grades of alcohol; embedded in xylol-paraffin. Serial sections, stained by hematoxylin and eosin. The resultant precipitate of prussian-blue has not been reproduced in the photomicrograph. Enlargement, 8 diameters. Drawing of blocked area in fig. 17 under higher magnification. The granules of prussian-blue are here repre- sented by the blue stenciling. The transit of the fluid, as shown by the granules, into the periaxial mesenchyme through the two membranous areas (ams and ami) in the roof of the fourth ventricle are well shown. Enlargement, 35 diameters. Puate VII. Photomicrograph from a sagittal section of a fetal pig of 27 mm. The cerebro-spinal fluid in this specimen was replaced by a 1 per cent solution of potassium ferrocyanide and iron-ammonium citrate; the fetus was kept alive for 40 minutes; fixed in 10 per cent formol containing 1 per cent hydrochloric acid for 15 minutes; then over night in modified Bouin’s solution; dehydrated by 2 and 4 per cent grades of alcohol; embedded in xylol-paraffin. The prussian-blue granules are not represented in this photo- micrograph. Enlargement, 8 diameters. . Drawing of squared area in fig. 19. The center of the field is occupied by the optic nerve; around it the developing extrinsic optic muscles are shown. The precipitate of prussian-blue occurs in the perineural mesenchyme. Enlargement, 190 diameters. . Photomicrograph of rectangular area in fig. 19. The passage of the ferrocyanide solution into the sinus transversus (str) is represented by the precipitated blue granules. Enlargement, 133 diameters. . Photomicrograph of a transverse section of a pig embryo of 23 mm. The cerebro-spinal fluid was replaced in this embryo with a 1 per cent solution of potassium ferrocyanide and iron-ammonium citrate. The embryo was kept alive for 50 minutes and was then fixed over night in 10 per cent formol containing 1 per cent hydrochloric acid. The granules of prussian-blue are not shown in this reproduction. Enlarge- ment, 13 diameters. Drawing of squared area in fig. 22. The area membranacea superior (ams) is shown, surrounded on either side by tufts of the chorioid plexus (chp) and the typical ventricular ependyma. The transit of the solution is shown, as represented by the resultant granules, through the area, with the subsequent spread into the periaxial mesenchyme. Enlargement, 125 diameters. Fig. 24. Fria. 25. Fia, 26. Fia. 27. Fia. 28. Fia. 29. Fie. 30. Fia. 31. Fig. 32. Fia. 33. Fig. 34. Fia. 35. Fic. 36. Fia. 37. Fig. 38. Fia. 39. Fia. 40. Fra. 41. Fig. 42. Fia. 43. Fig. 44, Fia. 45. Fie. 46. Fia. 47. EXPLANATION OF PLATES. 113 Prats VII. Photomicrograph of a transverse section of a pig embryo of 8mm. Fixed in modified Bouin’s solution over night, dehydrated by 2 and 4 per cent grades of alcohol, embedded in xylol-paraffin. Enlargement, 30 diameters. Photomicrograph, retouched, of the blocked area in fig. 24. The character of the cells (epc) composing the roof of the fourth ventricle (4ve) is shown in this reproduction. Enlargement, 165 diameters. Photomicrograph of a sagittal section from a pig embryo of ll mm. Fixed in modified Bouin’s solution over night, dehydrated by 2 and 4 per cent grades of alcohol, embedded in xylol-paraffin. Enlargement, 11 diameters. Photomicrograph of the blocked area in fig. 26. The area membranacea superior (ams) in the roof of the fourth ventricle is shown sharply delimited from the two processes of typical ependyma (epe). Enlarge- ment, 67 diameters. Photomicrograph of a more lateral section of the pig embryo of 11 mm. given in fig. 26. Enlargement, 11 diameters. Photomicrograph, under higher magnification, of the blocked area in fig. 28. The lateral border of the area membranacea superior (ams) of the roof of the fourth ventricle is given. Enlargement, 50 diameters. Photomicrograph of a sagittal section from a pig embryo of 13 mm. Fixed in modified Bouin’s solution, dehydrated by 2 and 4 per cent grades of alcohol, and embedded in xylol-paraffin, Enlargement, 8 diameters. Photomicrograph, under higher magnification, of the squared area in fig. 30. The reproduction comprises a sagittal section of the area membranacea superior (ams) of the roof of the fourth ventricle. Enlargement, 67 diameters. Photomicrograph of a sagittal section of a pig embryo of 14mm. Fixed in modified Bouin’s solution, dehy- drated by 2 and 4 per cent grades, and embedded in xylol-paraffin. Enlargement, 11 diameters. Photomicrograph of the blocked area in fig. 32 under higher magnification. The area membranacea superior (ams) in the roof of the fourth ventricle is reproduced. Enlargement, 75 diameters. Puate IX. Photomicrograph of a transverse section of a pigembryoof 18mm. Fixed in Carnoy’s fluid (6:3: 1), dehy- drated by 2 and 4 per cent changes of alcohol, and embedded in xylol-paraffin. Enlargement, 13 diameters. Photomicrograph, under higher magnification, of the blocked area in fig. 34. ‘The area membranacea superior (ams) is here given, flanked on either side by typical ependyma (epe). Enlargement, 170 diameters. Photomicrograph of a transverse section of a pig embryo of 18 mm. Fixed in modified Bouin’s fluid, dehy- drated by 2 and 4 per cent changes, and embedded in xylol-paraffin. Enlargement, 13 diameters. Photomicrograph of rectangular area outlined in fig.36. The extent of the area membranacea superior (ams), with its adherent coagulum of albuminous material, is well differentiated from the adjacent typical ven- tricular ependyma (epe). Enlargement, 100 diameters. Photomicrograph of a transverse section of a pig embryo of 19 mm. Fixed in modified Bouin’s solution, dehydrated by 2 and 4 per cent grades, and embedded in xylol-paraffin. Enlargement, 13 diameters. Photomicrograph, under higher power, of the rectangular area in fig. 38. A small break in the integrity of the lining ependyma of the roof of the fourth ventricle, representing the irregular boundary of the area membranacea superior (ams), is given. Enlargement, 290 diameters. Photomicrograph of a transverse section of a human embryo of 4 mm. (No. 836 of collection of Carnegie Institution of Washington). Enlargement, 33 diameters. Photomicrograph, retouched, of the blocked area in fig. 40. The epithelial-like cells (epc) composing the roof of the fourth ventricle (4ve) are here shown separated from the denser nervous tissue. Enlargement, 100 diameters. PuaTe X. Photomicrograph of transverse section of pig embryo of 19 mm. Fixed over night in modified Bouin’s solution, dehydrated by 2 and 4 per cent changes of alcohol, and embedded in xylol-paraffin. Enlarge- ment, 13 diameters. Photomicrograph of squared area in figure 42, under higher magnification. The area membranacea superior (ams) with the attached coagulum of albumen is reproduced. Enlargement, 115 diameters. Photomicrograph of sagittal section of pig embryo of 23mm. Fixed in modified Bouin’s fluid, dehydrated by 2 and 4 per cent changes, and embedded in xylol-paraffin. Enlargement, 5 diameters. Photomicrograph, under higher magnification, of squared area in fig. 44. The area membranacea superior (ams) is here shown, delimited by the cells of the chorioid plexus (chp) on one side and by the further ependymal prolongation (epe) of the cerebellar lip. Enlargement, 88 diameters. Photomicrograph of sagittal section of pig embryo of 32mm. Fixed in modified Bouin’s solution, dehydrated by 2 and 4 per cent changes, and embedded in xylol-paraffin. Certain portions of the dura mater (dmc) are indicated. Enlargement, 5 diameters. Photomicrograph of blocked area in fig. 46, under higher magnification. The small remaining area mem- branacea superior (ams) is quite surrounded by encroaching ependyma in the chorioidal folds. En- largement, 88 diameters. 114 Fic. Fic. Fic. : Fic. Fic. £ Fic. Fis. Fic. é Fic. Fic. : Fig. Fic. 5 Fig. Fic. Fic. Fic. Fic. Fic. Fic. Fia. Fic. Fia. Fia. 62. 63. 68. 69. 70. Fic. 71 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. Piate X—Continued. . Photomicrograph of transverse section of human embryo of 7 mm. (No. 617 of the collection of the Carnegie Institution of Washington). Enlargement, 10 diameters. . Photomicrograph of squared area in fig. 48, under higher magnification. The epithelial-like cells (epc) composing the roof of the fourth ventricle at this stage are well shown. Enlargement, 100 diameters. . Photomicrograph of transverse section of human embryo of 7 mm. (No. 617 in the Carnegie Institution of Washington). Enlargement, 10 diameters. . Photomicrograph of blocked area in fig. 50. The marked invagination of the roof of the fourth ventricle (4ve) with the lining of epithelial-like cells (epc) is given. Enlargement, 33 diameters. . Photomicrograph of transverse section of human embryo of 9 mm. (No. 721 in the collection of the Carnegie Institution of Washington). Enlargement, 10 diameters. . Photomicrograph of squared area outlined in fig. 52. The pale, large cells (epc) comprising the roof of the fourth ventricle characterize the reproduction. Enlargement, 50 diameters. . Photomicrograph of sagittal section of human embryo of 11 mm. (No. 544 in the collection of the Carnegie Institution of Washington). Enlargement, 6 diameters. . Photomicrograph of blocked area in fig. 54. The apparent break in the continuity of the roof of the fourth ventricle with exudation of the ventricular albumen into the mesenchyme is brought out. Enlargement, 50 diameters. Puate XI. . Photomicrograph of sagittal section of human embryo of 14 mm. measured on the slide (No. 144 of the col- lection of the Carnegie Institution of Washington). Enlargement, 8 diameters. . Photomicrograph, under higher magnification, of blocked area in fig. 56. The greater part of the ventricular wall shown is composed of the area membranacea superior (ams), bounded below by typical ventricular ependyma (epe). Enlargement, 67 diameters. . Photomicrograph of sagittal section of human embryo of 17 mm. (No. '576 of the collection of the Carnegie Institution of Washington). Enlargement, 10 diameters. . Photomicrograph of rectangular area in fig. 58, showing the area membranacea superior (ams) of the roof of the fourth ventricle. Enlargement, 50 diameters. . Photomicrograph of sagittal section of human embryo of 17 mm. (No. 576 of the collection of the Carnegie Institution of Washington). Enlargement, 7 diameters. . Photomicrograph of the blocked area in fig. 60 under higher magnification. The aggregation of epithelial-like cells (epc) on the lateral border of the area membranacea superior is here portrayed. Enlargement, 67 diameters. Photomicrograph of transverse section of human embryo of 18 mm. (No. 409 of the collection of the Carnegie Institution of Washington). Enlargement, 7 diameters. Photomicrograph, under higher power, of squared field in fig.62. The peculiar inversion of the roof of the fourth ventricle (4ve) indicated in fig. 62, has resulted in a marked dislocation of the area membranacea superior (ams), shown in this figure. Enlargement, 75 diameters. Puate XII. . Photomicrograph, retouched, of a transverse section of a human embryo of 21 mm. (No. 460 of the collection of the Carnegie Institution of Washington). The field taken consists of a portion of the fourth ventricle with the lining of typical ependyma (epe) on either side. The area membranacea superior (ams) is shown between the two lips of ependyma. Enlargement, 33 diameters. 5. Photomicrograph, retouched, of a similar section to that given in fig. 64, but taken from a more anterior plane from the same embryo. The field shown is analogous in every way to that in the preceding figure. . Photomicrograph of a transverse section of an embryo chick of 121 hours’ incubation. Fixed in Bouin’s solu- tion. Enlargement, 15 diameters. . Retouched photomicrograph, under higher magnification, of the blocked area in fig. 66. The area mem- branacea superior (ams) is here given, delimited sharply from the lips of ependyma (epe) which line the roof of the fourth ventricle. Enlargement, 133 diameters. Photomicrograph of a more caudal section from the same embryo as portrayed in fig. 66. Enlargement, 15 diameters. Retouched photomicrograph, under higher magnification, of the blocked area in fig. 68. The area mem- branacea superior (ams) is shown at the point of its greatest transverse diameter. Enlargement, 88 diameters. Photomicrograph of a sagittal section of a pig embryo of 15 mm, Fixed in modified Bouin’s solution, dehy- drated by 2 and 4 per cent grades of alcohol, and embedded in xylol-paraffin. Enlargement, 8 diameters. . Photomicrograph, under higher magnification, of blocked area in fig. 70. The earliest evidence of the area membranacea inferior (amt) in the roof of the fourth ventricle is here shown. Enlargement, 125 diameters. Fia. 72. Fia. 73. Fia. 74. Fia. 75. Fia. 76. Fia. 77. Fia. 78. Fia. 79. Fia. 80. Fra, 81. Fig. 82. Fic. 83. Fia. 84. Fig. 85. Fia. 86. Fia. 87. Fia. 88. Fia. 89. Fig, 90. Fie. 91. Fia. 92. Fia. 93. Fia. 94. Fia. 95. Fra. 96. EXPLANATION OF PLATES. 115 Puate XIII. Photomicrograph of sagittal section of a pig embryo of 18 mm. Fixed in modified Bouin’s solution, dehy- drated by 2 and 4 per cent grades of alcohol, and embedded in xylol-paraffin. Enlargement, 11 diameters. Photomicrograph, under higher power, of the rectangular area outlined in fig. 72. The enlarging area mem- branacea inferior (amz) is shown in the midst of the typical lining ependyma of the roof. Enlargement, 100 diameters. Photomicrograph of sagittal section of a pig embryo of 23 mm. Fixed in modified Bouin’s solution, dehy- drated by 2 and 4 per cent grades of alcohol, and embedded in xylol-paraffin. Enlargement, 6 diameters. Photomicrograph of blocked area in fig. 74. The area membranacea inferior (ami) is, at this stage, quite extensive, as shown in the reproduction; the early stages in the development of the cisterna cerebello- medullaris may also be seen. Enlargement, 75 diameters. Photomicrograph of sagittal section of a pig embryo of 32 mm. Fixed in modified Bouin’s solution, dehy- drated by 2 and 4 per cent grades of aleohol, and embedded in xylol-paraffin. Enlargement, 7 diameters. Photomicrograph, under higher magnification, of blocked area in fig. 76. The unsupported character of the area membranacea inferior and the formation of the cisterna cerebello-medullaris is here reproduced. Enlargement, 67 diameters. Piate XIV. Photomicrograph of a sagittal section of a pig embryo of 32mm. Fixed in modified Bouin’s solution, dehy- drated by 2 and 4 per cent grades of alcohol, and embedded in xylol-paraffin. Enlargement, 7 diameters. Photomicrograph of the blocked area in fig. 78, under higher magnification. The intact area membranacea inferior (ami), unsupported by any mass of tissue, is shown separating the ventricular cavity from the developing cisterna cerebello-medullaris. Enlargement, 67 diameters. Photomicrograph of a sagittal section of a human embryo of 16 mm. (No. 406 in the collection of the Carnegie Institution of Washington). Enlargement, 7 diameters. Photomicrograph of the area outlined in fig. 80, but under higher magnification. An early stage in the differ- entiation of the area membranacea inferior (ami) is given. Enlargement, 50 diameters. Photomicrograph of a sagittal section of a human embryo of 17 mm. (No. 576 in the collection of the Carnegie Institution of Washington). Enlargement, 6 diameters. Photomicrograph, under higher power, of the area blocked in fig. 82. The chorioid plexuses of the fourth ventricle lie in the central portion of the field; above is the thick cell-layer on the lateral side of the area membranacea superior (ams), while below the upper limit of the area membranacea inferior (ami) appears. Enlargement, 67 diameters. Photomicrograph of a transverse section of a human embryo of 18 mm. (No. 409 in the collection of the Carnegie Institution of Washington). Enlargement, 5 diameters. Photomicrograph of the blocked area in fig. 84. The cellular character, and especially the clumping of cells, of the area membranacea inferior (ami) is shown. Enlargement, 25 diameters. Photomicrograph of a sagittal section of a human embryo of 19 mm. (No, 431 in the collection of the Carnegie Institution of Washington). Enlargement, 5 diameters. Photomicrograph of the blocked area outlined in fig. 86. The area membranacea inferior (ami) appears separating the fourth ventricle from the developing cisterna cerebello-medullaris. Enlargement, 25 diameters. Puate XV. Photomicrograph from a sagittal section of a human embryo of 17 mm. (No. 576 of the collection of the Carnegie Institution of Washington), representing an enlargement of the second blocked area in fig. 58. The erea membranacea inferior (ami) appears sharply delimited from the adjoining typical ependyma. Enlargement, 67 diameters. They Photomicrograph of a sagittal section of a human embryo of 23 mm. (No. 453 of the collection of the Carnegie Institution of Washington). Enlargement, 6 diameters. Photomicrograph of the blocked area in fig. 89. The area membranacea superior (ams) appears in the stage of closure, while the area membranacea inferior (ami) is becoming well differentiated from the typical ependyma lining the other portions of the fourth ventricle. Enlargement, 26 diameters. Photomicrograph of a sagittal section of a human embryo of 26 mm. (No. 1008 of the collection of the Carnegie Institution of Washington). Enlargement, 4.5 diameters. Photomicrograph, under higher magnification, of the blocked area in fig.91. The area membranacea superior has been almost completely closed by the dense ependyma of the superior half of the roof of the fourth ventricle, while the inferior area (ami) has become a membrane lacking wholly the character of ependyma. Enlargement, 23 diameters. Photomicrograph of a sagittal section of a human embryo of 35 mm. (No. 199 of the collection of the Carnegie Institution of Washington). Enlargement, 3 diameters. Photomicrograph, under higher powers, of the blocked areas in fig. 93. The formation of the cisterna cere- bello-medullaris is shown in relation to the ventricular roof. Enlargement, 23 diameters. Drawing of cells of the chorioid plexus from the lateral ventricles of a fetal pig of 132mm. The specimen was fixed in absolute alcohol, and stained by Best’s carmine stain for glycogen. The glycogen occurs in the _ form of globules within the epithelial cells. Enlargement, 950 diameters. Drawings of the cells of the chorioid plexus from the lateral ventricles of a fetal pig of 36 mm. The specimen was fixed in absolute alcohol and stained by Best’s carmine method. The glycogen appears in the epi- thelial eclls in the form of basilar plaques. Enlargement, 950 diameters. 116 DEVELOPMENT OF CEREBRO-SPINAL SPACES IN PIG AND IN MAN. Puate XVI. Fie. 97. Photomicrograph of a transverse section of a pig embryo of 19 mm. Fixed in modified Bouin’s fluid, dehy- Fic. Fic. Fia. 98. . 99. . 100. . 101. 102. . 103. 104. . 105. . 106. . 107. . 108. . 109. . 110. eb B . 112. . 113. . 114. . 115. . 116. . 117. drated by 2 and 4 per cent grades of alcohol, and embedded in xylol-paraffin. Enlargement, 10 diameters. Photomicrograph, under higher magnification, of the blocked area in fig. 97. The double condensations of mesenchyme to form pia mater (pmc) and cerebral blastema (cbl) appear separated by a region of mesen- chyme which is breaking down. ‘This central area of mesenchyme, with the marked albumen-content, is to become the arachnoid spaces. Enlargement, 133 diameters. Photomicrograph of a transverse section of a pig embryo of 20mm. Fixed in modified Bouin’s fluid, dehy- drated by 2 and 4 per cent changes of alcohol, and embedded in xylol-paraffin. Enlargement,10 diameters. Photomicrograph, under higher powers, of the blocked areas in fig. 99. The relations of the pial condensation (pmc) of mesenchyme to the nervous system, as well as the infiltration of the arachnoid mesenchyme (sas) with albumen, is reproduced. Enlargement, 133 diameters. Photomicrograph, under higher magnification, of the blocked area in fig. 22. The reproduction is included here to show the double condensation (cbl) of mesenchyme which goes to form ultimately bone and pos- sibly a portion of the dura. Enlargement, 132 diameters. Photomicrograph of a transverse section of a pig embryo of 18 mm. The embryo was one in which the cerebro-spinal fluid was replaced by the ferrocyanide solution. Subsequently the embryo was fixed in 10 per cent formol containing 1 per cent hydrochloric acid for a few minutes to precipitate the prussian- blue. It was then transferred to modified Bouin’s solution, dehydrated by 2 and 4 per cent grades of alcohol, and embedded in xylol-paraffin. The granules of prussian-blue are not represented in this figure. Enlargement, 10 diameters. Photomicrograph of the squared area in fig. 102. The relation of the thinning mesenchyme in the arachnoid areas to the caudal cranial nerves is shown. The granules of prussian-blue, scattered through the area of thin mesenchyme (sas), are nof reproduced. Enlargement, 40 diameters. Photomicrograph of a coronal section of a tissue block which includes the meninges and cerebral cortex in the region of the sinus sagittalis superior. The block was obtained from a fetal pig of 80 mm., fixed in Zenker’s fluid, and stained, after embedding in celloidin, by Mallory’s technique for connective tissue. Enlargement, 27 diameters. Puate XVII. Photomicrograph of a coronal section of a tissue block including cerebral cortex and meninges in the region of the sinus sagittalissuperior. The block was obtained from a fetal pig of 10 em., fixed in Zenker’s fluid, and stained by Mallory’s technique for connective tissue. Enlargement, 13 diameters. Photomicrograph of a coronal section, similar to that in figs. 104 and 105, except in that it was obtained from afetal pig of 17cm. The same technical procedures employed in the other specimens were used in this. Enlargement, 27 diameters. Photomicrograph of a similar section to those of the foregoing figures. ‘The specimen was obtained from a fetal pig of 20 cm. and was treated in the manner outlined above. Enlargement, 20 diameters. Drawing of the cell pattern from the inner surface of the dura mater of a fetal pig of 5em. The specimen was prepared by the reduction of a dilute solution of silver nitrate in sunlight. The preparation was subsequently stained by hematoxylin. Enlargement, 190 diameters. Drawing of a preparation, similar to that of fig. 108, but obtained from the inner surface of the dura mater of a fetal pig of 75 mm. Enlargement, 285 diameters. Drawing of a preparation, similar to those of figs. 108 and 109, obtained from the inner surface of the dura mater of a fetal pig of 90 mm. Enlargement, 285 diameters. Drawing of a preparation from the inner surface of the dura mater of a fetal pig of 16 cm. The specimen was made in the same manner as outlined in fig. 108. Enlargement, 285 diameters. Photomicrograph of a sagittal section of a pig embryo of 17 mm. An injection of an 0.5 per cent solution of nitrate of silver was made into the central canal of the spinal cord; the silver was reduced in sunlight and the embryo fixed in formalin. Enlargement, 13 diameters. Photomicrograph, under higher powers, of the blocked areas in fig. 112. The accumulation of the reduced silver (psn) against the area membranacea superior is represented in black. Enlargement, 117 diameters. Photomicrograph of a transverse section of a pig embryo of 19mm. An injection of 0.5 per cent solution of silver nitrate was made into the central canal of this embryo and the silver immediately reduced in sunlight. The embryo was fixed in formalin, carefully dehydrated, and embedded in xylol-paraffin. Enlargement, 10 diameters. Photomicrograph, under higher magnification, of the blocked area in fig. 114. The collection of reduced silver (psn) against the cells at the inferior end of the area membranacea superior is illustrated. Enlarge- ment, 100 diameters. Photomicrograph of a tranverse section of a pig embryo of 16 mm. The central canal of the spinal cord of this embryo was injected with a 1 per cent ferrocyanide and citrate solution under mild syringe-pressure ; the embryo was then fixed in 10 per cent formol containing 1 per cent hydrochloric acid. Enlargement, 10 diameters. Photomicrograph of the blocked area in fig. 116, under higher magnification. The accumulation of the precipitated injection fluid against the area membranacea superior is represented in black. A slight extraventricular spread of the fluid, which is found in this as in all embryos of this stage, can not be made out in the reproduction. Enlargement, 67 diameters. PLATE | WEED WEED PLATE Il << | - ~ 5 3 Fi a a 2 ; i PLATE 1 a w wi = out 5 5 2 + - 2 ." AN ee es y , “ ee AEE abies an Oty FO tchge oy AHoen &Co Lith Balti “ PLATE IV WEED AHoen @ Co Lith Baltimor ar | wh WEED . PLATE \ \ — Len | Ree a ig F < aay J. F. Didusch fecit AHioen &Co sth Kaltimore —_ | | 4 ) an PLATE VI ype - an a: SF, tt ghd’ 3 Wig, ami -“€ 2 29 2° ¢ bei i ae s Oo ois Bye ec lih ai fags ~ 8+ a> wee + oe S S$ 0. ag re cb J. F. Didusch fecit 18 AHloen &Co, Lith Baltanore. e¥ PLATE Vil WEED b Baltinore £ Hi § 4 23 J. F. Didusch fecit mo! AY a es = ¥> a > WEED PLATE VIII PLATE IX WEED ams PERT 1 . ae ‘epe WEED x< Ww KE < = a WEED PLATE Xl PLATE XII WEED v v WEED PLATE Xill ami PLATE XIv PLATE XV we ert — heer SEEN A COE TDOORE i pmc WEED - - *" yee pe Ss — 5 alee < . a eee a oe e - 3 Re PLATE XVI bl WEED PLATE XVil 115 113 CONTRIBUTIONS TO EMBRYOLOGY VoutumeE VI, Nos. 15, 16, 17, 18, 19 PUBLISHED BY THE CARNEGIE INSTITUTION OF WASHINGTON WASHINGTON, 1917 CARNEGIE INSTITUTION OF WASHINGTON PuBLIcATION No. 226 PRESS OF GIBSON BROTHERS WASHINGTON CONTENTS. PAGES. No. 15. Cyclopia in the human embryo. By FRANKLIN P. Matt (3 plates, 7 text-figures).. 5-33 16. Quantitative studies on mitochondria in nerve-cells. By MapGe DeG. THurLow (GT TaN EE HIPS) foe Ves Bohn ccs Eien oA Res A ROP em CTSA RI sr, 35-44 17. Development of connective-tissue fibers in tissue cultures of chick embryos. By MARGAR ED RinmD slapd (2 plates) seas ce cres soci seca es siree eee rialeie= 45-60 18. Origin and development of the primitive vessels of the chick and of the pig. By PEHORENCE VR OABIN (i plates: Grsext-hpures)=. acs --- ee \2ee aee- eee s - 61-124 . A human embryo of twenty-four pairs of somites. By FRANKLIN PARADISE JOHNSON (Stplares#Oibextcheoures).. = serjccociee see se oe hea essen e+ seek 125-168 wow oa i : ‘ | a x % Sone We aieaaT: a apna. ¢ «eae Padi es a? | » . y , vy Y : f ae CONTRIBUTIONS TO EMBRYOLOGY, No. 15. CYCLOPIA IN THE HUMAN EMBRYO, By FRANKLIN P. Matt. With three plates and seven figures. APU LG > CYCLOPIA IN THE HUMAN EMBRYO. By Frankuin P. Matt. The progress made in recent years on the study of teratology has been so marked that it is now possible to reconsider the whole subject and to place it upon a permanent scientific basis. For this progress we are indebted almost exclusively to the experimental embryologists. Problems which formerly seemed impossible of solution—for example, the formation of the double monsters—have yielded as by magic to the embryologist, who made experimental studies upon the living egg. Perhaps the best example that can be brought forward to illustrate this point is the question of the cause of cyclopia. As soon as it was possible to experiment on eggs in such a way that practically all of them developed into cyclopean monsters the explanation of this condition was at hand. For this work we are indebted entirely to Stockard. Before reviewing the four specimens which I have to report it may be well to give an account of the theories regarding the origin of the eyclopean condition. There are two chief theories, both resting upon an embryological basis. The first of these is that the eggs begin to develop normally and that subsequently, on account of an imperfect development of the head, the eyes coalesce to form a single eye. This theory can be traced back to Meckel. The second is that the eyes arise normally from the midventral line of the brain as a single structure, which in the course of development divides into two eyes. This view was first advanced by Huschke, who believed cyclopia to be due to an arrest of the development of the brain at the time the eyes are forming. Although Huschke’s opinion seemed to be quite sound at the time it was advanced, it did not attach itself firmly to literature, nor could we well accept it at present as resting upon a sound embryo- logical basis. The figures which he gives in illustration shows first an early stage of development of the brain, with a marked forebrain, and then an embryo with two eye-vesicles hanging to the forebrain. He apparently confounded the whole forebrain with the eye primordium. Meckel’s studies rest upon much sounder embryological and anatomical evidence, and his views gradually made their way into the literature of teratology. Until a decade ago it was practically impossible to find any description of cyclopia in which Meckel’s studies were not reflected in the background. According to Ahlfeld, Meckel states that cyclopia is characterized by a coalescence of the eye- balls as well as of the orbital cavities. In case the orbital cavities unite very early in development they distend evenly in a lateral direction. The tissues which normally separate these cavities are absent or are pushed aside. In fact, the structures which give the frame to the nose are most rudimentary, or absent, while the nose itself is represented as a membranous snout, varying in form and located above the confluent eyes. The mouth is frequently involved in this type S CYCLOPIA IN THE HUMAN EMBRYO. of monster and is usually rudimentary, while in some instances it, as well as the snout, is missing altogether. Since the eyeballs are developed from pouches which arise from the forebrain, it follows that the primary cause of this anomaly is not to be sought in the development of the skull, but in the development of the brain itself. We find in these cases that the width of the forebrain and midbrain diminishes in the course of their development, corresponding to the union of the orbital cavities and the eyeballs, making the brain appear at term much like that of an embryo of the twelfth week. In addition to the atrophy of these parts, the formation of the hemispheres as a single body is especially noticeable—that is, they have not been divided into two lobes. This division often is only slightly indicated. The ventricles have united to form a single large cavity. In most of the cases at birth the quantity of fluid within the ventricle is increased, so that as a rule we have a large, bladder-like body in place of the forebrain. In this way is the fact explained that in spite of the rudimentary development of the brain there is no diminution of the size of the fore part of the head, as most cases of eyclopia are accompanied with hydrocephalus. There are cases, however, in which there is no hydrocephalus, which naturally result in a small head. This is most pronounced in cases of eyclopia in double monsters in which the head contains two brains. In these cases a symmetrical development of the brain is very rarely found. The rudimentary brain can no doubt be held responsible for the most pronounced specimens of cyclopean faces. In may be taken for granted that the nerves which are to supply the deformed eye and face are simple in their development, cor- responding in amount with the degree of the anomaly. This general description of the anatomy of the eye and face in cases of cyclopia is one which will be found in most teratologies, and in all of these accounts it would appear as though the authors mean to say that the eyes must arise from the fore- brain and that they subsequently unite into a single compound eye, more or less hourglass-shaped, due to an arrest of the growth of the brain which in some way interfered with the development of the forehead and eventually left the nose above the cyclopean eye. Teratologists are inclined to believe that the accom- panying hydrocephalus is to be viewed as the primary cause of the anomaly, although in many instances they try to trace this back to amniotic bands, which, however, are not found in human specimens of eyclopia, and of course such bands could play no réle in the formation of this anomaly in animals which develop without an amnion. Furthermore, the explanation of the formation of monsters by means of amniotic bands is alluded to in recent teratologies only as one of the myths of teratology. In my paper on monsters some ten years ago I gave a review of the experi- mental work upon cyclopia as it appeared at that time. These statements I shall recapitulate in part in order to bring out more clearly the recent progress made in the study of cyclopia. In numerous experiments upon frog’s eggs, Born, in 1897, occasionally pro- duced monsters by splitting the head through its sagittal midplane after the medullary plate was formed, and then readjusting the two halves. The pieces CYCLOPIA IN THE HUMAN EMBRYO. 7] united at once, but in a few instances a double eye was formed. Later Spemann, making similar experiments, also produced cyclopean embryos. In some of Spemann’s experiments triton eggs were ligated in the sagittal plane during seg- mentation, and frequently embryos with double heads resulted, one or both being eyclopean. Spemann believes this experiment proves that in its differentiation the cyclopean eye is defective from its beginning and is not produced by con- erescence of two eyes which started to develop normally. Levy also produced cyclopean monsters by cutting off the front of the head of triton larve. In the course of two weeks the two eyes approached each other and formed a double eye, but they did not fuse. However, the pigment layer was destroyed, or absent, at the point of contact. The two optic cups touched each other, but did not unite. In 1906 Harrison produced a new variety of cyclopia by removing the entire brain from frog embryos. In these specimens the eye moved to the back of the head and appeared to unite in a single vesicle in the region usually occupied by the pineal eye. These specimens are still unpublished. By pricking the extreme anterior end of the embryonic shield in Fundulus eggs, Lewis found that many of the eggs developed into cyclopean monsters. All grades of defective eye were formed—from a double eye and hourglass-shaped eye with two lenses to oblong eyes with two lenses or with but a single lens. The optic cups blended absolutely, thus apparently showing the mode of develop- ment of these eyes. Lewis also found that in many of the embryos the brain had not been injured at all, but that the prick had destroyed the nose only. This experiment seems to show conclusively that it is the absence of tissues between the eye primordia which allows them to come together and unite, and that a rudimentary brain is unnecessary. In his remarkable experiments on the artificial production of a single median eye in the fish embryo by means of sea-water solutions of magnesium chloride, Stockard found that 50 per cent of the embryos developed cyclopia. In these embryos the optic cups were fused at an early developmental stage, much as was the case in Lewis’s specimens, in which the embryonic shield had first been pricked. The union of the two cups formed a large compound eye, which in turn derived its lens from the epidermis immediately overlying it in the midline of the embryo. How the magnesium acts upon the embryo is not clear from Stockard’s descrip- tion. No doubt it will be found that it retards the growth of the frontal process, much as in Lewis’s experiments. The salt, however, acted upon the whole body of the embryos, for their development was retarded, thus making them smaller than usual, and their circulation was feeble, but they did not die. In these em- bryos, as in Lewis’s specimens, the growth of the brain was normal. The remark- able experiments of Stockard set at rest all germinal theories of cyclopia and prove that every egg has in it the power to develop cyclopean monsters. These experiments, as well as the numerous pathological embryos with deformed heads and faces which I have studied, prove at any rate that in the formation of many monsters there is an extensive destruction and shifting of tissues. This is also well illustrated in the production of club-foot in the human 10 CYCLOPIA IN THE HUMAN EMBRYO. embryo. It has frequently been noticed that tadpoles whose growth has been arrested develop stubby or club tails and fins—a condition which corresponds well with club-shaped extremities in man. Our collection contains 18 embryos with deformed legs and feet, with catalogue numbers less than 400, ranging from the very earliest period until the fetus is well formed. The leg-buds are irregular in shape and are filled with condensed mesenchyme; sometimes they are stubby on one side of the body and normal on the other. The study of the larger embryos shows that there is a variety of inflammation of the tissues which is especially well marked in the tendons and around the cartilages. In general this condition may be accounted for by an arrest of development due to impaired nutrition. At any rate, embryos that are not developing well—experimental larve and human embryos with other malformations—often have club-shaped arms, legs, fins, or tails. The inference to be drawn from the above summary is that after the eyes have become well formed they do not pass out to the side of the head as in normal development, but approach each other and more or less unite, and thus form cyclopia. Recent embryological studies of Stockard and of Spemann show con- clusively that this view can not be correct, for it is found that the eyclopean con- dition can be followed back through earlier and earlier embryos, and that all varieties of cyclopia are present while the eyes are still firmly attached to the brain. ‘It is now maintained by Stockard that, from its very beginning, the eye primordium is in the midventral line of the brain, and that in cyclopean embryos there is an arrest of its development, the eye remaining median or dividing in part, forming the hourglass-shaped cyclopean eye with two lenses, ete. This view is combated more or less by Spemann; but I must confess that it is difficult for me clearly to understand his view as given in his various papers. Through his well-known magnesium experiments, Stockard has been able to procure an abundance of material for the study of the early development of cyclopia. He proves first of all that the condition of cyclopia is present in the earliest stages in which it would be possible to recognize it. At no stage are there two normal eyes which subsequently blend to form a single eye. The cyclopean condition is present in the eyes while they are still closely attached to the brain. Stockard observes, secondly, that the cyclopean eye is rarely equal in extent and size to the sum of two normal eyes combined. A cyclopean eye is, as a rule, very slightly if any larger than one normal lateral eye, and in fact it is often much reduced or actually minute in size as compared with a normal eye. According to Stockard, this fact indicates most decidedly that the eye material, as such, has been injured or arrested in development and differentiation. He believes that we are scarcely warranted in assuming, as have various authors at different times, that eyclopia is due to a fusion of the eyes after they have arisen from the brain and that the earlier in development the fusion occurs the more intimately asso- ciated the two eye components become. This view, according to Stockard, has been proved incorrect by actual observation on cyclopean monsters, where it is found that the eyclopean condition of the eye—whether large and hourglass- shaped or of small size resembling a normal eye—is present from the earliest CYCLOPIA IN THE HUMAN EMBRYO. 11 appearance of the optic vesicle from the brain. In other words, the several degrees of the cyclopean eye come off from the brain in their final condition. The idea of the fusion of the eye parts, Stockard continues, was deep-rooted, however, and exists in the recent views of Spemann in a refined form. Spemann ‘believes, as others have previously suggested, that cyclopia is due to an absence of non-ophthalmiec tissue in the median region of the medullary plate or groove. This lack of median tissue allows the eye primordia, which he holds to be lateral in position, near the borders of the medullary plate, to come together and fuse in the median plane and later give rise to a cyclopean eye. Cyclopia, according to this view, occurs in a more or less passive manner, and is actually a fusion of the eye primordia of the two sides during development. Stockard adds that he is certain that this fusion explanation, which has now been forced entirely back into the medullary plate, is as false as its bolder predecessor, which assumed the fusion to take place outside of the brain-tissues after the optie vesicles or cups had arisen. He says that Spemann did not at first advocate this late-fusion view, but claimed (from his experiment on Triton) that the cyclopean eye arose out of the medullary tissues in its final condition; subsequently, however, he assumed the rdle of a most ardent supporter of the view that the fusion of the optic pri- mordia takes place within the medullary plate. It may be added that there is no known instance of the formation of cyclopia by experimental methods after the eyes are fairly well formed in normal develop- ment. All the experiments in which cyclopia has been produced were made upon the embryo at a stage before the eyes could be recognized under the microscope. One must recall that Stockard’s magnesium experiment is effective only when it is done before the embryo is 15 hours old. In fact, Stockard found that the best results were obtained if the eggs were placed in magnesium-chloride solution immediately after fertilization. If the eggs are not placed in the solution until 15 hours after fertilization, before the germ-ring forms and begins its downward growth from the yolk-mass, no cyclopia occurs. Cyclopia is less frequent in eggs which are treated at later stages than in eggs immersed in magnesium-chloride solution during the fourth and eighth cell stage. It appears, then, that the critical stage at which ecyclopia is best produced with magnesium is shortly before the germ-ring is formed. According to Stockard, a 15-hour embryo has the germ-ring beginning to form and descend over the yolk-sphere; the embryonic shield is searcely indicated, but appears soon afterward. Embryos of later stages sub- jected to the same treatment develop normally, or at least do not show cyclopia, while embryos younger than 15 hours, and at as early a stage as the first cleavage, are much more readily affected in such a manner as to cause the cyclopean defect. The optic vesicles appear at about 30 hours after fertilization, but the stimulus must be applied at a time sufficiently long before this process occurs, since a num- ber of important steps in eye formation are doubtless taking place before the visible signs of optic vesicles are present. It is interesting to note that the Lewis pricking experiment is made at a stage in which cyclopia can no longer be produced by placing the eggs in a magnesium 12 CYCLOPIA IN THE HUMAN EMBRYO. solution. According to Lewis, the experiment should be made on the second day. Although he does not give his experiment in hours, his illustrations show the stage of development. According to these the embryonic shield is well formed. The experiments of Lewis were first described in my monograph on monsters, but they have since been reported in detail by their author. As has been stated, Lewis produced cyclopia in Fundulus by pricking the middle of the anterior end of the embryonic shield two days after fertilization. In the course of a few days it became apparent that in some of the eggs operated upon the eyes had developed normally, while in others they had become cyclopean. Most of the specimens were killed after 15 days. Pricking of the embryonic shield was accompanied by the escape of a sight amount of tissue, and as there is little or no regeneration of the central nervous system in F'undulus, the defect caused at the time of pricking subsequently became more and more apparent as development proceeded. Both Lewis and Stockard have found that cyclopean Fundulus embryos usually develop with a normal brain, thus no doubt accounting for the vitality of this special cyclops. Furthermore, it appears that the eye primordium in Fundulus is more circumscribed than in many other animals. In a number of his experiments Lewis found that the material withdrawn with the needle-point came from one side of the anterior end of the embryonic shield, with a resulting abnormality of the eye on that side. In a specific case, at the time of hatching, the right eye of the specimen consisted of a small bit of retina connecting with the otherwise almost normal brain-wall. The left eye was apparently normal, as were also the brain and the nasal pit. In other specimens, in which the operation was about medial and was done at the time the embryonic shield was beginning to form, the embryos developed with the two eyes in contact, with two optic nerves and two lenses. Among other specimens there is one with a eyclopean eye, having a layer of pigment narrowing between the two eyeballs. In specimens operated upon at a little later stage there is a median cyclopean eye with two lenses, one pupil, and one cup cavity. Using Lewis’s language, the large optic cup shows in sections a very beautiful median eye with complete con- tinuity of the layers of the retina of two components about a single large cup cavity of a single lens. According to Lewis, the explanation of these various abnormalities is in a way comparatively simple, if we assume that in the early embryonic-shield stage the various parts of the central nervous system and the eyes are probably already predetermined, and, secondly, that there is very little or no power of regeneration in this tissue. Numerous experiments on regeneration indicate very clearly that there is little or no regeneration of the tissue (at least of that of the central nervous system) extruded at the time of the operation. The repair which takes place after the operation consists merely of a rapid closing together of the parts left remaining, and thus a healing of the wound occurs without regeneration of lost parts. ‘This closing of the wound is accomplished in a few minutes, and primordia are thus brought into contact which normally are quite widely separated—those of the two eyes, for example. The subsequent differentiation adjusts itself to the CYCLOPIA IN THE HUMAN EMBRYO. 13 new relations of these primordia with the resulting abnormal forms. Thus, as one examines these developing embryos, from the time the eye primordia are first visible in the living specimens under the binocular microscope, they appear to have the same amount of fusion or loss of eye that is clearly to be found in the same individual at later stages and at the time of hatching. So we can explain these cyclopean forms by a fusion of the primordia of the two eyes immediately after the operation, even though at this time no primordia are visible. Differentia- tion of the eye-tissues evidently occurs some time before it is visible by our crude microscopic methods. Briefly summarizing the experiments of Stockard and Lewis, it may be said that Stockard produced cyclopia by immersing Fundulus eggs in a magnesium solution before the formation of the germ-ring, while Lewis operated upon the embryonic shield after it had arisen from the germ-ring. According to Stockard, the magnesium solution possesses a decidedly anesthetic effect and inhibits the growth of the optic out-pocketings; and therefore the condition of cyclopia must be present before the formation of the optic eup—which he believes to be median— the anesthetic effect preventing the medial cup from dividing, thus bringing about the cyclopean condition. According to Lewis, the optic primordia are brought together through the removal of a small amount of tissue which normally lies between them. The primordia then unite and produce all degrees of the cyclopean condition. For practical purposes either theory will suffice to explain the condition as found in man, and there is at present no evidence which can decide which of the two is correct, for I may add that (as Dr. Lewis informs me) the optic primordia arise very close to the ventral midline of the brain, being separated by only a few cells. Stockard has recently attempted to define more accurately the eye primordia in Amblystoma by operating upon the medullary plate. First of all, he found that pricking the medullary plate, as Lewis pricked the germ-shield in Fundulus, had no effect whatever upon the growth of the eyes. They invariably grew in a normal way. He then removed various parts of the medullary plate and found that the removal of a median strip about one-fourth to one-third the width of the medullary plate resulted in eyeless embryos. The entire eye primordium apparently lies within this median strip. When a narrower strip was removed the embryos de- veloped with one eye, with defective eyes, or with no eyes at all. From these experiments he concludes that the primordia of Amblystoma arise in the antero- median portion of the medullary plate, and not from two independent primordia, as is believed by Lewis. It may be added that the earlier papers of Lewis and of Stockard were written partly to demonstrate that cyclopia is not an hereditary but an acquired quality. This opinion is much at variance with that of Wilder, who upholds the hereditary theory. In this relation may be stated that there are two records of cyclopia in twins. One, by Ellis, is referred to by Ashfeld on page 283 and is also illustrated in figures 11 and 12, plate 47, of his Atlas. The other is by Van Duyse, and is referred to by Schwalbe and Josephy on page 210. The Van Duyse case is inter- 14 CYCLOPIA IN THE HUMAN EMBRYO. esting, as both parents and grandparents were perfectly healthy and monsters were not known to have occurred in the family. The mother had been pregnant eight times, four of the pregnancies ending in abortions. The first child had harelip, the second had cleft palate, the third was normal, and the fourth pregnancy resulted in the eyelopean twins. In my own experience I can report an even more remarkable case. In 1900 a pig uterus was brought to me which contained a number of normal embryos and three cyclopean embryos, all of about the same stage of development. The cyclopean pigs measured about 40 mm. in length, and each of them had a marked depression in the front of the head and a single pig- mented eye with a snout over it. Unfortunately I did not keep the uterus of this specimen, so that it was impossible for us to examine it with care. The somewhat lengthy discussion on the differentiation of the eye from the medullary plate is justifiable, because at least one of the specimens I have to report is practically a perfect one, which enables me to discuss the origin of the cyclopean eye in a somewhat connected way, from the condition found in normal embryos. After a description of this specimen I purpose to compare the eyes and brain with the same structures in several younger embryos in the Carnegie collection, as well as with those found in the literature. I shall begin with the smallest speci- men to be described, namely, No. 559. Empryo, CR 6.5, NoRMAL IN Form, witH CycLopiA, CARNEGIE CoLLectTion, No. 559. This interesting specimen was sent to me by Dr. Merrill, of Stillwater, Minne- sota, on December 21, 1911. Dr. Merrill writes that the specimen came from a white American, who is the mother of one child, 9 years old. The patient gives a subsequent history of three of four abortions which took place early in preg- nancy. The last menstrual period before the present abortion occurred on October 27, 1911. The abortion followed on December 20. No particulars are obtainable to account for the abortion and there is no evidence of its having been produced by mechanical means. The patient has a history of irregular men- struation and has been treated for metritis and endometritis. It is impossible to obtain any history of venereal disease. The unopened ovum, measuring 20 by 15 by 12, came to us fixed in formalin. It is almost entirely covered with villi which branch three of four times and are about 3 mm. long. On one side there is a small area without villi, covered only by the transparent chorionic membrane. Through this ean be seen a well-formed embryo, apparently normal, measuring about 8 or 9 mm. in length and filling about one-half of the ovum. The remaining half is filled with dense reticular magma. The umbilical vesicle is spherical, measuring about 3.5 mm. in diameter. The appearance of the ovum before and after opening is shown on plate 2, figures 2 and 5. The embryo was removed by cutting the umbilical cord near its attach- ment to the chorion. Photographs were then made at both sides of the embryo, at one diameter enlargement, care being taken to get the exact profile pictured. Numerous other photographs were taken, and it then became apparent that we were dealing with an embryo with a very curious deformity of the head. We CYCLOPIA IN THE HUMAN EMBRYO. 15 also made profile outlines of the two sides of the specimen, being careful to have them in geometrical projection. The branchial region of the two sides of the head were then very carefully drawn. These drawings are reproduced in plate 1, figures 2 and 4. The specimen was dehydrated by placing the entire specimen in several grades of alcohol. It was opened a year later, January 1913, at which time the photographs were made. The specimens were prepared for embedding in February 1914. At this time the embryo measured in absolute alcohol GL 8.6 mm., CR 6.5mm. In xylol the GL measurement was reduced to 8.2 mm. It remained in several changes of xylol for 30 minutes and then was rapidly transferred through several dishes of paraffin, the entire time of this operation being one hour. The aim was to embed the specimen as quickly as possible in order to avoid any undue shrinkage. It was then cut into transverse sections 204 thick. These were stained upon glass slides in hematoxylin and Congo red. It was now found that we had an unusually good series of practically prefect sections, none of them being distorted. Many cell divisions were found in the tissues of different parts of the embryo. We now readily realized the condition of the head, as we found in the sections a perfect series of an exquisite cyclopean embryo. A month later the half of the chorion which was removed to expose the embryo was also cut into serial sections 20 thick and stained with hematoxylin and eosin. Further exam- ination of the part of the chorion from which the embryo was peeled showed the amnion close to the chorionic wall, at the point where the cord had been cut. At the point of juncture there is no complete cord, but in its place are numerous single blood-vessels, making the chorionic attachment of the cord appear like the hairs of a camel’s-hair brush. This part of the chorion was now stained in toto with alum cochineal and cut into serial sections, in order to determine the exact nature of the tissue of the cord as it spreads out into the chorion; for the anomaly here seen had not been encountered by us before. A superficial survey of the villi of the chorion shows it to be apparently normal, but the interior of the ovum, on account of the great amount of magma encircling the embryo, indicates that the ovum is pathological. Furthermore, the chorion is much too small for a normal embryo of this size. As a rule, pathological embryos are contained in ova which are larger than normal. The attachment of the cord to the chorion is also anomalous and a superficial glance at the head of the embryo shows that it is atrophic. The whole front of the head is occupied by the mid- brain. There is no lateral bulging to correspond with the cerebral vesicle. The small pigmented eyes are buried deeply in the head and there is a pronounced frontal process in front of them. On the right side of the head, just in front of the eye, was a small protruding villus-like body which subsequently proved to be the snout (plate 1, fig. 2; plate 2, fig. 7; and plate 3, fig. 5). A more detailed description of the anatomy of the eye region will be given in considering the sec- tions of this specimen. In order to interpret the sections properly a plaster-of- paris model of the brain and head was made at a scale of 50 diameters. Later it was found that this model was on too small a scale to include the muscles of the eye, and a second model of the eye region was made at a seale of 100 diameters. 16 CYCLOPIA IN THE HUMAN EMBRYO. The two halves of the chorion having been cut into serial sections, it was possi- ble to ascertain with greater precision the attachment of the umbilical cord as well as the amniotic adhesions spoken of above. It was found that the cord was attached to one half and that there were delicate amniotic adhesions in the other half of the specimen. The attachment of the cord was by means of blood-vessels passing directly from the embryo to the chorion, while the adhesions in the second half were by means of loose strands of mesenchyme cells binding the amnion to the chorion. The tissue of the chorion appears much like that in normal ova. It is of about the same quantity and is possibly a little more fibrous. The mesenchyme of the villa as well infiltrated with embryonal blood-cells, and their trophoblast is quite scanty. At points the villi are stuck together with maternal blood, in which are found islands of syncytial cells, showing that the infiltration of blood took place before the time of the abortion. The mesenchyme of a few of the villi takes on an intense hematoxylin stain, which indicates that it is degenerating. In fact, the cells of the mesenchyme in many of the villi have mostly disintegrated. The reticular magma stains intensely with eosin. Scattered through the dense network composing it are numerous large protoplasmic cells containing nuclei. In certain places the cells accumulate in large masses, forming colonies. No doubt these are the so-called Hofbauer cells, so well described by Essick. The embryo had been cut into a very perfect series of transverse sections, which show that very little unequal shrinkage took place while it was being embedded. Nearly the entire wall of the central nervous system is in apposition with the surrounding mesenchyme. However, there are occasional separations along the dorsal midline in the head region, the most pronounced one being around the midbrain, but this is not marked. The thin roof-plate of the hindbrain has collapsed only to a slight extent. All in all, the preservation, embedding, mount- ing, and staining of this specimen is quite perfect. There are numerous cell divisions in the brain-tube around the central canal as well as in the retina of the eyclopean eye. Furthermore, these cell divisions are found also in the atrophic cerebral vesicle, showing that at the time of the abortion the atrophic cerebral vesicle, as well as the cyclopean eye, was growing actively. The form of the head is well shown in the figures. In order to study the head with greater care a model was made of the external form at a scale of 50 diameters. This model shows the shrinkage of the head while the specimen was being embedded. It was made so that the entire head could be removed from the body in order to give a face view of the embryo, which could not be obtained from the specimen before it was cut into sections (plate 1, fig. 3). The model also shows the mid- brain to be very prominent, the frontal process being pronounced but small. The eyes are shown deep in the head, and the snout protrudes above the mouth from a point immediately below the process and just in front of the eye. The body otherwise appears to be normal in form, and a microscopic survey of the sections shows that the tissues are also normal in structure. CYCLOPIA IN THE HUMAN EMBRYO. 17 The lateral view of the head as given in the illustrations on plate 1 may be compared with a normal embryo of the same size; for this purpose I will take the Huber embryo No. 3, pictured by Streeter in figure 86, Volume II, of the Manual of Human Embryology. In the Huber specimen the floor of the forebrain is un- usually large, as may be observed by comparing the above-named figure (86) with Streeter’s figure 83, which is taken from His’s embryo Br;. This region is also somewhat smaller in the Huber embryo than in a model of the brain of our No. 163 made by Dr. Lewis. No. 163 is 9 mm. long, and a profile drawing of its brain is shown in figure 428 in the second volume of the Manual. Careful com- parison of the model of the brain of the cyclopean embryo No. 559 with models of the brain of normal embryos shows clearly that there is a decided ventral median defect of the brain of this embryo, from the mammillary bodies to the front end of the brain. This defect naturally takes away the tissues between the éyes and between the cerebral vesicles. In other words, the floor-plate, as shown in figure 55 in the Manual, reaching from the mammillary bodies to the neuropore, has been cut out. In the cyclopean specimen the hypophysis is also absent. The eye- stems have been taken out, the olfactory lobes are absent, and the brain is reduced to a single vesicle, as is the case in older specimens of cyclopia. Such an extreme destruction of the base of the brain rarely occurs in cyclopean Fundulus embryos, as in this animal, according to Stockard and Lewis, the brain is frequently entirely normal, the eyes alone being deformed; but in man marked brain defects have always been found to accompany cases of cyclopia. The optic vesicles in No. 559 form an hourglass-shaped body with two lenses, as shown in plate 1, figure 1, and plate 3, figure 4. The tissues are beautifully preserved and apparently normal in structure. The primary chambers of the eyes communicate freely with each other (plate 3, fig. 4), and through a common eye-stalk, which in turn communicates with the ventricle of the brain (plate 3, fig. 2). The tapetum nigrum covers the optic vesicles and crosses the midline on the dorsal side of the eyes—that is, between the eyes and the common cerebral vesicle. The tapetum stops abruptly at the optic stem and passes down slightly along the anterior wall which joins the two retinas. The choroidal fissures reach clear through the front of the eyes, running almost together as they approach the common eye-stem, as shown in plate 1, figure 1. The topography of the optic stem, ganglion layer of the retina, and tapetum is in the order given, starting from the midline. in the normal embryo, as is probably the relation of their primor- dia in the normal neural plate, judging by the work of Eycleshymer and of Lewis. Eycleshymer showed that very early in development the eye appears, in amphibia, as two pigmented spots lying quite close to the midline in the anterior end of the medullary plate. If these groups of pigmented cells are destined to form the . tapetum, then the ganglion layer of the retinas would form nearer the midline, while the cells which cross the midline would probably form the optic stem. That Eycleshymer’s view is correct is indicated by the work of Locy in his studies upon the shark and by Keibel in his studies upon the pig. According to these authors, the eye primordia arise from small depressions near the midline of the 18 CYCLOPIA IN THE HUMAN EMBRYO. anterior end of the medullary plate. According to Lewis, certain groups of cells in the medullary plate are predetermined to form the tapetum, the retina, and the optic stem. Lewis’s theory has been objected to by Bell, but it has been amply confirmed by Spemann. At any rate, the arrangement of the structures in our cyclopean embryo indicates that the optic stems have been cut out and that the primordia of the retina and tapetum of the two sides have united, blending abso- lutely with each other across the midventral line. The tissues of the midbrain and most of those of the interbrain appear to be normal, but our knowledge of the normal brain at this period of development is so scanty that it is dangerous to make any defi- nite statement. Single groups of cells may be wanting or may be blending without our noticing the change. Such a blending is clear only when it involves a sharply cireumscribed structure like the eye. However, the tissues of the hypothalamus seem to be disarranged (plate 3, fig. 2), and those of the single united cerebral vesicle (plate 3, fig. 5) are certainly dissociated. In the cerebral vesicle the cells form a uniform layer, which is not beautifully stratified, as is the case in normal development. Over the most anterior part of the brain (plate 3, fig. 5), cross- ing the midline, is a crescent- shaped cap covering the outside : of the brain and reaching back Fia. 1.— Semi diagrananttiae section through the SA tarbeain and cyclopean to the hypothalamus just below eye of embryo No. $9, X60. The guniel nave are masked mitt the point of attachment of the mouth. common optic stem (plate 3, fig. 3). This cap is composed of pale cells of uniform size, undoubtedly belonging to the neural tube. It is located on the part of the brain which gives rise to the olfactory lobes in normal development; and it may represent these lobes in a degenerated form. It was necessary to make a model (enlarged 100 diameters) of the eye region in order to study carefully the anatomy of the structures of the orbit. In this model the nerves, from the third to the seventh, were worked out to their termina- tions. The muscle masses, as far as they could be determined, are also included in this model. CYCLOPIA IN THE HUMAN EMBRYO. 19 The first branch of the fifth nerve is much thinner than the second and third, and passes directly from the Gasserian ganglion back of the eye on either side of the optic stalk; the branches anastomose with each other through several delicate filaments back of the eyes, and then a larger bundle and several very small ones enter the snout, to be lost there. The nerves are shown in section in the figures on plate 3 and in reconstruction in plate 1 and figure 1. The fourth nerve takes its usual route and finally ends in a very large pre- muscle mass lateral to the eye and to the first branch of the fifth nerve. It may be noted that this arrangement of the fourth and first branches of the fifth appears to be the reverse of the normal distribution according to Lewis’s reconstruction of our embryo No. 163 (fig. 368 of the Manual). In the Lewis reconstruction all of the muscle of the primordium of the eye is blended into a single muscle mass, while in the eyclopean embryo the premuscle mass of the superior oblique muscle is entirely separated from the remaining premuscle mass of the orbit. The sixth nerve takes its usual course and ends independently in the hour- glass-shaped premuscle mass which crosses the midline between the cyclopean eye and the hindbrain (fig. 1). It is interesting to know that the transverse median muscle mass, as well as the median anastomosis of the third nerve, occurs at the point through which the pharynx gives rise to the hypophysis. In this embryo the chorda ends in the pharynx behind this muscle mass and the pontine flexure of the hindbrain. It appears as though, on account of the great amount of kink- ing, the region of the infundibulum of the interbrain were pushed away from the pharynx, thus making it impossible for the hypophysis to reach it. As a result of this the third nerve and its muscle masses cross the midline. It may be that the curious cytological changes in the muscle masses (plate 3, fig. 1, Oc.) indicate that destructive changes are taking place in them, and that these small round nuclei correspond with the Hofbauer cells as described by Essick in his studies of the transitory cavities in the corpus striatum of the human embryo. The primordium of the eye-muscles show some very remarkable cytological changes. As the sixth nerve approaches the muscle mass of the lateral rectus it is at once observed that this muscle falls into two sharply defined groups of cells, namely, a median group which appears to be normal, and a lateral mass of smaller round cells, the nuclei of which stain intensely. The same grouping is present in the muscle mass of the third nerve. Near the midline the cells appear to be normal, and laterally they are again composed of small round cells. The premuscle mass at the end of the fourth nerve—that is, the superior oblique muscle—can be outlined only with difficulty. The third nerve shows most remarkable changes in this specimen. It passes along its usual course until it reaches the common eye-stem, over which it circles, for the nerves from the two sides anastomose here, without any diminution in size, within the common median primordium of the eye-muscle (fig. 1). This pro- nounced anastomosis is also found in another cyclopean embryo, No. 201, in our collection, as shown in figure 3. 20 CYCLOPIA IN THE HUMAN EMBRYO. Wilder pictures and describes a eyclopean pig (Wilder’s fig. 1, plate 2) in which the third nerve arises as a pair in the usual way, which unites after passing through the superior orbital fissure. The union is in the neighborhood of the orbital muscles. He also described the orbit of the eyclopean eye in a large double- headed fetus. By dissection of the head he found that the two third nerves, one coming from each brain, unite with each other and form a common trunk stretched transversely across the midline back of the eyeball. From this anastomosis small twigs arise to supply the muscles of the eyes (Wilder’s fig. 6, plate 3). In his work on monsters (page 282), Ahlfeld states that Delle Chiaie described a specimen having a similar anomaly, which was published in Naples in 1840. Delle Chiaie gives an excellent illustration of this specimen, with a diagrammatic section of the head, showing the eye and its attachments. This picture is copied by Ahlfeld on plate 46, figure 18. It is somewhat difficult to identify all the structures given, but he apparently pictures the two third nerves anastomosing before they reach the single eye. He also pictures a branch of the fifth nerve passing into the snout, which, as in our specimen, contains a cavity. I have been able to find one more specimen in the literature in which there is an anastomosis of the two third nerves within the orbit in cyclopia. This is in the excellent description of Dr. Black on the nervous system of a eyclopean specimen at birth. Black alludes to the third nerve in a single sentence. He says (on page 204) that in the region of the central tendon the third nerve divides into a number of small branches, each of which com- municates with its fellow on the opposite side. I have given these references, as they are the only ones which I can find in the literature, and they invariably accompany the description of the orbit in cyclopia. The anatomy of cyclopia in monsters is rarely given; and it may be remarked that until we have numerous good descriptions (like that of Black) of the eyes, central nerves, and face in cyclopia, we shall not understand fully the anatomy of this most interesting type of specimen. In a Janus monster (No. 1178, a and 6b) at birth with cyclopia on one side, Dr. Theodora Finney has demonstrated a large anastomosis behind the orbital branches of the third nerve before they are distributed to the eye-muscles. An account of her specimen with a figure is given on page 30. It remains to attempt to correlate what has been said above regarding cyclopia with the form of the brain and the optie vesicle in early human embryos. Before undertaking this attention is called to two papers by Tandler on the form of the early brain in Tarsius and in Platydactylus. Tandler’s papers are especially noteworthy for the reason that the topography of the forebrain has been deter- mined with a greater precision than has been carried out in the human embryo, except possibly in the most recent work of His. There is sufficient material at hand to make similar studies upon the human brain, but until this is done I must content myself with what has already been published, alluding occasionally to several of the specimens in our own collection. A specimen 3.2 mm. long, with 13 or 14 myotomes, was described with much care by His in his last publication upon the brain. He has illustrated this speci- men by a sagittal median section through the body as well as by the external and CYCLOPIA IN THE HUMAN EMBRYO. 21 internal forms of the brain. These illustrations are given in the His monograph as figures 2 and 33, and they are also copied by Streeter in his article on the brain in the Manual. It may be stated that figure 33 shows the neuropore nearly closed, the optic stalk being still represented as a wide-open canal which reaches to the midventral line. Dorsal to the optic stem is a slightly marked pocket which reaches to the neuropore, and it is believed by His that this represents the beginning of the cerebral hemisphere. It is interesting to note that Keibel and Else state in their Normal Plates (page 100) that the cerebral vesicles are just beginning in embryos 4.5 to 5 mm. long. It may be that His exaggerates this pocket slightly in his model, but it is of great value to us to have his opinion regard- ing the location of the primordia of the cerebral hemispheres in the brain-tube before the neuropore is closed. According to Watt, the cerebral hemispheres arise much more dorsal than is indicated by His in his figures. Keibel and Else give excellent illustrations of the form of the brain up to the time of the closure of the neural tube. No doubt the Kroemer-Pfannenstiel embryo, which contains five or six myotomes, represents a normal stage with medullary plates wide open, and in this embryo there is no indication whatever of an eye primordium. The same is true in the Kollmann embryo, containing 14 myotomes, which is illustrated by Keibel and Else as figure 4. This specimen also seems to me to represent a normal embryo, as we have in our collection a stage that is practically identical with it. Our embryo No. 391 contains seven pairs of somites, and has been carefully studied and published by Dandy. A model of it, as well as sections through the head, has also been pictured by Evans in figures 408 and 409, Volume II, of the Manual, and it shows no out-pocketings in the anterior part of the brain to represent the eye primordia. However, when we reach Keibel and Else’s figure 5, which is taken from the Pfannenstiel III embryo, two marked diverticula are seen to arise from the front end of the neural tube, which Keibel, in his article on the eye in the Manual of Embryology, believes to represent the primordia of the eye. As Keibel and Else have reproduced numerous figures of sections through the head of this embryo, it is easy to ascertain the exact form of its neural tube. However, I am of the opinion that we can hardly view the neural tube in this embryo as normal, as it is not sufficiently advanced in development for an embryo of this stage and as it corresponds very much in form with the brain of our embryo No. 12, which I believe to be pathological. At this time the neural tube should be nearly closed, while in Keibel and Else’s specimen and in our No. 12 it is still wide open. The Pfannenstiel III embryo contains the same number of somites— that is, 14—as our embryo No. 12, and the form of the brain and of the eye-vesicles is very similar in both embryos. A picture of the external form of embryo No. 12 will be found in my article on the development of the intestines (plate 19, fig. 2). My reason for believing that the brain form in both of these embryos can not be viewed as normal is that in other young specimens published recently by Wallin and by Bremer quite a different form of the brain-tube is shown for this stage of development. The Wallin specimen, which contains 13 somites, has the brain- 22 CYCLOPIA IN THE HUMAN EMBRYO. “~ tube pretty well closed up, leaving a large neuropore in the front. The Bremer specimen is slightly in advance of this. We have also in our collection a similar embryo (No. 470) in which the brain form corresponds exactly with that in the specimens of Wallin and of Bremer. Furthermore, the twin specimens of Watt, which contain 17 or 18 somites, correspond very closely with the three above-named embryos. In the Watt specimen the neuropore is nearly closed and the eye-vesicles reach all the way across the front of the brain-tube. The Pfannenstiel III embryo and the Bremer embryo are found pictured by Bach and Seefelder, both in profile and in sections, but I do not think we should unreservedly accept their description of the begin- ning of the eye-vesicles as final for the human embryo. It seems to me that our knowledge of the form of the forebrain before closure of the neuropore is much in need of revision, and towards this revision we have assembled several new models of young embryos. The first is a model by Dr. Bartelmez of our No. 1201 (University of Chicago, No. 87) from an embryo with 8 pairs of somites, which seems to me to bear very much upon this question. The neural plate flanges out into a large tongue with a slightly hourglass-shaped de- pression running across the midventral line (plate 2, fig. 6.) The larger lateral depressions no doubt indicate the foveola, and the groove connecting them across the midline is in the position in which the optic stalk develops later on. It would probably be better if we accepted Froriep’s designation of torus opticus for this connection. The torus opticus seems very insignificant in this specimen, as shown in the illustration (¢. 0.), but when we consider to what extent the torus may be stretched, as illustrated by Bach and Seefelder (fig. 2, plate 13), we recog- nize the importance of this structure. I am inclined to believe that the form of the brain in the Bartelmez embryo must be viewed as normal, as it corresponds so well in a series with that of several older human embryos, namely, those recently described by Wallin and by Bremer and our No. 470. There is another ridge already indicated in the brain-tube of His’s speci- men EB reaching across the midline just below the neuropore. This is the torus transversus of Kupffer; and those who are interested in this structure are referred for greater details to the articles by Tandler, Kupffer, and Johnston. The neuropore is found just closed in our embryos No. 148, published by Mrs. Gage, and No. 836, which has been modeled by Dr. Evans, as well as a specimen of the same size modeled by Johnston. Dr. Johnston has been good enough to send me photographs of his model, so that I am able to compare it with our own. It is clear to me that the large “optic vesicle,” extending over the whole lateral wall of the front part of the neural tube, represents more than the optic vesicle, as it must also include the primordium of the cerebral hemispheres, since the torus transversus touches the lower border of the neuropore and the optic vesicles fall below this line. This large so-called optic vesicle must resolve itself into the optic vesicle and brain hemisphere in subsequent development. In this process the torus opticus gradually must become more pronounced. CYCLOPIA IN THE HUMAN EMBRYO. 23 It is somewhat easy to compare the medullary plate of the Bartelmez embryo No. 1201 with the medullary plate in amphibia. The lateral foveola corresponds with Eycleshymer’s pigmented spots, and if these areas were cut out eyeless embryos should be produced, as Stockard found in his experiments on Amblystoma. If it is admitted that the eye primordia communicate across the midline through the torus opticus, then Stockard’s experiments upon the medullary plate may be interpreted as follows: No important organ develops from the midline of the medullary plate, and this is represented only by a thin layer of cells. It has been called by His the floor-plate. The motor nuclei arise from the thickenings on either side of the floor-plate, and these are known as the basal plates. The narrow, thin floor- plate really forms the ventral midseptum of the spinal cord of the brain, and sub- sequently commissural fibers grow through it to form the raphe. If we view the basal plate from above, as shown in embryo No. 1201 (plate 2, fig. 6), we find that this raphe should extend forward to the neuropore, at which point the raphe fibers are the anterior commissure within the torus transversus. Back of this we have the torus opticus, and its commissural fibers are the fibers of the optic nerve. At an early period in its development the torus opticus must widen rapidly and push through the rest of the brain, for the optic stalk appears quite suddenly, and an injury to the medullary plate at this time would probably make itself felt more upon the optic stalk than upon the eye, for the short period in which it grows very rapidly is its critical period. If we cut out the optic stalk or the torus opticus, as Stockard and Lewis did in their experiments, then the foveola would remain together and form cyclopia. Stockard and Lewis also note that in their experi- ments they frequently obtained embryos with but one eye which appeared to be quite normal. This is to be expected when experiments are made upon two prim- ordia which he very close together; and when either the left or right eye primor- dium is removed, left or right-eyed embryos are produced, but not cyclopia. Lewis had to destroy only the midline of the embryonic shield in order to produce true cyclopia. It may be added that the anatomical changes found in our small cyclopean human embryo, as well as in all cyclopean human monsters, can be explained by removal of the structures represented along the line of the raphe of the meduilary plate reaching from the mammillary bodies to the neuropore. This includes the torus transversus, which naturally involves the olfactory region and the anterior commissure. Thus we can explain by a study of this specimen the anatomical changes of the brain found in human eyelopia. StunteD Empryo, GL 20, wirH Cyciopra, CARNEGIE CoLLecTion, No. 201. The second specimen of cyclopia in our collection is a pathological embryo, which is not very well preserved, measuring GL 20 mm. This specimen came from Dr. Schelly of Baltimore, who obtained it from an abortion on February 7, 1902. He brought it to Mr. Brédel, and subsequently, because it was patholog- ical, it was given to me. The embryo was incased in an ovum covered with a ragged decidua, together measuring 80 by 60 by 50 mm. Upon opening this 24 CYCLOPIA IN THE HUMAN EMBRYO. ovum it was found filled with a jelly-like fluid forming a type of magma described in my paper upon this subject. Sections were cut of the fleshy chorion and it was found that the wall is composed of a true chorionic membrane, villi, maternal blood, fibrin, decidua, blood sinuses, and trophoblast, with an extensive infiltration of leucocytes often accumulating in large masses or small abscesses. These layers are not in any regular order, but are intermingled, and show various stages of disintegration. The mesenchyme of the villi is fibrous, and many of these are invaded by trophoblast cells as well as by leucocytes. The trophoblast also invades the blood-clot, and maternal-blood sinuses are frequently found filled either with trophoblast cells or with leucocytes. There are certain groups of tissue in which the intermingling of the trophoblast cells within the fibrinoid substance appears, in sections, very much like earti- lage. Most of the decidua ad- jacent to active trophoblast on the tips of some of the villi has the usual fibrinoid layer char- acteristic of normal develop- ment. It may be added that the structures of the chorionic membrane, as well as those of the embryo, stain unusually well, which indicates that the tissues were active and alive at the time of the abortion. After the embryo was re- ceived it was photographed from one side (plate 2, fig. 1), and this record proved to be very useful In making several Fic. 2.—Reconstruction of the head of embryo No. 201. X25. The , 1 outline of the head was obtained from a photograph, the brain and eyes reconstructions. The embryo from a plaster reconstruction, and the nerves were added from the sec- was then eut into serial sections tions. C. H., cerebral hemisphere; M, midbrain; H, hindbrain; S, rudi- has ; . mentary snout; V, fifth nerve; V/JJJ, eighth nerve; F, external ear. 50 mm. thick; finally, in order to study more carefully the structures within the head, a reconstruction of its external form, as well as of the form of the brain and the eye, was made in plaster of paris at a scale of 40 diameters. Most extensive changes have taken place within the embryo. The brain is greatly deformed and is severed from the spinal cord through a growth of tissue in the region of the medulla back of the deformed ear. In fact, a part of the brain is included within the cap-like body on top of the head. The spinal cord begins again quite abruptly in the upper cervical region and ends with the same abruptness in the upper lumbar region. At its lower end there is a curious fibrous tumor measuring half the diameter of the cord. The cord, so far as it is developed, bo on CYCLOPIA IN THE HUMAN EMBRYO. appears to be normal, but it is markedly dissociated. Below the upper lumbar region the spinal cord is wholly wanting and the spinal canal is filled with meso- dermal tissue which is rich in blood-vessels. Where the cord is missing most of the spinal nerves still remain, and many dorsal ganglia can be made out, show- ing that the changes in the central nervous system in this region took place after the spinal nerves had been developed from it. Most of the epidermis is intact, but it is broken through at the back of the head, where there is found an extensive ulcer or necrotic mass, which is very rich in blood-vessels and involves the walls of the brain but does not reach into its ventricle. At the highest point of the head the epidermis has developed into a papilliform body, and below this there is a large necrotic area in which there is a ereat quantity of yellow pigment granules. The mouth is closed completely, although the alimentary canal, from the mouth to the stomach, is open and appears normal. The intestine is matted together; the cloaca and anus are obliterated. The epithelium of the upper portion of the intestine gives rise to marked growths into the matted mass. The thoracic region, liver, and vascular system have undergone practically no change. The extensive dissociation of the tissues throughout the embryo has caused an exten- sive destruction and arrest in the develop- ment of the muscular system. This is marked by all kinds of secondary changes in the con- nective tissue, especially in that of the skin, where the change is pronounced, as may be ri. 3.—Diagrammatic reconstruction of the eye and seen in plate 2, figure 4. In this region the Th. uvial‘nerves are marked with Roman numerals: change is so great that it obliterates entirely [ft rancho Ath age, § ar the external auditory canal. A reconstruction of the brain and upper part of the spinal canal enabled me to determine with greater precision the parts of the brain which are within the head of this embryo. Subsequently it was possible to find the remnants of the ganglion of the eighth nerve; then that of the fifth nerve was determined. Their position is shown in the profile diagram of the outlines of the brain and these nerves (fig. 2). It at once becomes apparent that the portion of the brain extend- ing up in the necrotic cap on the top of the head consists of the midbrain and hindbrain. With this idea in mind it was possible to answer the question in the serial sections, as the walls of the midbrain are thick and the ventricle relatively small, and the walls of the hindbrain are thin and its ventricle relative large. The pointed process which reaches towards the ganglion of the fifth therefore represents the pontine flexure of the medulla. It is now easy to interpret the 26 CYCLOPIA IN THE HUMAN EMBRYO. isolated brain-mass between the eye and the large mass just described. By com- paring these structures with a profile reconstruction of the forebrain of the cyclo- pean embryo No. 559 (plate 1, fig. 1), the larger mass just above the ganglion of the fifth undoubtedly represents the interbrain, as the free end of the optic nerve _ reaches just to its base but does not enter it. The crescent- shaped mass of the brain in front of the interbrain is the unpaired single cerebral vesicle, which communicates freely with the ventricle of the interbrain. The structure of the wall of this degenerated cerebral vesicle cor- | responds very closely with that of the small vesicle in embryo No. 559. With these structures established, as shown in the Fic. 4.—Section through the cyclopean eye of embryo No. 201. X40. profile reconstruction, it is pos- V', first branch of fifth nerve; S, snout. sible to identify some of the re- maining structures of the orbit. However, the tissues are very greatly dissociated, and for the present it is impossible for me to follow them farther than is given in the following description. First of all, the three branches of the fifth nerve can be fol- lowed to their termination. The first branch reaches up to the rudimentary snout, where it anastomoses with F its fellow from the opposite C= side—shown also in the dia- , gram (fig. 3). This snout, by the way, is represented by a slight elevation in the middle of the face above the eye, and sections through it show that it is composed of a relatively large, irregular mass of cells which are sepa- Iie. 5.—Section eciek the cyclopean eye and optic nerve of rated more or less from the embryo No. 201. X40. surrounding mesenchyme. Within the middle of this mass is a small pearl-like body about 0.1 mm. in diameter. Just behind this body the first branch of the fifth nerve anastomoses across the midline. The second branch of the fifth runs below the eye on either side and reaches nearly to the skin, where it spreads out into several large branches. The third CYCLOPIA IN THE HUMAN EMBRYO. rat branch of the fifth nerve runs deep into the neck and is separated from the second by a pronounced ossification center representing, of course, the maxillary bone. The two eyes are united, forming an hourglass-shaped body with a double retina, a single pigmented layer, and a single optic nerve which arises from the retinas as they approach each other (figs. 4 and 5). The tapetum is continuous over the superior surface of the eyes, but it is broken below, repeating the condition found in the eye of embryo No. 559. The optic nerve reaches to the base of the interbrain, where it ends abruptly. It is impossible to determine with precision the arrangement of the muscle masses of the orbit or of the nerves passing to them. That no trace of the sixth nerve could be made out is shown in figure 3, and this may be accounted for by the fact that the organ which gives rise to the sixth nerve has undergone extensive degeneration. However, the peripheral ends of the third and fourth nerves can be found, but they can not be traced back very far in the direction of their origin. The fourth nerve is thicker than normal and ends on the lateral side in an enlargement which may represent the superior oblique muscle. Below the optic nerve is a common muscle mass which crosses the midline, and to either side of this there are two independent muscle masses. Before the third nerve reaches these muscle masses lateral branches are given off, which pass to the second lateral muscle mass, as shown in the diagram. The two nerves then approach each other and communicate freely through the unpaired muscle mass, and then pass forward under the cyclopean eye and finally end just beneath the skin. A comparison of plate 1, figure 1, and text-figure 1 with text-figure 3 shows that the first branch of the fifth nerve in both embryos anastomoses across the midventral line in the region of the snout and that the two third nerves anastomose with each other through the main muscle primordium of the orbit. Both of these anastomoses must be viewed as secondary, for the two nerves must have been single when they arose from the brain. This observation favors the theory that the eye promordia must also have been bilateral—that is, they must have been separated by a narrow strip of non-ocular tissue in the normal medullary plate. Fetus CoMPRESSUS WITH CycLopIA, CARNEGIE CoLLEcTION No. 1165. This embryo was sent to me by Dr. Ralph 8. Perkins, of Exeter, New Hamp- shire. Only the embryo was received, which measures 43 mm. CR. It was found to be greatly distorted; the umbilical cord is of thread-like thinness, and the development of the different parts of the body seems to be unequal. Appar- ently some of the jomts are dislocated, but at present it is impossible to say whether or not these distortions are due to mechanical manipulation after the embryo was.aborted. This is possible, because the embryo had been wrapped in a towel some time before it was fixed in formalin, but a careful study of the sections demonstrates that the specimen is quite a typical fetus compressus. The embryo came from a white woman, 35 years old. Her first child is 16 years of age; the second died at the age of 2, and the third is 12 years old. These are all by her first marriage. The first pregnancy of her second marriage 28 CYCLOPIA IN THE HUMAN EMBRYO. ended in an abortion at 5 months, and then she gave birth to a child which is now 21 months old. The next pregnancy resulted in an abortion at 5 months, and the last one gave the specimen under consideration. Her last normal menstrual period began on February 25, 1915. The next period began on March 21 and continued for only one day; and this was followed by the abortion on May 2. There are no other data bearing upon this case except that 15 years ago the woman had an operation for suspension of the uterus. Upon careful inspection of the head of the specimen a mechanical injury just below the lower jaw was found, as shown in figure 6. The ear seems to be dis- torted or abnormal, and in place of the nose and eyes there is a depression in front of the face, and running from it is a cleft reaching to the mouth. Appar- ently we have here a fetus com- pressus with cyclopia and hare- lip. The head of the embryo was stained in toto in cochineal and embedded in paraffin. It was cut into serial sections 50 thick. The sections show that all the tissues are markedly dis- sociated, and in addition the brain is completely macerated. In fact, the brain-cavity appears like a bag filled with débris, which reaches down into the cervical region of the neck and terminates abruptly where the spinal canal is filled with a new formation of fibroustissue. The ; « A Fic. 6.—Direct drawing of the head of embryo No. 1165. 4.5. The primordial skull 1S composed of tissue of the lower jaw is injured. The depression from the eyclopean eye extends down into the mouth, forming hare-lip. vartilages which have under- gone some fibrous changes, and their borders are not sharply defined, but grade over into the surrounding connective tissue. The cartilages at the base of the skull appear to be enlarged and extended; but this point can not be established without making an elaborate reconstruction. In the cervical region the bodies of the vertebrae are displaced backward into the spinal canal, which in turn is largely filled up with the newly formed fibrous tissue as well as with numerous round cells. The tissues of the various ossification centers have undergone a curious change, reminding one of necrosis. It appears as though the ossification centers had died while the surrounding cartilage had continued growing. It is difficult to define precisely the muscles and nerves in all of the various sections, while at points certain muscle groups seem to retain their normal form. CYCLOPIA IN THE HUMAN EMBRYO. 29 Most of the epidermis is wanting and in the region of the face are large skin protuberances composed principally of round cells. Such protuberances form the lids of the cyclopean eye, as shown in plate 2, figure 3. The orbital cavity lies upon the cribriform primordia of the maxillary bones and is filled with a single group of pigmented cells, which is surrounded by an infiltration of round cells. Back of this pigmented mass are the primordia of the eye-muscles, but their dis- sociation is so complete that it is impossible to locate the individual muscles, nor can any of the nerves be made out with precision. Aside from the pigmented mass there are no remnants of the layers of the retina, these having undergone complete dissociation. In the upper part of the orbital mass is a curious gland- like structure badly dissociated, which may represent the lacrymal gland. We have, therefore, in this specimen the remnant of a single median eye represented by an irregular but rounded mass of the tapetum situated below the depression of the skin. In turn this depression is partly covered with folds of dissociated tissue which may be recognized as the eyelids of the eyclopean eye. CEPHALOTHORACOPAGUS MONOSYMMETROS WITH CYCLOPIA ON ONE SIDE, CARNEGIE CoLtecTion No. 1178 a anp b. The double female monster, 205 CR and 350 GL long, weighing 1,624 grams was sent to us by Dr. J. I. Butler, Rodgers Hospital, Tucson, Arizona, on May 14, 1915. The mother is a Chinese woman, age 24, who has given birth to three children at term and has had two abortions. Apparently the uterus is normal and there is no history of venereal diseases. There is nothing else in the history that bears upon this case. The specimen has been completely dissected by Dr. Theodora Finney, who has given me the notes for the following description of the muscles of the orbit and the nerves of the cyclopean eye. A more detailed account of the anatomy of this interesting specimen will be published by Dr. Finney at some subsequent date. The fetus is composed of two nearly complete bodies which lie with their anterior surfaces toward each other, and, as the name implies, are fused from the umbilicus up, forming one thoracic trunk and one head. There are two inde- pendent spinal columns, eight extremities, and two composite fronts, every sym- metrical part of which is formed half of one and half of the other individual. There are also two faces, one of which is well formed, while the other is a synote with a eyclopean eye and snout situated above it. In dealing with the cyclops, then, it must be noted that its left half is formed from the left side of one indi- vidual, while its right half is from the right side of the other individual. Internally the thoracic and abdominal viscera are double, with the exception of the esophagus, the stomach, and the upper part of the intestine, which are united with a single canal. There are two central nervous systems, separate and complete to above the level of the two hypophysi, where fusion occurs. As much tissue was lost from the region of the thalami in removing the brain, the mode of union of the base of the brain could not be determined. An optic chiasm, how- ever, belonging to the well-formed face, remains in sifu immediately behind the 30 CYCLOPIA IN THE HUMAN EMBRYO. orbits. This shows there is a true normal union for the two individuals at this point. In the cranial cavity behind the eyclopean eye one optic nerve-stalk, com- posed of two bundles pressed together, is observed. The dome of the cranium is filled by three cerebral bodies; two of these are recognizable hemispheres, though much shortened antero-posteriorly. Their position is normal, behind the well-formed face. They possess well-defined but shallow cortical sulci. The third cerebral division consists of a kidney-shaped lobe. Its cortex is smooth, except for two or three atypical creases near the poles. It lies transversely across the cyclopean side of the cranial cavity with its two poles directed infe- riorly, the convex portion between them strad- dling the single orbit. It represents fused cere- bral tissue obtained from both individuals. The cyclops has a well-formed eyeball to which four pairs of muscles are attached; their arrangement is shown in figure 7. These mus- cles can be identified by their nerve-supply as being the muscles of the upper and outer parts of the two fused eyes. These muscles are changed from their normal positions, so that they entirely surround the eye. The muscles are the supe- rior obliques, the levator palpebre, the superior and lateral recti. The two superior obliques lie near to the midline on the superior surface. Slightly lateral to these, though still on the superior surface, lie the two levator palpebre. ¥) it M+ Aik On the sides, in the place usually occupied by 6. 7-—Diagram of eyclopean eye and its append- Sia c= f 2 . ages of the Janus monster, No. 1178 a and b, the lateral recti, lie the superior recti, which are _ fromasketch and dissection.by Dr. Finney. For : : “)° the sake of clearness the superior oblique muscles shifted downward from their normal position are moved forward. The cranial nerves are = a ee ° . marked with Roman numerals. S. ob., superior through an are of 90°. About thesame amount Oblique muscles; L. p., levator palpebre; S. r. of shifting causes the lateral recti to lie close — superior rectus; Z. r., lateral rectus; M, rudi- mentary muscle-mass, probably the remains of together on the inferior surface of the eyeball. the inferior recti. It is noticed that the first ml : ; C F branch of the fifth nerve gives rise to a trunk Che inferior oblique muscles and the medial which anastamoses across the midline. The same recti are completely eliminated. The inferior — ** of the third and sixth nerves. recti are entirely absent at the bulbar end. There is a short bundle of muscles underneath the proximal end of the lateral recti, which probably represents remains of the inferior recti. The lacrymal glands have participated in the change of position and fusion. ‘Their tissues lie as a broad single gland-mass on the inferior surface of the bulb. In order to be sure that the identification of the nerves passing to the muscles of the eyclopean eye was made correctly, they were carefully compared with the normal nerves of the eyes of the well-developed face. This comparison left no doubt as to which nerves were being traced to the single eye, as the points of origin CYCLOPIA IN THE HUMAN EMBRYO. 31 from the brain-stem of the third, fourth, fifth, and sixth nerves were symmetrical for both faces. The arrangement of the nerves on the cyclopean side are as follows: 1. The olfactory nerves are absent. 2. The origin of the optic nerve was lost. Two small and flattened optic nerves, however, pass out together in the dura. These finally fuse into one stalk which ends in the bulb. This stalk, 2 mm. in diameter, is about the same size as the normal optic nerves of the well-formed face on the opposite side. 3. The two third nerves which belong to the cyclops are 0.5 mm. in diameter at their point of origin and throughout their course, while the third nerves on the opposite side which pass to the perfect face are twice that size. The cyclopean oculo-motor nerves pass into the dura, where they run toward each other to the place where the eye-muscles arise. Here these nerves lie within 3 mm. of each other. Branching occurs in this region.. Two of these branches fuse im- mediately. There are two other pairs of main branches which innervate the levator palpebre and the superior recti on each side of the single eye. There are some finer branches whose course could not be definitely ascertained. 4. The cyclopean fourth nerves are equal in size with those of the normal eye. They run as two fine threads to within a few millimeters of each other, when they turn anteriorly and run parallel on the surface of the superior oblique muscles, in which they terminate. 5. The two Gasserian ganglia of the cyclops are somewhat smaller than those of the normal face. Each has three divisions: ophthalmic, maxillary, and man- dibular.. The two ophthalmic divisions have each three main branches. One of these branches passes along the roof of the orbit and makes several y-shaped anastamoses with its fellow near the front of the eyeball. Another runs forward, parallel with its fellow, out into the skin, where they are both cut; so if anastamosis occurred it could not be determined. The third and last branch, one on each side of the eye, ends in the lacrymal gland. 6. The sixth nerves of the cyclops, about 0.8 mm. in diameter, are equal in size with the sixth nerves, passing to the well-developed face. They converge to the base of the orbit when they run parallel to each other on the upper side of the lateral recti muscles, in whose substance they terminate after making several x-shaped anastamoses. 6. ~1 © 10. . AHLFELD, . Bacu, L., and R. SEEFELDER. . DaARESTE, CAMILLE. . Exits, R. Ona rare form of twin monstrosity. BIBLIOGRAPHY. Friepricu. Die Missbildungen des Leipzig, F. W. Grunow. 1880, p. 277. Atlas zur Entwicklungs- geschichte des menschlichen Auges. Leipzig, 1911. Jou. Menschen. . Bett, E. T. Some experiments on the development and regeneration of the eye and the nasal organ in frog embryos. Arch. f. Entwicklungsmechn. d. Organ., Leipz., 1907, xxi, 457-478, 7 pl. . Brack, D. D. The central nervous system in a case of cyclopia in homo. Jour. Compar. Neurol., Phila., 1913, xxi, 193-257. . Bremer, J. L. Description of a 4mm. human embryo. Amer. Jour. Anst., Balt., 1906, v, 459-480. Danpy, W. E. A human embryo with seven pairs of somites measuring about 2 mm. in length. Amer. Jour. Anat., Phila., 1910, x, 85-108, 6 pl. Recherches sur la production arti- ficielle des monstruosités. Paris, C. Reinwald et Cie., 1877. Trans. Obst. Soe. Lond., 1866, vir, 160-164. . Essicx, C. R. Transitory cavities in the corpus stri- atum of the human embryo. Contributions to Embryology, No. 6. Carnegie Inst. Wash. Pub. No. 222, 1915. EyciesHymMer, A. C. The development of the optic vesicles in amphibia. Jour. Morphol., Bost., 1893, vin, 189-194. . Frorrep, A. Die Entwicklung des Auges der Wirbel- tiere. Handbuch d. vergleichenden u. experimen- tellen Entwicklungslehre der Wirbeltiere (O. Hertwig), Jena, 1905, 1, 2 Teil, 139-266. . Gace, Susanna P. A three weeks’ human embryo, with special reference to the brain and nephric system. Amer. Jour. Anat., Balt., 1906, Iv, 409-443, 5 pl. 13. His, W. Die Entwicklung des menschlichen Gehirns. Leipzig, S. Hirzel, 1904. 14. Huscuxe, E. Ueber die erste Entwicklung des Auges . Keret, F., and C. Evce. . Kuprrer. . Lewis, W. H. und die damit Arch. f. Anat. u. Physiol. 1832, v1, 1-47, 1 pl. zusammenhiingende Cyklopie. (Meckel’s), Leipz., . Jounston, J. B. The morphology of the forebrain vesicles in vertebrates. Jour. Compar. Neurol., Phila., 1909, xrx, 457-539. The evolution of the cerebral cortex. Record, Phila., 1910, rv, 143-166. Anat. . Kerset, F. Normentafel zur Entwicklungsgeschichte des Schweines. Jena, G. Fischer, 1897. The development of the sense-organs. In Manual of Human Embryology (Keibel and Mall), Phila. and Lond., 1912, 1, 180-290; also, Handbuch d. Entwicklungsgesch. d. Menschen (Keibel and Mall), Leipz., 9111, 1m, 179-281. Normentafeln zur Entwick- lungsgeschichte der Wirbeltiere, Heft 8. Jena, G. Fischer, 1908. Die Morphologie des Centralnervensystems. Handbuch d. vergleichenden u. experimentellen Entwicklungslehre d. Wirbeltiere (O. Hertwig), Jena, 1903, m1, 2 Teil, 1-272. The experimental production of cyclopia in the fish embryo (Fundulus heteroclitus). Anat. Record, Phila., 1909, m1, 175-181. Experiments on the origin and differentiation of the optic vesicle in amphibia. Amer. Jour. Anat., Balt., 1907, vu, 259-276, 1 pl. The development of the muscular system. In Manual of Human Embryology (Keibel and 32 33. 34. 35. 36. 37. - MALL, FRANKLIN P. . NAEGELI, Orro. . Scuwaxse, E., and H. Joseruy. . TANDLER, J. . VAN Duyse. . Warr, J. C. Mall), Phila. and Lond., 1910, 1, 454-522. Also: Handbuch. d. Entwicklungsgesch, d. Menschen (Keibel & Mall) Leipz., 1910, 1, 457-526. . Locy, W. A. Contributions to the structure and devel- opment of the vertebrate” head. Jour. Morphol., Bost., 1895, x1, 497-594, 5 pl. The optic vesicles of elasmobranchs and their serial relations to other structures on the cephalic plate. Jour. Morphol., Bost., 1894, rx, 115-122. A study of the causes underlying the origin of human monsters.” Jour. Morphol., Phila., 1908, xrx, 3-368. Ueber die Entwicklung des menschlichen Darmes und seiner Lage beim Erwachsenen. Arch. f. Anat. u. Entweklungsgesch., Leipz., 1897, Supplement (His Festschrift), 403-434, 10 pl. - Mecxet, J. F. Ueber die Prioritiit der{centralen Theile vor den peripherischen. Arch. f. Anar. u. Physiol. (Meckel’s), Leipz., 1826, 1, 310-315. Ueber eine neue mit Cyclopie ver- kniipfte Missbildung des Centralnervensystems. Arch. f. Entwiklungsmechn. d. Organ., Leipz., 1897, v, 168-218, 4 pl. Die Morphologie der Missbildungen, Jena, 1913, 3. Teil, 1. Abt., 205-246. . Spemann, H. Ueber die Entwickelung umgedrehter Hirnteile bei Amphibienembryonen. Zoologische Jahrb. Supplement 15, Bd. 3. Jena, 1912. . Srockarp, C. R. The artificial production of a single median cyclopean eye in the fish embryo by means of sea-water solution of magnesium chlorid. Arch. f. Entweklungsmechn. d. Organ., Leipz., 1907, xxii, 249-258. The artificial production of one-eyed monsters and other defects, which occur in nature, by the use of chemicals. Anat. Record, Phila., 1909, m1, 167-173. The development of artificially produced cyclopean fish—the “‘magnesium embryo.” Jour. Exper. Zool., Phila., 1909, vr, 285-338, 1 pl. The influence of alcohol and other anzestheties on embryonic development. Amer. Jour. Anat., Phila., 1910, x, 369-392. An experimental study on the position of the optic anlage in Amblystoma punctatum, with a discussion of certain eye defects. Amer. Jour. Anat., Phila., 1913-14, xv, 253-289. Srreerer, C. L. Development of the nervous system. Chap. 15, Manual of Human Embryology, Keibel and Mall, vol. 2, Phila., 1912. Beitriige zur Entwicklungsgeschichte des Vertebratengehirns. 1. Anatomische Hefte 1 Abt., Wiesb., 1907, xxxut, 553-665, 8 pl. Beitriige zur Entwicklungsgeschichte des Vertebratengehirns, Ibid., 1915, Lu, p. 85. Cyclopie avee crytophtalmos et kystes colobomateux. Arch. d’opht., Paris, 1909, xxrx, 65-77. . Wattry, I. E. A human embryo of thirteen somites. Amer. Jour. Anat., Phila., 1913-14, xv, 319-331. Description of two young twin human embryos with 17-19 paired somites, Contributions to Embryology, No. 2, Carnegie Inst. Wash. Pub. No. 222, 1915. . Witper, H. H. The morphology of Cosmobia; specula- tions concerning the significance of certain types of monsters. Amer. Jour. Anat., Phila., 1908, vin, 355-440, 4 pl. to Sed or EXPLANATION OF PLATES. Puate 1. . Plaster reconstruction of the brain and cyclopean eye of embryo No. 559. 25. Cranial nerves are marked with Roman numerals. o. v., optic vesicle; s, snout; m, mouth. . View of the right side of embryo No. 559. 9. Only the face region is worked out in detail. U. v., umbilical vesicle. . Face of embryo No. 559. 16. The drawing is made from a plaster-of-paris reconstruction. S, snout. . View of the left side of cyclopean embryo No. 559. 9. The drawing was made directly from the specimen in formalin. PLATE 2. . From the photograph of embryo No. 201. X 13. . Photograph of ovum, No. 559. &X 1. . Section through the cyclopean eye, No. 1165. 40. Z£, eye; e. l., eyelid. Behind the eye are seen the ocular muscles. . Section through the external ear of embryo No. 201. 75. ‘There is an invagination of the epidermis and tissues of the ear are dissociated. . Photograph of the ovum, No. 559, showing the embryo in position. 3. The exoccelom is filled with a dense magma. . Outline of brain of embryo No1201. X 100. From a plaster reconstruction made by Dr. Bartelmez. The wide- open flange in front contains two depressions, the optic vesicle, which unite through a common groove, the torus opticus, as marked on the drawing. The depression behind this flange no doubt represents the cavity of the midbrain and hindbrain. This specimen is No. 87 of the collection of the University of Chicago and con- tains eight pairs of myotomes. . Photograph of section through the snout of specimen No. 559. X60. The frontal process contains the common cerebral vesicle, c. v., and below the snout there is the upper jaw, wu. j. : PLATE 3. All the photographs are from the sections of the embryo No. 559. Figs. 1 and 2, 50; figs. 3, 4, 5, X40. . Section through the ocular muscle, showing the terminal fibrils of the third and sixth nerves. Oc. mus., ocular muscle; , notochord; m, mouth; v, fifth nerve. . Section through the eye at its attachment to the interbrain. YT. 0., torus opticus; v',v”’, v’’’, branches of the fifth nerve; zy‘, terminal filaments of the fourth nerve ending in the primordia of the superior oblique muscle. . Section through the lower tip of the cerebral hemisphere. The peculiar tissue surrounding the nerve-body may represent a degenerated olfactory region. About this is seen a section of the interbrain, and below a process containing the eyclopean eye. Fb, first branch of fifth nerve; m, mouth. . Section through the middle of the cyclopean eye. J. b., interbrain; Fb, first branch of fifth nerve; m, mouth. . Section through the cerebral hemispheres as they communicate with the interbrain. J. b., interbrain; c. h., cerebral hemisphere; s, snout; u. j., upper jaw. Over the lamina terminalis is seen the peculiar thickening of the out- side ef the body which may represent the degenerated olfactory region also shown in the flat section of figure 3. 33 ..- 2 t ae ?Te ie vie re ste ee Ny . em it lee i ott an py here xt eS Als * ut ives: = % MALL MALL PLA Fic. 1. PLATE 3 MALL Fie. 4. CONTRIBUTIONS TO EMBRYOLOGY, No. 16. QUANTITATIVE STUDIES ON MITOCHONDRIA IN NERVE-CELLS. By Mapcr DeG. Txurtow. One plate. =e QUANTITATIVE STUDIES ON MITOCHONDRIA IN NERVE-CELLS., By Mapncre DreG. Turow. The hope of being able to establish a sound foundation for investigation into cellular physiology has inspired most of the work on cell constants. Perhaps the best known of these is the nucleus-cytoplasmic ratio of Hertwig (1902), which has already proved of great value in the investigation of changes in nerve-cell activities. On account of the reawakening of interest in mitochondria, the atten- tion of investigators has been drawn in recent years to the cytoplasm. This is not surprising, as structural changes resulting from experimental variations are evident in the cytoplasm, for it is here that most of the products of differentiation are laid down and readjustments in response to changes in the environment take place. Though the study of mitochondria has been carried far along many different lines, up to the present time no attempt has been made to place these cytoplasmic structures upon a quantitative basis. With this object in view the present work was undertaken, making use of a favorable method of technique. No attempt was made to establish a ratio between mitochondria and cytoplasm on the basis of relative volumes, the number of mitochondria per unit volume of cytoplasm being the basis of comparison. This relationship has served well as an adequate foundation for comparison of various nerve-cells and is thought to be of particular value in the case of the nerve-cell, inasmuch as it possesses no other cytoplasmic constituent lending itself to quantitative study; for the Nissl substance differs so widely in form and density that it is absolutely impossible with our present methods of technique to estimate its amount with any degree of accuracy, and the significance and form relations of the neurofibrils are not clear. The original plan of the investigation was to determine whether by com- parisons of the mitochondrial content of known motor and sensory cells there was a distinctive difference between cells of these categories. With this purpose in view, quantitative estimations of mitochondria in the nuclei of origin of the cranial nerves were made. The results were disappointing, in that they showed that the mitochondrial content could not be used as a basis of classification for motor and sensory cells, but they did show something that was not known before, viz, that the number of mitochondria per unit volume was constant for the nucleus of any cranial nerve. MATERIAL AND METHOD. The animal selected was the white mouse, and the observations were confined to the nuclei of the cranial nerves. The nuclei of the [X and XI nerves were not included in the investigation, owing to the difficulty of ascertaining with absolute certainty what cells constituted these nuclei in the nucleus ambiguus. 38 QUANTITATIVE STUDIES ON MITOCHONDRIA IN NERVE-CELLS. The method of fixation and staining has been described by Cowdry (1916a). The mitochondria, which take the fuchsin, appear as discrete, bright-red granules sharply outlined against a background of light-green Nissl substance, and, with proper optical and lighting facilities, may be readily counted. Quantitative estimations were made by carefully counting the mitochondria occurring within a field of constant area. Such a field was obtained by placing within the ocular a glass disk upon which a single square had been ruled accord- ing to the method of Isaacs (1915). The actual area of the optical field covered by the square was determined by the stage micrometer and found to be 19.78 square micra. The thickness of the sections was 4 micra; hence the volume of the field counted could easily be determined, and from that the amount of mito- chondria per cubic millimeter was calculated. The lenses used were the Zeiss apochromatic 1.5 mm. objective and Zeiss No. 6 compensating eyepiece. Constant conditions of illumination were obtained by the use of the 40-watt Mazda lamp. A mechanical stage was employed. All counts and drawings were made under uniform conditions of magnification and illumination. In order to avcid inaccuracies due to possible minor differences in thickness of the sections, the same number of fields was counted in every nucleus occurring in any one section. For example, in the same section may be found cells of the cochlear and vestibular nuclei of the VIII, of the VI, VII, and the mesencephalic nucleus of the V. If four fields were counted in one section in mesencephalic cells, four fields of all the other nuclei were counted in that section. OBSERVATIONS. Studies were carried out upon five brains which had been cut into partial serial sections. Only such nuclei as were well stained in each series were investi- gated and the results tabulated (table 1). TABLE 1. Series | Series | Series | Series} Series Nucleus. 1259 | 1348 | 1237 | 1260 | 1257 Remarks. Mesencephalic of V.} 22.6 | 22.5 | 21.7 | 22.9 | 22.1 VI; csaies eee Dar Bl ee nes| Oat Lace ae eet , : VID i eee aye || tony ell ees: 1 OK oe eae. Series 1259, male, weight 9 grams, age 33 days. VIE (yest.)c2ccve eal al eee | eee lene eee Series 1348, female, weight 13.5 grams, age 61 days. VIII (vent. coch:)'-:|| 18281] 18. 0b bee ele ose see ean Series 1237, male, werght 10 grams, age 25 days. 9 X (motor),;..sac 92 cee Pn th | eae A ee tants Series 1260, male, weight 8.5 grams, age 31 days. 9 al ee 15 14.3 | 15.4 | 14.3 Series 1257, female, weight 9 grams, age 35 days. > 0s 0.019 oe » x 4.0 oil vipinieia ai > oatdimtel ee i> tate iets 17.3 PPT Pe, (Soames ose Ve aos 22.9 The above figures represent the average number of mitochondria present in the field counted, the volume of which was 79.12 cubic micra. It will be noted that there are many gaps in the series. This is due to the fact that, owing to the great difficulty of cutting perfect serial sections 4 micra in thickness, some of the nuclei are missing. Again, perfect definition of outline of the mitochondria is required before they can be counted, and since it is im- possible, even with the most expert technique in staining, to attain this ideal in every section, some of them could not be used. QUANTITATIVE STUDIES ON MITOCHONDRIA IN NERVE-CELLS. 39 At first 150 fields were counted in each nucleus and the average was taken, but as there was never any wide variation in the figures obtained for different fields the number was limited to 50, and later to 10. Most of the averages re- corded here were based on 10 field counts—never on less. Lists were kept of the number of mitochondria in each field as they were counted, and such variations as the following were noted: In 365 fields observed in the VI nucleus the numbers ranged from 20 to 26; in 10 from the mesencephalic the range was 20 to 24; in 10 from the VII it was 14 to17. This uniformity ran through all the nuclei of all the series, with a few exceptions that will be mentioned later. In order to determine the percentage error in the total, 10 fields were re- counted in several cases and the error estimated, and it never amounted to more than 1.3 per cent. This maximum occurred in the cells most closely crowded with mitochondria; in cells having few mitochondria the recounts showed no error. Table 2 contains the detailed results of specimen counts and recounts. TABLE 2. | | | = ee || Nucleus V || Nucleus ‘VE Nucleus VI. Nucleus (mesen- | (motor). | XII. ne halic). | ‘ 1 wo prone ] Remarks. Original Recount. Original Recount. Original || Original | count. | count. | count. count. 15 16 [25 24 =| 17 22 Original counts of four nuclei are given. The figures 15 16 || 25 26 ii 15 24 illustrate the uniformity of the mitochondrial content 16 15 .|| 21 22 | 15 22 || of the individual nuclei and are typical of all the nuclei 16 17 27 2657 | 15 25 | counted. 15 16 24 26 «|| 15 23 | In the case of nuclei VII and VI figures for the re- 14 15 | 28 27 14 23 | counts are also given (right-hand columns). These show 14 14. | 23 24 14 | 21 I little variation from the original count. In making the 16 17 | 22 20 14 | 24 || recounts, the slide was not moved, in order to have the 16 IT pal 23 16 22 | limits of the field unchanged, the recount being made 17 16 25 23 17 | 22 || immediately after the original count. I} Care was taken to avoid cells whose limits could not be clearly defined. Inasmuch as the cell processes and the neuroglia possess mitochondria, it was necessary to choose sharply outlined cells, in order to eliminate errors due to counting mitochondria outside the cell-body. It has been stated that the number of mitochondria in the cells of the same nucleus is quite constant, but that there are some exceptions. In one animal, in the VI nucleus some cells had fewer mitochondria than others, but as these appeared normal in other respects they were counted, the variation ranging in this instance from 13 to 21. The greater number, however, contained about 17 per field. In the case of the cochlear nucleus there was a group of cells dorsal and somewhat lateral to what appeared to be the main body of the nucleus. The dorsal group corresponds to current descriptions of the dorsal cochlear nucleus, and the larger group to the ventral cochlear nucleus. The cells of the dorsal nucleus contained practically no mitochondria, were slightly smaller, and pos- sessed a cytoplasm clearer than those of the ventral nucleus. Only the cells of the ventral nucleus were included in the count, for it was felt that cells which 40 QUANTITATIVE STUDIES ON MITOCHONDRIA IN NERVE-CELLS. showed such constant and specific morphological differences could not be included in the same functional category. Upon referring to plate 1 it will be seen that there are striking similarities between nuclei of different functional categories. For example, the mesencephalic nucleus (fig. 7) of the V (regarded as sensory by most authors—as Johnston, 1909, and Willems, reviewed by Donaldson, 1912) and the motor nucleus of the IV (fig. 4) have the same number of mitochondria per unit volume of cytoplasm; the visceral motor nucleus of the VII (fig. 1) and the somatic motor nucleus of the XII (fig. 5) have the same average; so, too, have the vestibular of the VIII (somatic sensory, fig. 2) and the motor of the III (fig. 6). Not only do nuclei of different categories agree in the number of mitochondria they contain, but those of the same category disagree, the numbers for the somatic motor nuclei being 14, 17, 20, and 22 mitochondria per unit volume of cytoplasm. Comparisons of the nuclei of the same general classification are made in table 3. TABLE 3. Somatic motor nuclei. Visceral motor nuclei. Somatic sensory nuclei. Average Average Average number of number of number of Cranial | mitochondria | Cranial | mitochondria Genial enves mitochondria nerves. per field for nerves. per field for anja: ® 1c per field for all cells all cells all cells counted. counted. counted. NUS a 17.3 >, Ce 14.4 Wiest 8 iniorecs thoes 22.3 LV 22.9 VII.. 15.3 MILD (vest.).c0 sce 17.6 MIL 20.5 VIII (vent. coch). 1828 XII... 14.8 No visceral sensory nuclei were studied. It may be seen that no distinction ‘an be made between motor and sensory nuclei on the basis of their mitochon- drial content. In order to have some idea of the enormous number of mitochondria in the cells of the nuclei of the cranial nerves and of the really tremendous variations in number of mitochondria in cells of the different nuclei whose number of mito- chondria per field vary only from 14.1 to 22.5, the results of some determinations as to the number of mitochondria per cubic millimeter of cytoplasm will be given. In nerve-cytoplasm containing 22.5 mitochondria per field, such as that of the cells of the mesencephalic nucleus (fig. 7), the number of mitochondria per cubic millimeter would be 284,378,159; the number for the cytoplasm of the cells of the nucleus of the X nerve (fig. 8), containing 14.1 per field, would be 178,210,313. This study has been very carefully controlled. In the first place, several observers have briefly called attention to the fact that mitochondria differ in form not only in different nerve-cells, but also to some extent in the same cell. Nichol- son (1916), working in this laboratory, has made a careful study of these morpho- logical variations in mitochondria in the nerve-cells of the white mouse, the same form which I have studied. He, however, worked with cells other than those of QUANTITATIVE STUDIES ON MITOCHONDRIA IN NERVE-CELLS. 41 the cranial nerves, where the mitochondria are, for the most part, granular, as seen by the illustrations. Working particularly with the anterior-horn cells and the large cells of the reticular formation, he describes mitochondria which are filamentous, the filaments varying in length, some being so short as to be almost identical with the granular forms, as one proceeds from the periphery to the nucleus. Naturally the question would arise as to whether, under these circumstances, quantitative variations such as those recorded in table 1 have any real significance. It is obvious that the method which I have adopted of counting the mitochondria would have to be accompanied by very accurate measurements in order to yield reliable information of the relative as well as actual amount of mitochondria in cells in which their size and shape differ to any appreciable extent. Filamentous mitochondria, though occurring in the cell processes, are rarely found in the cell- bodies of the cranial nerves and, since my observations are confined to the cell- bodies, these filamentous mitochondria do not constitute a source of error. A few of them are homogeneous throughout, but most can be resolved by careful focus- sing into rows of discrete granules, fairly uniform in size. For the work in hand this was a great advantage, for by counting the granules a more accurate index of the amount of mitochondria was obtained than would have been possible by counting the filament as a unit. Though the method of counting here used is not ideal in the case of cells characterized by great dimorphism on the part of their mitochondria, it does yield reliable results when care is taken to restrict its use to cells in which the size and shape of the contained mitochondria are practically uniform, as in the cells of the nuclei of the cranial nerves. Despite the fact that the granules were of the same size, if there were lack of uniformity in their distribution, an accurate estimation of the mitochondria for any one cell would be impossible unless all the mitochondria in that cell were counted. It is because of their practically uniform distribution that the amount in any one field can be used as typical for the whole cell. Counts were made of different fields of a cell in one section, also of fields selected from the same cell in successive serial sections; and the numbers were practically identical. It is true that there is usually a slight crowding of mitochondria in the axon hillock, with a tendency for them to be arranged in filaments or rows of granules along the long axis of the cell-process (figs. 3, 4, and 5). In the canalicular system, which in these preparations shows white, there are no mitochondria; where they do seem to occur in the canals the appearance is due to their presence in the thin layer of cytoplasm surrounding the canal. Especially in figures 1 and 5 there appear to be large areas free from mitochondria. This is explained by the fact that all the drawings were made in one optical plane; on a different focus mitochondria would have appeared in the cytoplasm, which is now free from them. No variations were noted in the density of distribution of mitochondria other than those just mentioned. Any minor unevenness in the distribution of mitochondria would be obviated by the fact that the square used in counting is relatively large, taking in an expanse of cytoplasm extending in most instances from the nucleus to the periphery of the cell. 42 QUANTITATIVE STUDIES ON MITOCHONDRIA IN NERVE-CELLS. Again, it has been shown that mitochondria vary in their solubilities in acetic acid (Regaud, 1910; Nicholson, 1916). In view of this fact objection might be made that the variation in mitochondrial content in different types of nerve- cells was an artefact produced by the solvent action of the reagents used. Such criticism of the results obtained in this investigation is invalid, for no acetic acid was used in the preparation of the specimens, nor‘was there any other solvent for mitochondria involved in the fixation or staining. The formalin which was used as a fixative for the mitochondria was neutral, and the long mordanting in the bichromate prevented their subsequent solution in alcohol, although their solu- bility in alcohol does not seem to be marked. Hence there is no error in tech- nique which could account for the striking variation in amount. DISCUSSION. Having established the certainty that there is, in the nerve-cells of the medulla of the species used, a definite amount of mitochondria per unit volume of cytoplasm, there remains to be determined the functional significance of such numerical variation. Other investigators have, in their work on the central nervous system, determined various cell ratios, among which might be mentioned the nucleus-plasma ratio. Dolley (1914) has found that the resting nerve-cells of corresponding type for the same species have a constant nucleus-plasma ratio which is altered temporarily through functional depression or permanently through functional senility. Having once established this constant, he could study path- ological changes following experimental conditions. Donaldson (1911) found varia- tions in the water content of the nerve-cells accompanying functional changes. He makes no further statement than this: the variation in water percentage of the nerve-cell is an index of functional activity. The relationship between number of mitochondria and volume of cytoplasm is another such constant. In the animals studied the number of mitochondria per unit volume of cytoplasm was found to be constant for corresponding cells, not only of the same animal, but also of different individuals of the same species (table 1). In nerve-cells of the same type, therefore, we have a cell constant which is definite for animals of the species studied and which can be used for observations of pathological conditions resulting from experiment. That mitochondria do react to conditions which affect the cell has been demonstrated by several authors, as Policard (1910 and 1912), Homans (1915), Scott (1916), and others. These observations may be given another application, viz, to the doctrine of neurone specificity. It would be reasonable to suppose that even if all the mito- chondria were identical, such definite and constant variations as are here recorded would be closely associated with a definite and constant functional differen- tiation. Combined, however, with this quantitative difference are qualitative differences (Nicholson, 1916), and this combination serves to strengthen the theory that the activity of the nerve-cells themselves differs in some way. It would be rather extreme to assume that cells differing specifically with respect to so con- Li QUANTITATIVE STUDIES ON MITOCHONDRIA IN NERVE-CELLS. 43 stant an element in their cytoplasm were functionally identical, for although as yet the role played by mitochondria in nerve-cells is unknown there is evidence that they play an important part in other cells. Cowdry (1916) has discussed briefly the literature bearing on the relations of mitochondria to cell metabolism. From such evidence it is safe to assume that they are not an unimportant con- stituent of nerve-cells and that their constancy in amount in the normal cell is definitely associated with its normal activity. CONCLUSION. There is a constant number of mitochondria per unit volume of cytoplasm in normal nerve-cells of a corresponding type in the mouse. This constant differs - for nerve-cells of different types. Sensory and motor cells can not be distinguished on the basis of their mitochondrial content. The significance of constant varia- tions can not be interpreted with our present meager knowledge of the rdle played by mitochondria, but there is support for the theory that nerve-cells are func- tionally differentiated in the evidence here advanced of their constant difference with respect to the number of mitochondria they contain. Finally, I desire to express my cordial thanks to Dr. E. V. Cowdry, who kindly suggested the problem and provided the material for the investigation. Batrimore, May 31, 1916. BIBLIOGRAPHY. Cowpry, E. V., 1916a. The structure of the chromophile | Homans, J., 1915. A study of experimental diabetes, etc. . cells of the nervous system. Contributions to Embry- ology, No. 11. Carnegie Inst. Wash. Pub. No. 224. , 1916b. The general functional significance of mito- chondria. Amer. Jour. Anat., Phila., xrx, 423-446. Dottey, D. H., 1914. On a law of species identity of the nucleus plasma norm for nerve-cell bodies of correspond- ing type. Jour. Compar. Neurol., Phila., xxiv, 445- 500, 1 pl. Donaupson, H. H., 1911. An interpretation of some dif- ferences in the percentage of water found in the central nervous system of the albino rat and due to conditions other than age. Jour. Compar. Neurol., Phila, xx1, 161-176. , 1912. Review of ‘Localisation motrice et kinesthé sique,” by Edouard Willems. Jour. Nerv. and Ment. Dis. INE ¥-,, KoCKEX, \Of—ce- Hertwic, R., 1902. Ueber das Wechselverhiltnis von Kern und Protoplasma. Sitzungsber. Gesellsch. f. Morphol. u. Physiol., Miinchen, xvut, 77-96. Jour. Med. Research, Bost., xxx1u, 1-51, 2 pl. Isaacs, R., 1915. A mechanical device to simplify drawing with the microscope. Anat. Rec. Phila., rx, 711-714. Jounston. J. B., 1909. The radix mesencephalica trigemini. Jour. Compar. Neurol., Phila, xrx, 593-644. Nicuotson, N. C., 1916. Morphological and microchemical variations in mitochondria in different types of cells of central nervous system. Amer. Jour. Anat., Phila., xx. Poticarp, A., 1910. Contribution A l’étude du mécanisme de la sécrétion urinaire. Arch. d’anat. micr., Paris, xu, 177-288, 1 pl. , 1912. Sensibilité des chondriosomes aux élévations de température. Compt. rend. Soc. de biol., txxn, 228-292. Recavup, C., 1910. Etudes sur la structure des tubes séminiféres, ete. Arch. d’anat. micr., Paris, x1, 291-433, 4 pl. : Scorr, W. J. M., 1916. Mitochondrial changes in the pancreas in experimental phosphorus poisoning. Amer. Jour. Anat., Phila., xrx. EXPLANATION OF PLATE. The following drawings were made with the aid of a camera lucida, a 1.5 mm. apochromatic Zeiss objective, and No. 6 compensating Zeiss ocular. All drawings are at a magnification of 1,650 diameters. The Nissl substance appears as a background of bluish-green masses, between which are the unstained canals of the canalicular system. Against this background the mitochondria appear as bright-red dots. All cells were drawn without changing the focus, so that sometimes the mitochondria are clumped within the Niss] substance and some- times within the canals, although this latter appearance is due to their presence in the thin layer of cytoplasm sur- rounding the canals. If the focus were changed sufficiently mitochondria would occupy the spaces now free. The counting, however, was done by focussing through the whole depth of the section. Some of the mitochondria seem to be smaller and less definite than others; this, in all the drawings, is due to the fact that they were slightly out of focus, yet sufficiently clear to be included in the drawing. Where they occur as chains of granules they are probably broken filaments. The granules, when brought clearly into focus, are of approximately the same size. Pate 1. 1. A typical cell of the motor nucleus of the VII cranial nerve. Note its similarity to the cell of the XII nucleus (fig. 5), both with respect to the appearance of the Nissl substance and the number of mitochondria, the number per unit volume of cytoplasm for both being 15. 2. A cell from the ventral cochlear nucleus of the VIII nerve. The Nissl substance here is a diffuse violet, and the mitochondria stain more intensely than those in the other nuclei. 3. A motor cell from the nucleus of the VI nerve. 4. A motor cell from the nucleus of the IV nerve. It will be noted that there appear to be more mitochondria in the cytoplasm of the cell of the mesencephalic nucleus (fig. 7) than in this, although the counts show that the number per field is practically the same. This is due to the fact that the cytoplasm of the mesencephalic cell is much more transparent, lacking the great clumps of relatively opaque Nissl substance which characterizes the IV nucleus cell; and so in a single optical plane, mitochondria may be seen to a greater depth in the mesencephalic cell. Since focussing was done throughout the depth of the section, the Nissl substance did not interfere with the accuracy of the counts. A motor cell from the nucleus of the XII nerve. 6. A motor cell from the nucleus of the III nerve. ; . A-sensory cell from the mesencephalic nucleus of the V nerve. The mitochondria appear closely crowded, even on one plane. 8. A cell from the dorsal motor nucleus of the X nerve. 44 “I Y PLATE 1 THURLOW (3 2 8 5 ‘ ; i 2 oe s [a CONTRIBUTIONS TO EMBRYOLOGY, No. 17. DEVELOPMENT OF CONNECTIVE-TISSUE FIBERS IN TISSUE CULTURES OF CHICK EMBRYOS, By Marcaret Reep Lewis. Two plates. - yi Tete ye — a * eae wy an te DEVELOPMENT OF CONNECTIVE-TISSUE FIBERS IN TISSUE CULTURES OF CHICK EMBRYOS. By Marearet Reep Lewis. Up to the present time the study of fixed and stained preparations of embryos has failed to decide the question of the origin of the connective-tissue fibers. This is partly because the methods used necessarily coagulate and distort the delicate cell processes and also the intercellular substances, and such results readily lend themselves to various interpretations by different investigators. In the following observations on tissue culture an attempt has been made to study the formation of the connective-tissue fibers directly within the living tissue, and not only within living tissue, but within tissue which has developed in an environment entirely free from fibrin or any substances other than those within the cell which coagulates upon fixation. The pieces of tissue to be explanted for the tissue cultures were washed through several changes of Locke’s solution until such fibrogen as was present had become coagulated within the pieces themselves before they were explanted. The medium thus remained free from fibrin, for no fibrin network was observed in the medium of any preparation. In order to see whether the substance forming fibrin would be dissolved out from the explanted piece and deposited as a fibrin network in the medium of the culture, a few cultures were made in which the explanted pieces of tissue were not carefully washed. No fibrin network was found in these cul- tures, even after many days. However, in such preparations a delicate network formed over the growth (not within the growth) upon fixation, showing that some substance had been dissolved out from the explanted piece, which coagulated upon fixation. Although this network did not resemble fibrin network, or the delicate processes from the cells, or even the fibrous tissue itself, all such prepara- tions were discarded in order to avoid any possible chance of error. By this method it is taken for granted that such connective-tissue fibrils as form in the tissue-culture growths arise from the cells, either as a secretion formed by the cells and deposited in the form of fibrils and fibers, or from the transforma- tion of the cytoplasm of the cell itself. As will be seen later, while there is evi- dence of a possible secretory activity of these mesenchyme cells, as Renaut (1904) and Renaut and Dubreuil (1906) have claimed, due to the presence of the “grains de segregation,” or the so-called vacuoles of Lewis and Lewis (1915), nevertheless, in these tissue cultures, the connective-tissue fibrils formed by a transformation of the cytoplasm of the cells. Tissue culture is not an entirely satisfactory method for the study of any highly differentiated tissue, owing to the fact that the cells which migrate out from the explanted piece and later increase by division attach themselves so closely to the cover-slip and become spread out in such a thin layer that the differ- 47 48 DEVELOPMENT OF CONNECTIVE-TISSUE FIBERS. entiated structure loses its characteristic appearance. Also, the cells of the new growth have a tendency to migrate away as individual cells instead of developing into a differentiated tissue composed of numerous cells. In all probability the cells do not de-differentiate and become more embryonic, as has been claimed by Champy (1913) and others, but simply lose their characteristic differentiated appearance, due to their changed shape and position. This is interestingly shown by a study of smooth muscle-cells (plate 2, tig. 6). Where the cells are attached closely to the cover-slip they no longer contract and the myofibrils appear as irregular bundles composed of numbers of delicate fibrils (plate 2, fig. 6). However, where the cell is not so closely attached to the cover-slip it continues to contract, and in this case the myofibrils are arranged into the characteristic fibrils. Taking the possible loss of the characteristic appearance of the differentiated structure into account, the very thin and largely spread-out living cells of the tissue culture furnish an excellent means for the study from day to day of certain structures of the cell. Just how much differentiation can take place in such cells in these tissue cultures is difficult to state. Certainly in a few cases, where the mesenchyme growth at 48 hours was composed of quite undifferentiated cells, this growth, when kept alive by frequent baths of fresh solution, did develop definite connec- tive-tissue fibrils. Muscle fibers have been observed to become more differen- tiated; but in the case both of the muscle fibers and connective tissue there is some continuation of function, as the muscle fibers frequently contract, and the connective-tissue growth also occasionally contracts back around the explanted piece and later grows out again. Fibrils did not develop in many of the cultures of connective tissue, owing to the fact that the cells remained spread out as individual cells until the death of the culture. In the few cultures that were kept alive for a sufficient length of time, and in which the connective-tissue fibers did develop, they could be clearly seen and studied in the living preparation from day to day, and their develop- ment could be traced from the earliest delicate fibril within the exoplasm of the cell to the more adult fibers, which appear to be free from the cells. PREVIOUS WRITINGS ON THE LIVING CONNECTIVE TISSUE. Whether the connective-tissue fibers arise within the cells or from an inter- cellular substance is still an open question. The weight of evidence seems to be in favor of a cellular origin, although certain text-books of histology present the question almost wholly from the intercellular point of view. There are many reviews of the literature on both sides of the question (Fleming, 1891; Spuler, 1896; Mall, 1901; Rothig, 1907), and also various text- books, and since the technique used by other investigators is so different from that employed in the following observations no effort will be made to take up in detail the various papers upon the origin of the connective-tissue fibers. While many observers have studied teased preparations of connective tissue, Boll (1872) was the first to study the development of the connective-tissue fibrils entirely from the living cell. Boll made his cultures by teasing out a few of the DEVELOPMENT OF CONNECTIVE-TISSUE FIBERS. 49 cells of the tissue to be studied and placing them in a hanging drop of amniotic fluid. Although he did not obtain any growth of the teased-out cells, he was able to observe the connective-tissue fibrils in connection with the cells throughout the different stages of the development of the connective-tissue fibers, and he became convinced, by this study of the living cells, that the fibrils had their origin within the cells and continued through the exoplasm of one or more cells. Boll studied carefully the following tissues: ? Arachnoid of chick embryo of 4 to 19 days’ incubation. Subcutaneous tissue of chick embryo of 7 to 17 days’ incubation. Cornea of chick embryo of 4 to 21 days’ incubation. Tendon of chick embryo of 7 to 21 days’ incubation. / He concluded that in all of these tissues the connective-tissue fibers originated / from the cells. In the study of the connective tissue by the tissue-culture method, prepara- tions. such as those studied by Boll were used, and also many others, in which the tissue was either teased out, or flattened out, or suspended in a hanging drop of Locke’s solution instead of amniotic fluid. Figures similar to those given by Boll were frequently observed (plate 1, figs. 2, 4, and 5), and his observations were corroborated. However, in all such preparations it is impossible to eliminate the possibility of fibrin or some other intercellular substance taking part in the forma- tion of the fibrils, and for this reason these observations will not be given below, although they undoubtedly show the connection of the fibrils with the cells. As no cultures containing fibrin have been studied, nothing can be said at this time in regard to Baitsell’s observations (1915), by means of which he shows that certain fibers which resemble the connective-tissue fibers may form in a fibrin clot in the presence of a piece of tissue of a chick embryo after various periods of time. In the development of the embryo there can be no question of fibrin playing any part in the formation of the fibrous tissue, since, so far as is known, fibrin is not present in the uninjured tissue. Whether the cells of the embryo possess the power of secreting a substance which may act in the same manner as the injured cell to produce the formation of fibrin, or whether the connective-tissue cells in the developing embryo act directly upon the plasma, are questions which Baitsell does not discuss. He quotes the following experi- ments of Loeb, from Adami: “When a drop of uncoagulated lymph is placed between two glass slides, the mere act of pulling one slide over the other leads to the appearance of fibrils, which grow in length and bulk; which like those of connective tissue are not only intracellular, but actually traverse cell bodies situated in their path; which show themselves first in im- mediate connection with the cells, the cells as we now hold-liberating an enzyme that determines the modification of the more soluble protein into a precipitated or coagulated modification. But the lines of the precipitation are evidently along the lines of strain.” These experiments of Loeb are in a way comparable to those of Baitsell, except that in Baitsell’s experiments the strain is brought about by the shrinkage of the plasma clot. It seems rather difficult to draw any conclusions in regard 50 DEVELOPMENT OF CONNECTIVE-TISSUE FIBERS. to the manner of the formation of the connective-tissue fibrils in the embryo from results which are so obviously due to injured cells as are those of Loeb’s experiments. However, there is a striking resemblance between the fibrous tissue obtained by Baitsell by means of a modification of the fibrin clot and the fibrous tissue of the embryo. Baitsell’s (1916) paper on wound-healing, in which he finds that very shortly after a wound has been made in the skin of a frog, fibrin fibers, which resemble connective-tissue fibrils, are deposited, and that these fibers persist and take part in the formation of the cutaneous tissue, opens the exceedingly interesting ques- tion as to whether what takes place in the process of wound-healing can be in any way comparable to the behavior of normal developing tissue. So far as can be gathered from Isaacs’s (1916) incomplete report of his obser- vations upon the living connective-tissue fibers, his results correspond more or less with those of Loeb—that is, that various strains cause the intercellular sub- stance to form fibrille. Just what part the cells play in this formation it is difficult to understand. Isaacs does not say that the cells form an enzyme, as Loeb claims, but states that the movements of the connective-tissue cells probably effect the distribution of the material through chemical or other action and cause the fibrillated structures of the adult fiber. From Isaacs’s brief report it is evident that he had performed numerous experiments with the living connective tissue, and it is hoped that his complete paper will clear up many points. Ferguson’s (1912) observations upon the living connective-tissue cells in the fins of fish embryos are extremely interesting, since by his method the connective- tissue cells were studied under entirely normal conditions. Since there has been some question as to whether fibers actually exist or whether they are merely coagulations of a colloid within the tissue due to abnormal conditions, it is inter- esting to note that Ferguson describes fibers as well as cells as existing in the living embryo. He found, by the aid of preparation stained by Bielschowsky’s method, that the fibers arise within the cell. Unfortunately he was not able to see the fibers in the embryonic cells of the living embryo and to determine whether they become separated from the cells, or how this takes place. His observations upon the movements of the connective-tissue cells show that the round and stellate cells may move up to and stretch along a fiber as a very thin, long spindle cell, and in a few cases he observed such a spindle cell to become stellate again. Ebeling (1913) has for a period of two years or more kept alive certain of the cultures started by Carrel. The growth of these cultures consists mainly of connective tissue, and Ebeling claims that connective tissue may have a permanent life outside the organism when properly cared for. The method of keeping the culture alive is as follows: The entire culture in its plasma clot is freed from the coyer-slip and washed in Locke’s solution to remove any waste products and is then cut into four or more pieces, and each of these pieces is again explanted into a drop of fresh plasma. This procedure is carried out every other day; although many of the cultures die, a few survive and grow, and are again explanted as described above. In all probability there is no differentiation of the connective tissue into fibrils or fibers, as Ebeling describes the growth as though it consisted DEVELOPMENT OF CONNECTIVE-TISSUE FIBERS. 51 of undifferentiated mesenchyme, which is what would be expected in any tissue that proliferates as rapidly as this tissue necessarily must. It would be interesting to see whether, if one of the cultures were kept alive without further explanation, it would again differentiate after a certain equilibrium of proliferation had been reached. Some workers have claimed that certain substances present in the medium of tissue cultures prevent the growth of the connective tissues. For example, Walton (1914) states that liver extract inhibits the growth of adult mammalian connective tissue in plasma cultures, and Russel (1914) claims that gentian violet, in solution of 1/20,000 in the medium of tissue culture, prevents the growth of connective tissue but not of endothelium. The reason for this neither writer explains, nor does either state what structure of the cell is affected by the sub- stance so that the cells do not grow out, or whether the medium may simply not attract the cells to migrate and that the cells themselves are uninjured. Thus a review of the literature on the living connective-tissue cells shows that the study of the living tissue has not presented decided proof as to whether the connective-tissue fibers arise from the cells or are formed from an intercellular substance. Evidence is presented on both sides, and the question remains as completely at a dead-lock as when the observations were confined to fixed and stained preparations. OBSERVATIONS IN GENERAL. A few general observations as to what takes place in the tissue cultures of con- nective tissue are given here in order to show what factors influence the growth of the fibers. Cultures from the subcutaneous tissue of chick embryos of various ages were made in the usual manner (W. H. and M. R. Lewis, 1915; M. R. Lewis, 1916). Lewis and Lewis have shown that while the cells in the new growth in tissue cultures are under somewhat abnormal conditions as regards environment and nourishment, nevertheless they are actively growing cells which undergo normal division and which grow out as definite types of cells—that is, nerve-cells, muscle- cells, heart-muscle cells, endoderm of the intestine, epithelial cells of the skin, and connective-tissue cells. As has been stated, no fibrin is present in the medium, and no substance which coagulates. The subcutaneous tissue can be removed as a thin, transparent sheath from the skin of chick embryos of 10 days or older. It proved difficult to isolate the subcutaneous tissue from embryos younger than 8 days; and in these embryos a piece of skin or one of the deeper skin fascias or the arachnoid tissue was used for explanation. The connective tissue of embryos less than 8 days old is com- posed of cells without definite fibrils; that of embryos of 11 days and over contains definite bundles of fibers. The new growth from an explanted piece of subcuta- neous tissue of embryos of 8, 9, and 10 days proves very satisfactory for the study of the development of the fibrils. The growth can be kept alive and healthy by frequent baths of fresh Locke’s solution, plus 10 per cent bouillon, plus 0.25 per cent dextrose; and fibrils begin to develop in the new growth in from 48 to 72 52 DEVELOPMENT OF CONNECTIVE-TISSUE FIBERS. a hours and continue to develop until the growth is about 6 days old or over. Cultures of the subcutaneous tissue from an 11 or 12 day chick embryo also prove very satisfactory for study, for not only is the new growth available for study, but the explanted piece itself is so thin that the cells and fibers can be observed even with the oil-immersion lens. The fibers in the explanted piece were not observed to grow either in length or bulk, and after a period of two weeks they remained much the same as when explanted. In no case was a fiber observed to pass out from an explanted piece over the new growth; such a fiber always remained curled up within the explanted piece. New fibrils begin to develop in the new growth from an explanted piece of tissue from an 8 to 12 day chick embryo shortly after the new growth is 24 hours old, and definite bundles of fibrils may be developed when the growth is 5 or 6 days old. These fibrils develop more quickly in growths from the older embryos of 10 to 12 days than in those from the younger embryos of 8 to 10 days. The new growth from the subcutaneous tissue is extremely sensitive and reacts to all sorts of changes in its environment, by contraction. Frequently while a membrane of connective tissue was under observation it would begin to contract from the outer edge of the growth and draw in towards the explanted piece. This contraction might stop at any period or it might continue until the entire new growth had contracted close to the explanted piece. The explanted piece was never observed to contract. The relaxation after such a contraction was exceedingly slow, and frequently a contraction that had taken no longer than 2 to 5 minutes required for relaxation from 1 to 6 hours. In fact, the process did not resemble relaxation, but rather a growing-out again of the new growth. Often, coincident with the contraction, there occurred a rolling-back of the edge of the growth, and in this case when the cells migrated out again many of them became changed in their relative positions. Thus it is evident that a decided strain is present during the development of the fibrils, though there is no fibrin and (so far as ean be seen) no substance which coagulates surrounding the cells. Whether this strain or tension (often exhibited by the contraction above described) may in any way influence the separation of the fibrils from the cytoplasm of the cell, it is impossible to state. It was not certain that a preparation which con- tained well-developed fibrils had not contracted during the development of the fibrils. However, it can be definitely stated that no substance formed into fibrils during contraction, as might have been expected from the experiments of Loeb and of Isaacs. The new growth, when relaxed after such a contraction, never contained any suddenly formed fibers or fibrils, and such fibrils as were present were in very much the same state of development as that in which they were before the contraction took place. Also, many membranes in which no fibrils ever developed possessed the power to contract, and did contract more than once. From these general observations it is evident that the fibers which form in the tissue cultures must arise from the cells; and since the cells are spread out in a thin layer the process of development of the fibers ean be observed in the living cell undisturbed by any manipulation. DEVELOPMENT OF CONNECTIVE-TISSUE FIBERS. 53 OBSERVATIONS IN DETAIL. The growth from a piece of chick embryo of 6 to 8 days’ incubation is usually in the form of a membrane closely attached to the cover-slip, and is composed of large, flat cells, either connected by numerous cytoplasmic processes (plate 1, fig. 1) or else crowded together so that the delicate processes from cell to cell are lost and a more definite cell-wall appears (plate 2, fig. 10). The growth from older chick embryos may also sometimes have the appearance of a membrane, especially where the cells are spread out in a thin layer along the cover-slip. When such a growth is treated with silver-nitrate stain the membrane becomes marked with more or less definite cell-walls, according to the amount of crowding of the cells (plate 2, fig. 10). Such a membrane has been described by Clark (1914), where the connective tissue is stimulated to grow out over a very smooth surface, which Clark interprets as showing that under certain conditions the connective-tissue cells may become transformed into endothelium. While the pattern which appears with the silver- nitrate stain is in many ways characteristic of endothelium, still growths from older chick embryos (8 to 10 days) in these tissue cultures exhibited the charac- teristic activities of connective-tissue cells, and in some cases fibrils were formed within the cytoplasm of the cells (plate 2, fig. 10). The growth from an 8 to 10 day chick embryo usually has the appearance of a reticulum of cells (plate 1, figs. 7 and 8). Some of these cells are of the large, flat, stellate type, having processes on all sides, in which may develop bundles of fibrils, which pass in more than one direction through the cells (plate 1, figs. 7, 8, and plate 2, fig. 10); others are cone-shaped— . e., while the cell-body may have several short processes, most of the cytoplasm is drawn out into one long process (plate 1, fig. 2, and plate 2, fig. 4). Both the granular and the clear cytoplasm is continued out into the one long process, which practically always extends in the direction from which the cell has migrated, and although in many cases it continues back as a delicate thread, passing as many as twelve or more cells, it has always a protoplasmic end, either free or closely attached to another cell. These long processes usually contain mitochondria and other granules scattered along their length, and never in any case have they been observed to change into connective-tissue fibers. In many “film prep- arations”’ of the subcutaneous tissue studied while alive, such long, delicate processes have been observed to extend along the side or through the middle of a bundle of fibrils. This is probably due to the fact that, through some stress, the cell has been drawn out into this shape, either from migration or manipulation, and the fibrils are those which were originally in the exoplasm of the cell. During the beginning migration (1 hour after explantation) of the cells in the explants from older chick embryos (10 to 15 days), when certain of the cells first begin to migrate it is seen that they are drawn out into exceedingly long and delicate processes which ramify in all directions, as though their cytoplasm had extended a greav 'ength along the fibers of the subcutaneous tissue (plate 1, fig. 3). As the cell continues to migrate towards the periphery of the explanted piece or out into the culture medium, these long processes are drawn into the cell, until finally 54 DEVELOPMENT OF CONNECTIVE-TISSUE FIBERS. it becomes stellate in form and later divides by mitosis and may again develop long and delicate processes among the cells of the new growth. From a study of this cell (plate 1, fig. 3) and of the cells shown in plate 1, figure 8, and plate 2, figure 4, it can be seen that inthe embryo, where the growth is in all direc- tions rather than in a flat plane (as in tissue cultures), a section must necessarily cut many of these delicate processes and cause the appearance of a network of isolated protoplasmic threads between the cells, because the connection of these threads with the cells to which they belong is not shown in the section. Typical spindle-shaped cells never appeared in pure cultures of connective tissue, but always in those which contained muscle-cells; and in every instance a typical spindle-cell could be identified as a muscle-cell. In certain of the explanted pieces spindle-cells were observed, but these appeared to be due to the pull which had been put upon the tissue during manip- ulation, for frequently parallel bands of fibers extended along these cells. In some preparations from a 10-day chick embryo the cells were connected by so many delicate processes that a network of these processes was formed between them, which would have been difficult to identify as cellular in origin had it not been for the fact that during the mitosis of one of these cells all the delicate network connected with the cell was partly drawn into it, and the space around the cell became free from network (plate 2, fig. 5). It thus became clear that the protoplasmic network between these cells was not extracellular in origin. The fibrils appeared first (after 24 hours’ growth) as slightly more refractive lines within the cytoplasm of the individual cells (plate 2, figs. 1 and 9). The mito- chondria were frequently stretched along these delicate lines; by careful study, however, it was seen that the mitochondria did not take part in the formation of the cellular fibrils, but that even though they stretched for a certain distance along a fibril they later separated from it. As the growth became older (48 to 72 hours) the cells become more and more densely connected by delicate processes with cells at a distance; and the refractive line of the primitive fibril appeared more and more within the cell and became partly gathered into bundles at one point or another (plate 2, figs. 1 and 2). The cellular cytoplasm became separated into an endoplasm—that is, the granular cytoplasm which contains mitochondria, fat, neutral red granules, ete.—which immediately surrounds the nucleus, and an exoplasm, or the clear, non-granular cytoplasm of the more remote surfaces of the cell (figs. 10 and 13). The delicate fibrils of the cytoplasm continued from one cell to another, usually through the exoplasm of the cell processes (plate 1, fig. 9 and plate 2, fig. 2) and appeared in the living cell as clear, slightly more refractive lines of exoplasm, extend- ing from one cell to another, and frequently across or through the exoplasm of one or more cells. As the fibrils developed from day to day the bundle became more definite and more independent of the cytoplasm of the cells, until finally it extended as a slender, clear fiber across several cells (plate 2, fig. 3), and except in cases where the DEVELOPMENT OF CONNECTIVE-TISSUE FIBERS. 55 individual fibrils can be traced into a cell, the bundle, or fiber itself, appeared quite independent of the cytoplasm of the cells (plate 2, figs. 2 and 3). No mitosis was observed in cells whose exoplasm was actively developing into fibrils during the time in which the exoplasm contained the fibrils. Mitosis, however, continued, and many cells were seen to undergo mitosis in regions where other cells were forming fibers. So far as can be determined from these observa- tions, the cell may again undergo mitosis after the bundle of fibers has become independent of the cell cytoplasm. Whether in such cases the cell actually sepa- rates itself from that part of its exoplasm which has been differentiated into fibrils or whether it simply divides the undifferentiated cytoplasm and mean- while remains attached to the differentiated exoplasm (or fibers) could not be determined. However, the cells which contributed fibrils to a fiber bundle grad- ually increased in number and extended over a wider territory, and the bundle became differentiated into a more and more definite fiber (plate 2, figs. 1 and 3). The study of the living cell, as well as of the fixed preparation, led to the idea that the fibers became more and more separated from the cells, although it is quite possible that they may merely continue through the exoplasm and become more definite, on account of the separation of the cells. Certainly in no case, in these tissue cultures, did the fiber become so well developed that the ending of the various fibrils which made up the fiber could not be traced into the exoplasm of a cell (plate 2, figs. 2 and 3). No completely differentiated fiber was observed through- out its development, although in a few instances, where the cultures were kept in a healthy condition for several weeks, fibers which resembled those of an 18-day chick embryo were developed. It seems probable that the development of these fibers was by a continuation of the process described above. Certain preparations which had been carefully studied during their growth and development were fixed and stained, and from these preparations most of the photographs and drawings have been made. A few of the living cells were drawn on successive days, and although it was frequently impossible to determine the exact cell drawn the day before, at least a cell in its near neighborhood was taken. 56 DEVELOPMENT OF CONNECTIVE-TISSUE FIBERS. MITOCHONDRIA AND THEIR RELATION TO THE CONNECTIVE- TISSUE FIBRILS. One of the most convincing arguments in favor of the view that the fibrils arise within the cytoplasm of the cells is the fact that frequently a few mitochon- dria are seen along a primitive bundle of fibrils (plate 2, fig. 2) and that occasionally a few are found isolated within a well-developed bundle of fibrils in the primitive fiber (plate 2, fig.3). So far as is known, mitochondria can not exist extracellularly. The mitochondria of the cells of the growth from a 6-day to 10-day chick embryo are usually of several types; that is, the granular, the short-rod, and the long-thread or filament type. The greatest number are filaments. Mitochondria are scattered throughout the cytoplasm and occasionally along the network of cell processes between the cells, and they may be arranged in a row along a cyto- plasmic process (plate 1, fig. 6). In a few instances a single filamentous mitochon- drium has been observed to lie along the length of such a process (plate 2, fig.3). A mitochondrium may be stretched along a fibril in such a way that in a fixed prep- aration it would be difficult to determine whether or not it took part in the forma- tion of the fiber. However, a study of the living cell shows that the mitochondria retain all their characteristic activities. They continue to bend, twist, and migrate, with the result that a mitochondrium, even though stretched for a time along a fibril so that it appears to be part of the fibril, very soon bends and later may move away. Mitochondria arranged in a row along a cell process do not necessarily remain there, but may migrate into the body of the cell again. In the older cultures the cell processes are usually free from mitochondria. In these cultures the mitochondria are more or less centralized around the nucleus— 7. e., Within the endoplasm of the cell. There is present in certain of the cells another structure, which stains in the manner characteristic of mitochondria with the various mitochondrial stains— red with Bensley’s anilin-fuchsin methylene green stain (plate 2, fig. 7), black with iron hematoxylin (plate 1, fig. 6), and purple with Benda’s method. This structure is in the form of a deposit along certain lines of the surface of the cell (plate 1, fig. 6), and is not present in the cell in its early development, but appears later along the edge or on the surface of the cell, and in certain cells, although not usually in those of subeutaneous connective tissue, frequently seems to be associated with the for- mation of fibrils. It seems probable that it is this structure rather than mitochondria which Meves (1910) had under observation when he described the formation of the fibrils of the tendon as taking place from the mitochondria after they had become arranged along the surface of the cell. In the stained preparation this structure definitely resembles the mitochondria, and it would be difficult to determine whether mitochondria take part in its formation. However, the living cell shows clearly that the structure is along the surface of the cell and that the mitochondria do not take part in its formation. Also, while the structure is fixed and stained by the same methods which fix and stain the mitochondria, it is not necessarily destroyed by the agents which destroy mitochondria, but may be present in DEVELOPMENT OF CONNECTIVE-TISSUE FIBERS. Sy preparations in which the mitochondria have been destroyed. It is very similar to the structure which forms the fibril of the muscle-cell (plate 2, fig. 6). Mislavsky (1913) was able to differentiate a plasma fibril as well as mito- chondria in the kidney tubule cells. He found that while the plasma fibrils stretched entirely across the cells as straight lines, the mitochondria did not pass to the walls of the cells. In the cultures studied mitochondria did not fuse into strands or become arranged in rows to form the connective-tissue fibrils. In all of these observa- tions, while the mitochondria at times remained caught within a bundle of fibrils, the fibrils themselves originated from the exoplasm of the cell. OTHER GRANULES AND “GRAINS DE SEGREGATION” OF THE CONNECTIVE-TISSUE CELL. - The connective-tissue cell ordinarily contains very few fat globules, and fre- quently none at all. When present they are small, round, highly refractive globules, which usually lie near the nucleus and which stain in the manner characteristic for fat. In addition to the mitochondria granules in the cells, there are a number of small, round granules, which can be distinguished from the granular type of mitochondria only by the rapidity of their movements and by certain vital dyes. These granules stain blue with pyrol-blue, purple with brilliant cresyl-blue, and red with neutral red. In the fixed preparation they frequently take certain of the mitochondrial stains, and especially do they take the same purple color as the mitochondria with Benda’s stain. The vacuoles of Lewis and Lewis (1915) correspond more or less with the grains de segregation of Renaut (1904, 1907) and Renaut and Dubrieul (1906), which these observers found to stain with neutral red and which they claimed formed fibrils through a secretory activity of the cell. These bodies are present in the connective-tissue cell, sometimes in large numbers (plate 2, figs. 2 and 3), but so far as could be determined they take no part in the formation of the fibrils. TENDON. Only a few growths of cells which could be definitely identified as tendon- cells took place, and in these growths the formation of the fibrils occurred in a manner somewhat different from that of the formation of the fibrils of the sub- cutaneous tissue. The tendon-cells were arranged as narrow, elongated cells, more or less parallel, and the fibrils developed as clear lines along the surface of the cells. These delicate lines joined into bundles from one cell to another in mark- edly parallel lines (plate 2, fig. 8). In preparations stained with Mallory’s con- nective-tissue stain the fibrils stained blue. 58 DEVELOPMENT OF CONNECTIVE-TISSUE FIBERS. AMNION. A study of the fibrils of the amnion was undertaken in order to see whether the observations of Péterfi (1914) could be corroborated in tissue cultures. Péterfi observed vacuoles within the epithelial cells of the amnion, and concluded from his preparations that these vacuoles fused together and became more numerous in the cells of the amnion of chick embryos of from 3 to 5 days’ incubation. Accord- ing to Péterfi, the walls of these vacuoles contain a substance which is different from the remainder of the cytoplasm, and as the walls fuse together they form a network of elastic fibers over the epithelial cells of the amnion of a chick embryo of 7 days’ incubation. My observations on the amnion in tissue cultures did not show this. The epithelial cells contained varying numbers of vacuoles, most of which stained with neutral red, though a few remained unstained. Fibrils are formed, but, so far as can be determined from these observations, they are formed from the exoplasm of the cell, regardless of the vacuole, in practically the same manner as are the fibrils of the connective-tissue cells (plate 2, fig. 9). CONCLUSIONS. 1. The connective-tissue fibrils begin to develop in the subcutaneous tissue of chick embryos of from 9 to 10 days’ incubation, and appear as well-developed fibers in the subcutaneous tissue of a 12-day chick embryo. The new growth from explanted pieces of subcutaneous tissue from chick embryos of 8 to 10 days’ incubation proved the most satisfactory for the study of the connective-tissue fibers. 2. The cut fibers which are present in the explanted piece of 11-day to 15-day chick embryo subcutaneous tissue do not grow either in length or bulk in the tissue cultures. 3. The new growth of connective tissue is exceedingly sensitive and reacts by a contraction of the cell from the outer edge in towards the explanted piece. This contraction does not cause the formation of fibers in the new growth. 4. Fibers are not present in the 24-hour growth from even 12-day to 15-day chick-embryo tissue, but develop in the cells of the new growth from delicate fibrils in the exoplasm of the cells after 24 hours. 5. The fibrils developed as delicate lines of the exoplasm of the cell; they became gathered into bundles which passed from cell to cell, and the bundles later passed over or through the exoplasm of several cells as a definite fiber. The fibers never became so adult that the individual fibrils which make up the fiber could not be traced into the cytoplasm of some cell, whether near or distant from the main body of the fiber. 6. Although the new growth, when closely attached to the smooth cover- slip, often takes the form of a membrane, and although this membrane exhibits the cell pattern which is characteristic for endothelium when treated with silver nitrate, nevertheless there is no evidence that these cells have become endothelial cells; they still retain the characteristics of connective-tissue cells, and many form fibrils. DEVELOPMENT OF CONNECTIVE-TISSUE FIBERS. 59 7. The mitochondria do not take part in the formation either of the fibrils or of the fibers in these cultures of connective tissue. 8. There was no evidence that the fibrils are formed by a secretory activity of the grains de segregation (vacuoles) of the connective tissue. 9. The fibrils of the epithelial cells of the amnion appeared to form in the same manner as those of the subcutaneous tissue—7. e., from the exoplasm of the cell, and not from the fusion of the walls of the vacuoles. LITERATURE. Apamt, J. G., 1908. Principles of pathology, Phila. and Lond., 1, 391. Bartsett, G. A., 1915. The origin and structure of a fibrous tissue which appears in living cultures of adult frog tissues. Jour. Exper. Med., Lancaster, Pa., xx1, 453- 479, 5 pl. , 1916. The origin and structure of a fibrous tissue formed in wound healing. Anat. Ree., x, 175. Bout, Fr., 1872. Untersuchungen iiber den Bau und die Entwicklung der Gewebe. Arch. f. mikr. Anat., Bonn, vim, 28-68, 1 pl. Cuampy, C., 1913. Note de biologie cytologique, . . . Le muscle lisse. Arch. de zool. expér. et gén., Notes et rev., Paris, Luu, 42. CuiarKeE, W. C., 1914. Experimental mesothelium. Record, Phila., vim, 95. Epewine, A. H., 1913. The permanent life of connective tissue outside of the organism. Jour. Exper. Med., Lancaster, Pa., xvi, 273-285, 2 pl. Ferauson, J. S., 1912. The behavior and relations of living connective-tissue cells in the fins of fish embryos, with special reference to the histogenesis of the collaginous or white fibers. Amer. Jour. Anat., Phila., xm, 129-149. Fiemminec, W., 1897. Ueber die Entwicklung des polla- genen Bindegewebes-fibrillen bei Amphibien und Sau- getieren. Arch. f. Anat. u. Entwcklngsgesch., Leipz., 171-190, 2 pl. Isaacs, R., 1916. An interpretation of connective tissue and neurogliar fibrille. Anat. Record, Phila., x, 206. Lewis, M. R., and W. H. Lewis, 1915. Mitochondria (and other cytoplasmic structures) in tissue cultures. Amer. Jour. Anat., Phila., xcm, 339-401. Lewis, M. R., 1916. Sea water as a medium for tissue . cultures. Anat. Record, Phila., x, 287-299. Anat. Matt, F. P., 1902. On the development of the connective tissues from the connective tissue syncytium. Amer. Jour. Anat., Balt., 1, 329-365. Meves, Fr., 1910. Ueber Strukturen in den Zellen des em- bryonalen Stiitzgewebes, sowie iber die Entstehung der Bindegewebes-fibrillen, insbesondere denjenigen der Sehne. Arch. f. mikr. Anat., Bonn, txxv, 149-208. Mistawsky, A. N., 1913. Plasmafibrillen und Chondrio- konten in den Stabchenepethelien der Niere. Arch. f. Mikr. Anat., 1. Abt., Bonn, txxxim, 361-370, 1 pl. PéterrF!, T., 1914. Beitrage zur Histologie des Amnions und zur Entstehung der fibrillaren Strukturen. Anat. Anz., Jena, xiv, 161-172. Renavt. J., 1904. Sur espéce nouvelle de cellules fixes du tissu conjonctif: les cellules rhagiocrines. Compt. rend. Société de biol., Paris, rv1, 916-919. , 1907. Les cellules connectives rhagiocrines. d’anat. micr., Paris, rx, 495-606, 3 pl. and G. Dusrevuit, 1906. Sur les cellules rhagio- crines libres du liquide des diverses séreuses. Compt. rend. Société de biol., Paris, tx, 34-37, 126-129. Roéruic, P., 1907. Entwickelung der elastischen Fasern. Anat. Hefte, 2. Abt., Wiesb., xv, 300-336. Russet, D. G., 1914. The effect of gentian violet on pro- tozoa and on tissues growing in vitro. Jour. Exper. Med., Lancaster, Pa., xx, 545-553, 1 pl. Sputer, A., 1896. Ueber die Entstehung der Bindegewebs- fibrillen und den feineren Bau den Bindegewebszellen. Anat. Hefte, 1. Abt., Wiesb., v1, 117-160, 6 pl. Watton, A. J., 1914. Effect of various tissue extracts upon the growth of adult mammalian cells in vitro. Jour. Exper. Med., Lancaster, Pa., xx, 554-572, 5 pl. Arch. 9 3. EXPLANATION OF PLATES. PLATE 1. . Photograph of the membrane of connective tissue of 48-hour growth from a tissue culture of a piece of stomach of a 6-day chick embryo. 4 oc. and 4 mm. lens. Zeiss. Photograph of cells and fibers in a film preparation of subcutaneous tissue of an 18-day chick embryo. Os. vap., iron hem. 4 oc. oil-imm. lens. Camera-lucida drawing of a living cell from a culture of subcutaneous tissue of 14-day chick embryo, 3-hour growth. 6 oc. 4 mm. lens. 4, 5. Camera-lucida drawings of living cells in a hanging-drop preparation, after Bcll, of subcutaneous tissue from a 7 | 10 14-day chick embryo in Locke’s solution. 4 oc. 4 mm. lens. 5. Retouched photograph of 48-hour growth from arachnoid tissue of 7-day chick embryo. Os. vapor, iron hem., 4 oc. oil-imm. lens. . Photograph of 48-hour growth from leg of 8-day chick embryo. Os. vap.,ironhem., and eosin. 4 oc. 4 mm. lens. . Photograph of 48-hour growth from subcutaneous tissue of 10-day chick embryo. Os. vap.,ironhem. 60c.4mm. lens. . Photograph of 48-hour growth from heart of 7-day chick embryo. Os. vap., iron hem. 4 oc. oil-imm. lens. PLATE 2. . Camera-lucida drawing of cell, showing primitive fibrils in the cytoplasm. 48-hour growth from subcutaneous tissue of 9-day chick embryo. Os. vap., iron hem., and eosin. 4 oc. oil-imm. lens. Drawn by Miss J. E. Lovett. . Camera-lucida drawing of cells and fibrils united into bundles. 72-hour growth from subcutaneous tissue of 11-day chick embryo. Os. vap., iron hem., and eosin. 4 oc. oil-imm. lens. Drawn by Miss J. E. Lovett. 3. Camera-lucida drawing showing cells with fibrils within the cytoplasm and also fibers. 120-hour growth from subcutaneous tissue of 11-day chick embryo. Os. vap., iron hem., and eosin. 4 oc. oil-imm. lens. Drawn by Miss J. E. Lovett. . Camera-lucida drawing of cells from 24-hour growth of leg of 8-day chick embryo. Os. vap., iron hem., and eosin. 4 oc. oil-imm. lens. . Camera-lucida drawing of cell undergoing mitosis in 48-hour growth from subcutaneous tissue of 11-day chick embryo. Os. vap., iron hem., and eosin. 4 oe. oil-imm. lens. j. Camera-lucida drawing of smooth muscle-cells, showing myofibrils from 48-hour growth of amnion of 5-day chick embryo. Os. vap., ironhem. 4 oc. oil-imm. lens. . Camera-lucida drawing of cell from 24-hour growth of 6-day chick embryo, showing deposit along surface of the cell, which is stained like mitochondria with Bensley’s aniline fuchsin, methylene green stain. 4 oc. oil-imm. lens. . Camera-lucida drawing of tendon-cells from 72-hour growth of muscle of 9-day chick embryo. Zenker’s fixation— Mallory’s stain. 4 oc. oil-imm. lens. . Camera-lucida drawing of epithelial cells of 48-hour growth from amnion of 5-day chick embryo. Iodine-vapor fixation—Mallory’s stain. 4 oc. oil-imm. lens. Camera-lucida drawing of thin membrane of connective tissue from 72-hour growth of 10-day chick embryo. Silver nitrate and Ehrlich hematoxylin. 4 oc. and oil-imm. lens. 60 UEwIs PLATE 1 ee ° u éw =. Saen + f A ~~ . .* ¥ Ss °, ‘ ** wa * f ~~ >s ba } . a» ’ \ LEWIS PLATE 2 AHOEN &CO BALTIMORE, J. E. Lovett et CONTRIBUTIONS TO EMBRYOLOGY No. 18. ORIGIN AND DEVELOPMENT OF THE PRIMITIVE VESSELS OF THE CHICK AND OF THE PIG, By FLorRENCE R. SaBIN. With seven plates and eight figures in the text. CONTENTS. Introduction. 225.).../aasce cst eee Ce EEE Eee sb ybtsie Sere eg ohs Oe eer ae Vascular system: of ithe chicls: -/. =tj5 5268 tad age Rhea es ante tena eee eee ee ees anor General account of the vascular system of the chick up to stage of 14 somites................ Origin of the cardinal veing 3324. S042. Ghe.8; din cece ogee deeb Berens were Tear Vascular system of the brain and the primary head-vein vena ote ahs Development:of the spinaliartertesscc 22023 5-2 «ais sisjars soci ste els 5 = SreGie esto nae tel ere ane eee eo0 The vaeoular myatem in ye Rng ie SmBEYO8 oe ee a a ee The form ‘of the hearts. 5.50.22 cthta cette bee antes ea oe Re ee eee ree a Res Ventral branches of the aorta, including the allantoic arteries and the Sentinal artery. . sees The umbilical yesselas 2.3.5 steaestsscetaten oe ae eee eke lx ate BOP AOC! Neural branches of the aorta and the primary head-vein........ aS Snsoee eo ono. es ; The cardinal veins:ini the pigs...30-0 oe. ae Saree eee eee LN Nephiritic: vessels in the pig 5 ¥0.5.2 45.5... pisses note pte a gee eee eee aoe ot Ce : 62 rae ORIGIN AND DEVELOPMENT OF THE PRIMITIVE VESSELS OF THE CHICK AND OF THE PIG, By FLorence R. Sasin. INTRODUCTION. In this paper is given an account of the primitive vessels of the chick and of the pig, as made out by injecting living embryos, and, in the case of the chick, as seen growing in the embryo. Such studies must necessarily be accompanied by the study of sections. In the case of the mammalian embryo I have made injections in earlier stages than had been done heretofore and in the case of the chick I have carried the method of injection to the earliest stage in which it is possible. Below the stage at which they can be injected, the vessels of the chick can be studied in the living blastoderm by a technique which has developed out of the method of tissue-culture introduced by Harrison. The chick thus offers unusually valuable material for the study of vascular problems, as it is possible to use both the method of injection and that of direct observation of the living embryo in the same stage. In the course of this study two fundamental ideas have been under considera- tion. The first concerns the most essential question in connection with the vascu- lar system, namely, the relation of differentiation and growth of endothelium. According to one theory there is a limited period for the differentiation of angio- blasts out of undifferentiated mesenchyme, and after this period all new blood-vessels arise from the growth or proliferation of older angioblasts. This theory seems to me to have the weight of evidence. The second theory is that angioblasts continue to differentiate out of mesenchyme indefinitely. If the former theory is correct and the period of differentiation of endothelium is a limited one, the fundamental prob- lem concerning the early blood-vessels is to determine which differentiate and which are formed from preceding vessels. In practically every embryo chick observed, up to a certain stage new angioblasts can be seen differentiating and joining the older angioblasts, but the phenomenon becomes less and less frequent as older stages are studied. In the living embryo the aorta itself can be seen to differentiate out of mesenchyme, and at the stage when the heart begins to beat every chick shows a few isolated angioblasts along the mesial border of the aorta, which will be seen to join the aorta if the specimen be watched for a short time. I have some evidence also that some of the primitive vessels along the neural tube differentiate out of mesenchyme, the process being observed in the living embryo. On the other hand, one can watch the growth of the entire wall of a vessel by cell-division in the living embryo and the formation of new vessels from the walls of old vessels; so that the study of the early blood-vessels is gradually becoming a more exact problem, namely, the determination for each vessel, whether it differentiates in situ or develops from preceding vessels. My present material is not adequate for the solution of this question, but throws some light upon it. O4 DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. The second question—which has proved of great interest—is the definition of the terms artery, vein, and capillary as they are used for the embryo. In the study of the vessels of the embryo particular stress should be laid on the time when circu- lation begins. That there is a very extensive development of blood-vessels before there is any circulation of the blood due to the beat of the heart was well known to the earlier embryologists; for example, to von Baer and later to His. Moreover, the heart beats for a considerable time before it starts any circulation. It is known that the blood-vessels spread over the body in definite and constant sheets of eapil- laries, and in these primitive vessels, after the circulation has begun, a vessel may serve as an artery for a time and then be reduced to a capillary plexus, in which the direction of the circulation is entirely different from that of the circulation of the original artery. Such a vessel, for example, is the subintestinal artery of the pig, - which arises in a capillary plexus around the caudal end of the primitive gut and carries blood out to the arteries of the yolk-sac, where it must again pass through a capillary bed before it returns to the heart. This artery becomes broken into a capillary plexus in the wall of the gut, which makes new connections with branches of the omphalo-mesenteric veins within the wall of the mesentery, so that its blood, instead of flowing away from the embryo to the membranes, flows within the embryo toward the heart. Again, a vessel may serve for a time as a vein in the return of blood to the heart and may subsequently receive new arterial connections and become an arterial plexus, with the direction of the flow of blood entirely changed. Such a vessel is the so-called vena capitis medialis. This is a primitive vessel along the hind- brain, which in the chick in the second and third days of incubation serves as a vein for the forebrain and midbrain, but as an arterial capillary trunk for the hind- brain; that is, it carries mixed blood and is the only vessel of the hindbrain, representing its entire capillary bed. Early in the fourth day it receives new arterial connections, a new vein develops to carry the venous blood for the fore- brain and midbrain, and the primitive vascular channel of the hindbrain breaks into a capillary plexus in which the direction of the current of blood is at right angles to the direction of the original current. From these two examples it must be clear that in the study of the primitive vascular system it is very important to understand the function of the vessels at each stage of development, and any presentation of the vascular system which overlooks this point and is dominated wholly by the pattern of the vessels of the adult becomes difficult to follow and may be misleading. In the question of nomenclature a decision has to be made between two theories—that is, whether the vessels are to be named according to the function they perform at any given stage or whether they are to be named according to the vessels for which they form the primordia. If the latter method is chosen it must be remembered that a given vessel of an embryo often disappears entirely in giving rise to new ves- sels—for example, the primitive vessel of the hindbrain. In this study I shall use terms as consistently as possible, in the following manner: By the term artery, in reference to an organ, I mean a vessel which brings blood to that organ but does not form any part of its capillary bed, and I have colored such vessels red. By the term vein I mean a vessel which carries blood from DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 65 an organ to the heart, provided it does not serve as the capillary bed for that organ or break up into another capillary plexus. Such vessels I have colored blue. All other vessels I have indicated in gray, and shall try to trace the complicated changes which such vessels undergo, serving at times as arteries, veins, or capillaries, or as vessels with a double function. This method of nomenclature is therefore based on the function of the vessel at the stage when it exists. It takes into account the very shifting course of the blood, as the vessels develop in the embryo better than does the method of making a too early identification of the adult vessels in those of the embryo. This usage of terms serves to restrict especially the term vein as applied to the embryo. The meaning of the terms artery and vein, as applied to the embryo, will become more clear in the discussion of individual vessels. METHODS. As has been stated, in this study I have followed two methods: first, that of the injection of embryos, and second, the method of studying the living blasto- derm in the case of the chick in a hanging-drop preparation. A general account of the methods of injecting embryos will be found in my paper on the ayzgos veins published as ‘‘Contribution to Embryology, No. 7,”’ by the Carnegie Institution of Washington in 1913. All of the injections of young embryos are made by blowing ink into the vessels through a very fine canula. To inject the young chick the shell is opened and the embryo exposed to a strong light under a binocular microscope. A few drops of warm Locke’s solution are placed on the blastoderm, and the vitelline membrane is removed. By the time the chick has 14 somites the sinus terminalis, or marginalis, is well developed in the edge of the area vasculosa and can easily be punctured with a fine canula. Nevertheless it is not easy to obtain complete injections of the blood-vessels of the chick through the veins until the embryo has about 16 somites. In stages between 9 and 16 somites more complete injections can be made by puncturing the aorta directly. This is a very interesting point in connection with the time of the beginning of the circulation. I shall show that, though the heart com- mences to beat about the time the tenth somite is forming, the circulation does not begin until about the stage of 16 somites. From the time the circulation begins it is easy to get complete injections by blowing a little ink into the vitelline veins and allowing the heart to pump the ink through the vessels of the embryo. If total specimens are desired it is well to dilute the ink one-half, so that the superficial vessels will not become so dense as to obscure the deep ones. I shall discuss in this paper the effects of injection in the embryo before the circulation has begun. The earliest chick embryo which I have injected was one of 9 somites; and I believe this stage to be about the youngest to which the method is applicable. At the stage of 6 somites the dorsal aorta is in the stage of a plexus of angioblasts, many of which are still solid cells. This plexus of cells gradually acquires a lumen and becomes the aorta during the stages of from 6 to 9 somites. All stages up to 18 or 19 somites can be studied to great advantage in hanging-drop preparations. 66 DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. Direct injections into the aorta can be made in the following manner: When the embryo is placed in a strong light the myotomes are of course very plainly visible, and along their lateral border can be seen a faint opaque streak, which is the intermediate cell-mass or nephrotome. Between the nephrotome and the lateral border of the myotomes is a thinner line which is more transparent. The canula is then introduced between the lateral border of the myotomes and the intermediate cell-mass, with the point toward the head of the embryo. If the canula enters the aorta, and only a slight pressure is used, there need be no extravasation at the point of puncture. After the embryo has been injected it is fixed by dropping Bouin’s mixture on the specimen while it is still on the yolk. It is kept flooded with the fixing agent, and is not removed from the yolk until it is well hardened. In regard to the injection of young mammalian embryos there are a few special points in technique which are of interest. In order to identify the young embryo pig the mucosa is examined carefully for long strings of chorion, which are so inconspicuous that they are more readily found by running the finger over the mucosa than by sight. These strings of chorion are then very carefully coiled on a glass slide or piece of filter paper until the embryo is found. In the case of the pig, I have never succeeded in puncturing the veins on the yolk-sac or the umbilical vein. The latter is so large that it might be punctured if it contained enough corpuscles to render it visible. The aorta, on the other hand, is readily punctured opposite the mid-body region. Here in early stages the two aorte seem slightly dilated, or later are fused into a single vessel. The needle is introduced ventral to the myotomes. The injection mass in every case was india ink. Silver nitrate seems to damage the tissue much more markedly in very young embryos than in later stages. After injection the pigs were fixed in Carnoy’s mixture of absolute alcohol 6 parts, chloroform 3 parts, and glacial acetic acid 1 part. They were then placed in 80 per cent alcohol, dehydrated in graded alcohols, and cleared by the Spalteholz method of benzine followed by oil of wintergreen. The study of the blastoderm of the living chick embryo in a hanging-drop preparation depends on the methods originated by Harrison and developed by a large group of workers—Burrows, Carrel, M. R. Lewis and W. H. Lewis, and others. In 1912 McWhorter and Whipple applied the method to the study of the growing blastoderm of the chick, which was its first application to the entire embryo, so far as lam aware. These investigators mounted the blastoderm in clotted plasma and used the method to test the question as to whether blood-vessels arise from fusion of isolated vesicles. In 1913 Brachet published a study on the growth of a mammalian embryo in a hanging-drop preparation. In studying the blastoderm of the chick by the method of the hanging-drop I have followed the technique of Margaret Reed Lewis, of growing the embryo in Locke’s solution. In this way the embryo can be kept growing for several hours, and the cells which are nearest the cover-slip can be seen with great clear- ness and followed with an oil-immersion lens. The embryo is removed from the yolk and placed in a dish of warm Locke’s solution and the vitelline membrane DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 67 and most of the yolk removed. It is more difficult to study the blastoderm with the dorsal surface against the cover-slip than from the ventral aspect, because the ectoderm does not adhere to the glass as well as does the endoderm, and it is necessary to have the embryo very flat in order to see the cells with higher powers. In regard to the blood-vessels, it is of course preferable to study the embryo from the ventral aspect, since the blood-vessels are nearer the endoderm than the ectoderm. In the study of the developing blood-islands it would be very advan- tageous to have the dorsal surface of the embryo against the cover-slip, as the blood-islands are farther dorsal and because the cells of the ectoderm over the area opaqua are much thinner than the cells of the endoderm over the same area. The cells of the endoderm of the area opaqua are so thick and so filled with glob- ules of yolk that one can seldom focus through them in the living embryo. From the ventral aspect there is also an area of the axis of the embryo that it is very difficult to study with high-power lenses, namely, the portion of the axis just caudal to the head-fold, because the heart lifts the embryo from the cover-slip and the cells can then be studied only with dry lenses. All of the chicks have been fixed in Bouin’s mixture of saturated aqueous picrie acid 75 parts, formalin (40 per cent) 20 parts, and glacial acetic acid 5 parts, for about 12 hours. They are then placed directly in 60 per cent alcohol. In the ease of the chicks which have been growing on the cover-slip it is very necessary to have the embryo stick to the cover-slip throughout the fixation and dehydration. If the specimens are to be mounted in toto, they are mounted on the same cover-slip on which they were growing. If they are to be embedded and cut they can be removed from the cover-slip after they have been cleared in the oil of wintergreen. The specimens do not become as brittle in the oil as in xylol. In the blastoderms which are kept on the cover-slips it is possible to watch the effects of dehydration much more accurately than with free specimens. The edge of the specimen around the outer margin of the area opaqua clings very closely to the cover-slip; in fact, in mounting the embryo strands of tissue are pulled out which dry slightly and help the specimen to remain fixed. If the specimens are put into alcohol weaker than 60 per cent this outer margin will stick to the cover-slip, but the entire area pellu- cida will become free from the cover-slip and swell into a bleb. The space beneath fills in with fluid, and in the subsequent dehydration there is an uneven shrinkage which distorts the tissues. Thus, weak alcohol or water, or a dilute stain, macerates and swells the tissues and the subsequent shrinkage distorts the cells. On the other hand, if the specimens are placed directly in alcohol as strong as 60 per cent, a plexus of cells, which has been studied in the living specimen, can be readily identified in the fixed specimens. If the specimens are to be stained in toto they must be placed in a stain sufficiently strong so that the tissues will not macerate before they react to the stain. From such an experience one should avoid washing embryos in water and should also avoid the use of the lower grades of alcohol. I have used several changes of 60 and 65 per cent alcohol to eliminate the excess of picric acid. The dehydration can be done by changes of 5 per cent. If it is carried out too rapidly the specimens will crack, but the shrinkage with the higher grades OS DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. of aleohol is by no means as marked as in the case of older embryos. The early embryos are injured much more by maceration due to weak alcohol than by shrink- age due to too rapid dehydration in the stronger grades. The specimens are all cleared by the Spalteholz method of benzine followed by oil of wintergreen. Speci- mens can be embedded from oil of wintergreen if they are passed directly from the oil to a mixture of the oil and paraffin. The tissue does not become too brittle to cut even after remaining in the oil for a year or two. VASCULAR SYSTEM OF THE CHICK. GENERAL ACCOUNT OF THE VASCULAR SYSTEM OF THE CHICK UP TO STAGE OF 14 SOMITES. In the study of the origin of the vessels of the chick I shall begin the detailed account with the stage of 6 somites. The study of the blastoderm in a hanging-drop preparation offers a valuable method for a study of the early stages. In the stages of the early somites there is a plexus in the area opaqua which, by the older embry- ologists, Pander, von Baer, Remak, and later by His, was identified as the fore- runner of the blood-vessels. Basing his studies.on those of von Baer and Remak, His gave a description of the origin of blood-vessels which remains the foundation of our knowledge upon this subject (1868, pages 95 to 100). He described the first appearance of blood-vessels, or, as he later termed them, angioblasts, as occurring just before the appearance of the somites. He stated that the vessels began as a plexus of angular or spindle-shaped solid cells in the area opaqua. These cells from the beginning were in the form of a plexus (Gleich von Anfang an ein geschlossenes Mosaik, page 98). The plexus was at first made up of solid cells without a lumen, and grew by processes of solid spindle-shaped cells, exactly similar to those which formed the original network. This plexus was in a definite layer—the vascular layer (das Gefissblatt) of Pander. The vascular layer, His said, consisted not only of solid angular cells, but also of elements having a yellow color, or, in other words, it gave rise not only to blood- vessels but also to blood-cells. He regarded it as of the greatest importance that the first appearance of vessels was in the area vasculosa before the heart formed, and that these vessels arose entirely independently of any circulation. He then noted that the plexus of solid cells became transformed into vessels, the exact method of the transformation being impossible to determine; but that as the solid cells became the walls of vessels, their cytoplasm became less granular and their nuclei flatter. He then described the approach of the blood-vessels toward the axis of the embryo by means of the same type of solid processes which formed the original plexus and found that they approached the axis in two zones: first, oppo- site the myotomes, and secondly, along the splanchnopleure in the region opposite the future emphalo-mesenteric veins—that is, over the ventral surface of the two amnio-cardiac vesicles. His noted that over the region of these amnio-cardiac vesicles there was a double sheet of vessels which approached the axis of the embryo, a more scanty sheet DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 69 in the somatopleure, and a more abundant sheet in the splanchnopleure. Because of the development of the head-fold and the heart, it was impossible that the approach of the vascular plexus should be uniform over the entire length of the embryo. For example, the most cephalic part of the head became cut off from direct lateral connection with the embryonic membranes, and the vessels which approach the heart gradually rotated from the direct transverse direction to an oblique angle. He then noted that the dorsal aorta developed in the mesial edge of the plexus of angioblasts of the area vasculosa along the line of the lateral edge of the myotomes. From these observations he concluded that the vessels of the embryo are derived from the vessels of the membranes, and that the portion of the axis which can not be seen to receive the plexus of primitive angioblasts from the membranes receives its vessels by a growth of the plexus which has already invaded the embryo at other places. In following the differentiation of the vascular area by improved methods whereby one can watch the living cells growing under an oil-immersion lens, it is astonishing how accurate is this description of His, which must have been made by far cruder methods. To his description must be added that with finer methods it is seen not only that the plexus out of which the aorta develops is the border of the common plexus of the entire area vasculosa, but that new cells differentiate along the axis of the embryo as well, so that angioblasts differentiate over the entire zone from the outer edge of the area opaqua to the margin of the future aorta along the lateral border of myotomes. Thus His’s description must be extended to include a dif- ferentiation of new angioblasts in the axis of the embryo itself. In the living blastoderm over the area opaqua, the endoderm-cells are so thick and so filled with yolk that the development of the blood-vessels and the blood-cells beneath them can be followed only with great difficulty. In the area pellucida, on the other hand, the endoderm is thin, and during the periods when the endoderm cells are not dividing they are so clear that it is easy to focus through them. At the stage of 6 somites the head-fold is well formed and the amnio-cardiac vesicles have met in the mid-ventral line. Along the axis of the embryo there is a zone of dense tissue radiating from the primitive streak and from the embryo cephalic to the streak. This denser mass of tissue divides the area pellucida into an inner thicker zone containing the axis of the embryo and an outer thinner zone. In sections it is clearly seen that this denser zone is due to the further development of the ccelom nearer the embryo. Over the cephalic part of the denser zone the ecelom has a wide lumen, and both its ventral mesoderm and the endoderm are thicker than the same membranes farther lateralward. This is very plainly shown in Duval’s Atlas, plate x1v, figure 218, in the zone extend- ing outward from his letter b. Farther caudalward in this dense band, opposite the undifferentiated myotomes and the primitive streak, there is no cavity of the ecelom, and its dorsal and ventral mesoderm are fused and form a dense mass of cells. This entire thicker zone is difficult to study in the living chick, but the whole outer margin of the area pellucida is clear and the cells are so thin that 70 DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. one can readily focus through them from the endoderm to the ectoderm, and see every cell of the entire zone. This is true, however, only when the endoderm is not dividing. The endoderm cells divide as a whole, and during the entire phase of cell division they are so opaque that it is impossible to focus through them. The phase requires about an hour, and to study the vessels beneath it is necessary to await until the cells become clear again. In the entire outer margin of the area pellucida, at the stage of 6 somites, there are two plexuses. The dorsal plexus, which is the developing ccelom of this area, appears to be composed of very large and flat vessels. Distinctly ventral to this plexus of the ccelom is another plexus, much less abundant and made up of solid bands of cells, which are angioblasts. An exceedingly important point, which can be determined with great distinctness in the living specimen, is that the plexus of angioblasts connects by many tiny filaments with the plexus of the mesoderm of the ccelom, but never connects by filaments with the endoderm. In sections the angioblasts of the vascular layer often touch the endoderm, but in the living embryo they are always separate. The living specimens also bring out very sharply the fact that the entire layer of angioblasts is distinctly ventral to the plexus of the mesoderm; in other words, the term vascular layer of Pander is an appropriate one, for the filaments of the angioblasts can be seen to dip down from the vascular layer to the mesoderm beneath. In the flat living specimen, and in sections which have been made from a specimen which was growing out flat on a cover-slip, there is no intermingling of the mesoderm and the vascular layer, such as is seen in Duval’s plate xvi, figure 264. Such an apparent inter- mingling of the two layers is due to shrinkage. In other words, the angioblasts differentiate out of the mesoderm and form a new layer, which is throughout ventral to the mesoderm. These two plexuses were well known to His, who recognized them in their relations to the ccelom on the one hand and to the angio- blasts on the other in his work published in 1868, but described them more fully in the Lecithoblast und Angioblast published in 1900. His stated that the two plexuses were at times very hard to analyze. The plexus of angioblasts is, then, distinguished first by its more ventral position, and secondly by the fact that the cytoplasm of the angioblasts is slightly more granular and reacts slightly more intensely to basic dyes than does the mesoderm. The following criterion, however, is the one which I have found most useful. In the living specimens there seems to be a sort of rhythm in cell division. I have already referred to the fact that the entire endoderm may divide and become so opaque that none of the cells beneath can be seen. At other times the entire plexus of angioblasts over a very extensive zone will pass into the phase of cell division. In this condition the cytoplasm of the plexus of angioblasts becomes very highly refractile and opaque, so that it can be distinguished from the plexus of the coelom with great ease, even with low powers of the microscope. The protoplasm shows this change for about an hour before the chromosomes pass on to the spindle; so, in order to obtain the nuclear figures characteristic of cell division, one must watch until a few areas in the plexus begin to clear and DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 71 then fix the specimen. The time required for the nuclear changes is much less than the time taken for the cytoplasmic changes. According to M. R. Lewis, the nuclear phase lasts about 5 minutes, while the cytoplasmic change takes about an hour. The facts that not every nucleus divides at the same moment and that the cytoplasmic changes have not been recognized explain the failure to note the rhythm of cell division. Using the criteria for distinguishing angioblasts which have just been indi- cated, I will now describe what has been made out concerning the vascular system at the stage of 6 somites, both in the living specimen and in sections which have been made from a blastoderm in which the cells had been charted in the total specimen before the sections were cut. For this description the axis of the embryo may be divided into four zones: (1) that part of the head which is covered by the head-fold, as seen from the ventral aspect; (2) the head between the head- fold and the first myotome; (3) the zone of the myotomes; (4) the zone caudal to the myotomes. As has been described, there is a dense band of tissue on either side of the axis of the embryo which divides the area pellucida into an inner dense zone and an outer thinner zone. The area opaqua, on the other hand, is denser along its outer margin. Beginning with the area opaqua, in its outer margin there is a large marginal plexus of vessels partly filled with blood-cells which cling in large masses to the dorsal wall of the vessels. The blood-cells can be distinguished from the angioblasts by the fact that in the edges of the masses they tend to separate from the mass and have a definitely round contour. Angio- blasts never have a round contour. In this marginal zone the ecelom is clearly seen, with its dorsal and ventral mesoderm, and the ventral wall of the blood- vessels is very plainly distinguished from the endoderm; but the dorsal wall of the blood-vessels is closely attached to the ventral mesoderm, and in places can not be distinguished from it. The inner margin of the area opaqua and the outer margin of the area pellu- cida have two definite plexuses: the dorsal plexus of the ecelom and the scantier ventral plexus of solid angioblasts. Over the dense area on either side of the myotomes the ccelom is no longer in the form of a plexus, but has a complete lumen; for there the body-cavity is well formed. The plexus of angioblasts covering this area is continuous with a plexus of angioblasts along the lateral margin of the myotomes. Caudal to the sixth myotome, the plexus extends for a short distance along the undifferentiated mesoderm, curving a little to the side. Very interesting appearances are to be made out near the first myotome. Extending forward from the lateral border of the first myotome, the chain of angioblasts representing the aorta can be seen up to the margin of the head-fold, when it disappears under the fold. Opposite the first myotome, and extending forward from its mesial border, there is also a chain of angioblasts along the hindbrain, and this chain of angio- blasts connects with the aorta above the first and between the first and second myotomes. The chain of cells along the margin of the hindbrain I should not recog- nize as angioblasts in sections; but in the living blastoderm they have exactly the appearance of the angioblasts of the aorta and connect with them by slender filaments. ~J] bo DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. In the region of the head, which can not be analyzed in the living blastoderm, the angioblasts representing the heart are well known and easily identified. The two cardiac primordia have met in the mid-ventral line and can be followed a short distance into a ventral cephalic aorta, which gradually becomes too indefinite for recognition. The dorsal cephalic aorta is very clear opposite the region of the heart, gradually disappearing farther forward. Thus, within the embryo there are chains of angioblasts representing the heart, most of the ventral cephalic aortz, and a part of the dorsal cephalic aortee. Opposite the region of the heart the two dorsal aortz are definite, tiny vessels which emerge from under the head-fold and are continued partly as a plexus of solid angioblasts and partly as a vessel along the ventro-lateral border of the myotomes. The entire plexus which is exposed on the ventral aspect connects with the plexus of angioblasts of the area pellucida. In this account I wish to emphasize the very early appearance of angioblasts along the hindbrain—the forerunner of the so-called vena capitis medialis, which I prefer to call the primitive vessel of the hindbrain. I have not yet a sufficient number of observations to prove, first, whether the transitory vessel of the hindbrain does differentiate in situ while the aorta is differentiating, and secondly, whether it is established earlier than the vessels of the forebrain; but both of these propositions seem to me to be very probable. In this study I have found Williams’s (1910) very careful description of the vas- cular system of the early chick embryo of great value. His specimen of 6 somites is clearly a little farther advanced than mine. He found that at 6 somites the aorte were established, but were still small and irregular. He then observed a vessel along the neural tube (hindbrain) connected with the aorta in the first and second interspaces, the vessel in the first interspace being nearly as large as the aorta itself. It is now important to consider how the plexus of angioblasts increases. This occurs first by cell division and secondly by the differentiation of new angioblasts. Cell division in the plexus of angioblasts is very extensive, for in watching the living specimens it is seen that large areas of the plexus divide at the same time, and in these cycles of cell division every cell of the plexus divides. Besides this very exten- sive cell division new angioblasts differentiate and join the plexus. This process can best be observed along the mesial border of the dorsal aorta itself, near the lowest myotome. Here practically every blastoderm between 6 to 10 somites will show one or two isolated angioblasts which are very readily marked from the dense mesoderm beneath. Out in the zone of the developing ccelom the distinction is by no means so easy. These angioblasts are either single, spindle-shaped cells or clumps of two or three cells. When observed they are seen to put out tiny filaments toward the wall of the aorta, which at once responds by putting out a filament toward the young cells. These tiny filaments meet halfway, and the new angioblasts thus join the wall of the aorta. They gradually approach the wall and become incorporated into the vessel. As the new cells become a part of the wall their protoplasm becomes less granular and they acquire a lumen. The exact process by which angioblasts acquire a lumen is extremely difficult to determine, and concerning this point nothing has yet been added to the original deseription of His. DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 73 These observations on the origin of the aorta, as well as the observations indicating that the transitory vessel of the hindbrain differentiates from angio- blasts in situ, at once lead to the general question of the origin of the vascular system All are agreed—on the foundation of the work of von Baer, Remak, and His—that certain cells of the embryo differentiate to form angioblasts or vasoformative cells in the early stages of embryonic life, and that these angioblasts increase by cell division. There has, however, been a wide divergence of opinion as to whether the differentiation of new angioblasts continues throughout life or whether there is a limit to the period of differentiation, after which all the new angioblasts must come from the growth of preceding endothelium. It is in relation to these two theories that I am making these studies on the living blastoderm. It is, I think, clear that the study of blood-vessels in the stages of their differentiation does not prove that they continue to differentiate out of mesoderm throughout life, any more than the finding of several primordia for the thymus proves that new thymus glands continue to arise throughout life. The question of the origin of the blood-vessels is now an exact one—namely, which vessels arise in the embryo (as does the aorta, at least, in part) by differentiation of angioblasts, and which grow from previous vessels. In other words, how long does the period of differentiation of angioblasts continue? His formulated the theory that the embryo itself is invaded by angioblasts from the yolk-sac. This theory was based on the following observations: First, that along the myotomes in the early stages angioblasts can be seen streaming toward the axis of the embryo from the outer margin of the area pellucida; second, that he observed no such streaming of angioblasts toward the axis of the embryo in the zone between the head-fold and the first myotome (here, as a matter of fact, a few angioblasts can be found in early stages, but are much scantier in number than lower down); and third, that the most cephalic part of the head does not receive angioblasts from the membranes. From these observations he concluded that the vessels of the axis of the embryo must arise from a growth of the angioblasts which could be seen to enter the embryo at certain places. Although these observations of His are for the most part correct, that a differen- tiation of new angioblasts does take place along the axis of the embryo was shown by two series of experiments. First, those of Hahn, who cut out the membranes of one side of a chick in the stage of the primitive streak and obtained a few specimens in which the membranes were entirely lacking, but the aorta was formed on the injured side. Second, the experiments of Reagan, in which he cut off a part of the head of the chick in the stages just before and just after the head-fold is visible, and allowed the isolated parts to remain in the egg and develop. In these isolated fragments he obtained vessels. The fact that angioblasts do differentiate in the axis of the embryo is con- clusively proved by my observations, having watched certain cells differentiate and join the aorta in the living blastoderm. In what I have called the second zone of the axis of the embryo—that is, the zone between the head-fold and the first: myotome—the process can not be followed with such minute detail as is 74 DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. possible opposite the myotomes, because in the former case the heart lifts this zone of the embryo from the cover-slip; but every specimen shows chains of angioblasts which are apparently differentiating in situ in this area. None of the experiments or observations here recorded take into account the ultimate point of origin of the cells which differentiate into angioblasts. By the time the chick has 9 somites the dorsal aorta is readily seen behind the head-fold as a complete vessel if the living embryo be viewed from the ventral aspect. Opposite the upper myotomes the aorta is directly ventral to the myo- tomes, but it gradually curves outward, so that opposite the ninth myotome and the undifferentiated mesenchyme it lies along the lateral border of the myotomes. Along its entire lateral border it is connected with the plexus of the area pellucida. As has been shown by Evans, the entire caudal portion of the aorta is a part of the capillary plexus of the area pellucida. In summing up the question of the origin of the aorta it may be said that it. differ- entiates as a part of the plexus of angio- blasts, extending over the entire area vas- culosa, and is increased by the addition of new angioblasts along the axial line of ,~/ the embryo. ; By the time the chick has 9 somites the aorta can be injected; it forms from the plexus of angioblasts while the seventh, Fic. 1.—Transverse section of an injected chick of 12 : a . A somites, passing through the middle of the mesencephalon, eighth, and ninth somites are forming. to show the vascular plexus on the mesencephalon. On sre - the left, the dotted area shows how far the ink passed While the dorsal aorta of the region of through a dorsal artery from the aorta into the plexus on : < 2 : 2 ahs the midbrain. The section is from the same series as the my otomes 1s best seen 1m the living those in figures 2 and 3, and it is to be compared with the chick, the cephalic aorta is best ob- total preparations shown on plate 1, figure 2, and on ) plate 2, figure 1. The section is 504 thick and is un- served in an injection. As can be seen stained. 133. A. me., artery to the plexus on the = = a 5 E mesencephalon; Ao. d. c., aorta dorsalis cephalica; Ao. In plate il. figure 3, the heart is a simple ». c., aorta ventralis cephalica; Me., mesencephalon; P, tube. In some specimens, even with pharynx; Pl. me., plexus on the mesencephalon. 10 somites, it is in the exact mid-ventral line; in others, as in plate 1, figure 3, it is slightly to the right. In some of my injections the ventral aorta has numerous mesial and lateral sprouts; in this particular specimen these sprouts are more numerous along the dorsal cephalic aorta. In one of my injections the heart itself shows a little of the primitive plexus. The dorsal cephalic aorta shown in plate 1, figure 3, is still in the form of a plexus; from the arch of the aorta two very constant sprouts extend to the ventral surface of the forebrain. The development of these sprouts is well shown in a figure by Evans from a duck embryo with 13 somites (fig. 398) in the ‘‘Manual of Human Embryology”? (Keibel and Mall). The mesial sprouts do not form permanent vessels; but in one very interesting abnormal embryo which I injected these mesial sprouts had formed anastomoses across the mid-line. They are thus, in this specimen, analogous to the vessels DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 75 which cause the fusing of the two aorte lower down. Opposite the region of the heart some of the lateral sprouts extend out in the somatopleure, as shown in plate 2, figure 1, and in sections in Duval’s plate xvu, figure 276. Opposite the midbrain some of these lateral sprouts may connect with the superficial plexus. The dorsal cephalic aorta itself, as seen in plate 1, figure 3, is very large. From the dorsal aspect it is broad and flat and is placed in a nearly exact trans- verse axis instead of in the oblique position which it subsequently assumes. The next stages in development, including the relations of the primitive cere- bral vessels and the cardinal system of veins up to the stage of 14 somites, I shall describe with the aid of two total preparations from chicks of 12 and 14 somites and three sections from the stage of 12 somites (figure 2 of plate 1, figure 1 of plate 2, and text-figs. 1, 2, and 3). At the stage of 12 somites the aorta is very readily injected. The vessels to the brain, however, though they connect with the aorta, are diffi- cult to inject. In plate 1, figure 2, is shown the usual result of injecting a small quantity of ink into the omphalo-mesenteric veins at the stage of 12 somites; the ink passes through the heart and the aorta into the capillaries, which are the fore- runners of the omphalo-mesenteric Fic. 2.—Transverse section of an injected chick of 12 somites, passing . able through the first interspace to show the relations of the primitive arteries. This is true, even though vessel of the hindbrain, the transverse vein of the first interspace, vessels to the entire brain—that is, {24 anterior cardinal vein. The section is rom the same series to the forebrain, midbrain (text-fig, sation shown on pat 1. Sure 2 and on late 2 Sure 1 The 12 and hindbrain (text-fig. 2)—are salis cephalica; C, ccelom; P, pharnyx; V. regs, v. eardinalis ante- . rior; V. om., v. omphalo-mesenterica; V. so., vein of the somato- present and connect with the aorta, pleure; V. ¢., v. transversa of the first interspace; Va. p. r., vasa although the common cardinal vein primitiva rhombencephali; Ven. c., ventriculus cordis. is present down to the twelfth interspace (text-fig. 3) and the entire lateral border of the aorta opposite the myotomes is connected with the plexus of the area vascu- losa. In plate 1, figure 2, the only branch of the aorta injected is an unusual dorsal branch opposite the tenth somite, passing out into the somatopleure. In order to fill these different branches of the aorta in the stages shown in figure 2 of plate 1 and figure 1 of plate 2 before the circulation has begun, it is necessary to introduce the needle into the aorta and inject, as it were, backwards. In this way the pressure in the aorta is probably raised, the heart being sufficiently stimulated by the ink to force the injection mass into the tiny channels that would otherwise remain empty. Indeed, after the circulation has begun, if only a very small quantity of ink enters the heart it will return to the area vasculosa without inject- ing the branches of the aorta within the embryo. These fill up only as the injection is continued and the heart becomes well filled with ink. 76 DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. That there are vessels within the embryo at the stage of 12 somites which can be injected from the aorta is proved by three sections from an injected chick of this stage (text-figs. 1 to3). These sections are best followed by comparing them with the specimen shown in plate 2, figure 1, from a chick with 14 somites. The section shown in text-figure 1 passes through the midbrain and shows a plexus of vessels on the midbrain fully as large as the aorta itself. On the left side of the section (right side of the embryo) is a slender artery containing ink, connecting this plexus with the aorta. This plexus of large vessels on the midbrain, shown in text-figure 1, also connects with a single longitudinal vessel along the hindbrain at this stage. These neural vessels, which at this stage are connected with the aorta, have no vent, which probably explains the great difficulty in injecting them. They are full of fluid, and though the ink enters them from the aorta, it does not penetrate far (text-fig. 1). This point is, I think, interesting in connection with the time of the beginning of circulation. As is well known, the heart begins to beat early in the second day. I have made a number of observations which show that it beats at the stage of 10 somites. In one instance I injected an embryo of 10 somites in which the heart was not beating, and when a small amount of the ink entered the heart it was stimulated to beat. In © another instance I had been watching aS <== =. an isolated blastoderm of 9 somites for Fic. 3.—Transverse section of an injected chick of 12 somites passing through the twelfth interspace to show the rela- over an hour when the heart began to tion of the posterior cardinal veins to the aorta. The : section is from the same series as figures 1 and 2, and is beat. This occurred just as the tenth to be compared with the total preparations shown on ; 5 3 d : plate 1, figure 2, and on plate 2, figure 1. The section is somite was beginning to appear. It is 50 thick and is unstained. X133. Ae., aorta; N, therefore quite certain that the stage of nePhrteme: V. « p. v. cardinalis posterior. 10 somites marks the beginning of the heart-beat. At the time the heart begins to beat its venous end connects with the exten- sive capillary plexus of the area pellucida in which the omphalo-mesenteric veins arise, and the entire aorta opposite the myotomes is connected with the capillary plexus in which the omphalo-mesenteric arteries arise. In other words, there is a plexus of vessels covering the entire area opaqua and area pellucida which con- nects with the venous end of the heart and with the entire dorsal aorta of the embryo opposite the zone of the myotomes. In the area pellucida this plexus of vessels is filled with fluid, but there are very few free cells in the vessels. After the heart begins to beat most of the isolated blastoderms show occasional wander- ing cells of various types that float into the vessels of the area pellucida, showing that these vessels are full of fluid; and when one of these cells approaches the heart in the omphalo-mesenteric veins it oscillates back and forth with each beat. It is thus very strikingly apparent that the circulation does not begin for a con- siderable time after the heart begins to beat. It is difficult to note the exact time of the beginning of the circulation while the chick is on the yolk, for the few red blood-corpuscles that are forced into the aorta are inconspicuous with the powers of the microscope that can be used. In the isolated blastoderms the earliest chick in which I have seen the circulation begin was one of 17 somites. At the beginning of circulation a few corpuscles are shot into the aorta with each DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. (i) beat of the heart. The mounting of the blastoderm on a cover-slip, however, interferes with the circulation much more than with the heart-beat, because the flattening of the blastoderm tends to flatten the vessels and thereby impede the circulation. This is often strikingly shown when through mechanical difficulties the circulation is entirely cut off on one side of an isolated blastoderm and not on the other. It is therefore probable that the circulation begins in the chick about at the stage of 15 or 16 somites. It is interesting to note that it is at this stage that the duct of Cuvier breaks through into the omphalo-mesenteric veins, whereby the dorsal aorta and the veins of the embryo become connected with the venous end of the heart. It is thus clear that at the stage of 12 somites, when the head of the embryo contains a complete aorta and a neural system of vessels which consists of a plexus of large vessels on the forebrain and midbrain, and a single channel on the hind- brain, there is no circulation through these vessels due to the beat of the heart. _ The connections of the vessels of the brain with the aorta are of importance. The arteries connecting the vessels of the forebrain with the aorta consist of a group of vessels just at the primitive arch of the aorta. These are shown in plate 2, figure 1, and have been thoroughly demonstrated by Evans. These arteries connect with the neural vessels at the base of the optic cup, in the groove repre- senting the line between the telencephalon and the diencephalon. Subsequently this group of vessels divides into two arteries, one of which encircles the optic stalk and the other extends caudalward along the ventral border of the thalamus and the midbrain (plate 6). Opposite the midbrain there is a group of tiny arteries connecting the plexus with the aorta, one of which is shown injected in a chick of 12 somites in text-figure 1. It is very clear (in the section of text-figure 1) that the vessels to the neural plexus are direct, dorsal branches of the aorta. The vessel along the hindbrain connects with the aorta by two groups of tiny branches, one cephalic and the other caudal to the otic vesicle. These branches are also for the most part direct dorsal branches. In one of my sections, however, two arteries to the vessel of the hindbrain are placed with reference to the aorta, as are the vessels on the left side in text-figure 2—that is, one is dorsal and the other dorso lateral. These connections between the aorta and the primitive vessel of the hindbrain are shown, injected, by Evans, in his figure 393 in the “Manual of Human Embry- ology”? (Keibel and Mall). As far as the vessels which connect the vascular channel of the hindbrain with the aorta are concerned, it has been shown that they differentiate as angio- blasts at the stage of 6 somites, while the aorta and the neural vessels are differ- entiating. The origin of the primary plexus of deep vessels on the surface of the forebrain and midbrain requires more careful study during the stages of from 6 to 12 somites. It is probable that these vessels differentiate, and that their connections witii the aorta differentiate, as does the preliminary vascular channel of the hindbrain. The development of the deep neural vessels and the origin of the superficial plexus of vessels opposite the brain, as well as the origin of the primary head-vein, will be taken up subsequently. 78 DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. ORIGIN OF THE CARDINAL VEINS. It is now important to consider the cardinal veins—how they arise, how they become related to the primitive neural vessels, and how they become con- nected with the heart through the duct of Cuvier. The general relations of the cardinal veins are best shown in plate 2, figure 1, from a chick of 14 somites, but their origin can be traced back to the stage of 9 somites. They form as a longi- tudinal anastomosis which connects diverticula of the aorta that project dorsal- ward between the somites. In 1906 these dorsal diverticula were described by Grafe, who stated that the cardinal veins arose from sinus-like projections from the aorta. That the cardinal veins arise from dorsal intersegmental branches of the aorta was shown by Rabl in 1892 and by Hoffmann in selachians in 1893. The condition of the aorta just before the diverticula arise is of importance. Up to the stage of 9 somites it is clear that the entire aorta which can be seen from the ventral aspect in the living blastoderm is connected with the plexus of the area vasculosa through so-called ventral branches which extend lateralward. Even cephalic to the first myotome a few chains of angioblasts connect the aorta with the plexus of the area vasculosa. However, these tiny branches all along the lateral border of the aorta are seldom injected, except opposite the caudal end of the aorta (plate 1, fig. 2). When the chick has 9 somites a new set of aortic branches begins to form, which are very distinct from the lateral vessels. In the living blastoderm of from 9 to 12 somites it can be seen that diverticula of the aorta project dorsalward into the interspaces. The more cephalic of these diverticula are dorso-lateral, as shown on the right side in text-figure 2, from a section through the first inter- space; the more caudal ones are distinctly dorsal, as seen for the twelfth interspace in text-figure 3. This is due to the fact that at the stage of 12 somites the aorta is obliquely placed with reference to the lateral margin of the myotomes. As shown in text-figure 2, in the first interspace the lateral margin of the aorta is in the lateral line, while in the twelfth interspace, as shown in text-figure 3, the aorta is directly under the lateral line. The first two of these diverticula have been seen at the stage of 9 somites; and they are present in all of the interspaces at the stage of 12 somites. In a total preparation of a chick of 12 somites the ink lodges in these dorsal diverticula and forms dark streaks across the aorta from the dorsal aspect; these streaks are very characteristic, but are difficult to indicate in a draw- ing. The specimen of plate 1, figure 2, shows such streaks across the aorta in the interspaces. The diverticula begin at the time when the first two somites lie within the arch formed by the two omphalo-mesenteric veins where they join the heart. In this connection I have tried to determine whether there is a constant relation in regard to the time when the cardiac or head fold reaches the level of the first somite; and in this regard the figures in His’s ‘‘ Untersuchungen ueber die erste Anlage des Wirbelthierleibes” (1868), plate x11, and those in Liliy’s ‘‘ Develop- ment of the Chick” (1908) are the most helpful. In general, at the stage of 9 somites the position of the first somite is about as shown in Lilly’s figure 61, page DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 79 106; but I have chicks of 10 somites in which the first somite is farther from the head-fold than in the usual specimen of 9 somites. As a rule the head-fold is along the cephalic border of the first somite when the embryo has 12 somites; but in some specimens, such as in His’s plate xu, figure 20, there is at this stage an interval between the first somite and the head-fold. After the cephalic curve of the midbrain has formed, as shown in plate 2, figure 2, the embryos are not as flat from the direct dorsal aspect and the maine can not be tested with the same definiteness. The longitudinal vessel which connects these diverticula in the lateral line of the embryo is the common cardinal vein (plate 2, fig. 1). The cardinal vein has two fundamental relations—on the one hand to the primitive vascular channel of the hindbrain and on the other hand to the venous end of the heart. As is shown in plate 2, figure 1, and in the section in text-figure 2, the cardinal vein becomes connected with the neural vessel by two cross-anastomoses in the first and. second interspaces. Of these vessels the one in the first interspace is the larger and more important. The cardinal vein itself is not shown on the left side in text-figure 2 (right side of the embryo), since the transverse vein of the first interspace is slightly oblique, as is plainly seen in plate 2, figure 1. The transverse vein of the first interspace has been described and illustrated by Evans; and has been traced back to the stage of 6 somites by Williams. In the chick it is an important channel in the second and third days of incubation, for it is the channel by which all of the blood for the brain drains into the cardinal vein and thence to the heart. The transverse vein of the first interspace is char- acteristic of the chick. It does not form in the pig where the transitory vessel of the hindbrain connects with the cardinal vein in front of the first somite instead of in the first interspace. At the stage of 12 somites the dorsal diverticula of the aorta are present in all the interspaces, but there is not yet a continuous vein connecting them opposite the lower interspaces. The cardinal veins begin to form at a very early stage, when the zone along which they form is close to the aorta (text-fig. 3), so that the primitive common cardinal vein is an accompanying vein to the aorta. It is this accompanying vein of the aorta which connects with the venous end of the heart, forming the duct of Cuvier. So close is its relation to the aorta that the duct of Cuvier may be regarded as a direct connection between the dorsal aorta and the omphalo-mesenteric veins. The position of the duct of Cuvier is well dean n, and is shown in plate 2 figure 1. At the stage of 14 somites, as shown in this figure, the common cardinal vein opposite the second, third, and fourth somites is in the form of a plexus; and it will be noted that there is a vessel extending lateralwards from this plexus opposite the cephalic border of the omphalo-mesenteric veins, and a similar vessel opposite the caudal border of the vein. These two vessels are in the somato- pleure dorsal to the omphalo-mesenterice veins. This is very clearly shown in the section in text-figure 2. The more cephalic of these two vessels (V. so.) develops, as I shall show for the pig, into veins which drain the body-wall over SO DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. the region of the heart cephalic to the duct of Cuvier. They receive their blood from lateral branches of the aorta (of which the lateral artery opposite the heart, shown in plate 2, fig. 1, may be one) and are analogous to the branches of the umbilical veins below the duct of Cuvier. Of the veins of the somatopleure, those which are opposite the caudal border of the omphalo-mesenterie veins join the omphalo-mesenteric vein in the septum transversum of His, as shown on the right side of text-figure 2. The connection, which has not taken place in the specimen in plate 2, figure 1, at the stage of 14 somites, occurs at the stage of 15 somites, as was shown by Evans. In making injections at the stage of 15 or 16 somites it sometimes happens that the ink first injected does not fill the neural vessels, but runs from the aorta into the duct of Cuvier through direct aortie branches, such as that shown in the third interspace in plate 2, figure 2. One of the most interesting points in connection with the duct of Cuvier is that it forms just about the time or just before the time when the circulation begins, which is probably of great importance from the standpoint of the physi- ology of the embryo. Thus plate 1, figures 2 and 3, and plate 2, figure 1, represent the blood-vessels of the chick before the circulation has begun, while figure 2 of plate 2 and figure 1 of plate 3 represent a series of chicks in which the circulation has commenced. Inasmuch as the duct of Cuvier has not connected with the omphalo-mesenteric veins (sinus venosus) at the stage shown in plate 2, figure 1, the longitudinal plexus and vessel of the lateral line at this stage is a common cardinal vein which will be divided into an anterior and a posterior division by the position of the duct of Cuvier. From a comparison of figures 1 and 2 of plate 2, figure 1 of plate 3, and plate 6 it is clear that the anterior cardinal vein must increase in length at the expense of the posterior cardinal vein as the heart shifts caudalward. In these figures it is plainly shown that the cardinal system opposite the duet of Cuvier continues in the form of an extensive plexus (see also plate 1, fig. 1, of the pig) and that the plexus ultimately gives rise to the umbilical veins. This completes the general account of the blood-vessels of the chick before the circulation has begun; that is, up to the stage of plate 2, figure 1. I shall take up, under two headings, the study of the further development of the primitive blood-vessels in the stages in which the blood is circulating; first, the vessels of the brain and their relation to the primary head-vein; second, the vessels of the spinal cord. The primitive vessels of the nephrotomes will be taken up in con- nection with the pig embryos. It is of course evident that the two divisions overlap, for the vessels of the brain begin in the period before the circulation commences. VASCULAR SYSTEM OF THE BRAIN AND THE PRIMARY HEAD-VEIN. As has been shown, the neural vessels begin to form very early, before there is any circulation, and indeed before the heart has begun to beat. The primitive vessel of the hindbrain differentiates at the stage of 6 somites as a chain of angio- blasts along the border of the hindbrain, and at the time it is differentiating DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 81 connects with the aorta by chains of angioblasts which are forerunners of direct dorsal branches of the aorta. Exactly when the angioblasts along the forebrain and the midbrain can be identified has not been determined, but at the stage of 12 somites there is a plexus of large vessels along the lateral surface of the forebrain and midbrain extending to the ventral surface of the forebrain at the base of the optic vesicle and anas- tomosing with the primary vascular channel of the hindbrain. This plexus connects with the aorta just at the base of the optic vesicle, as was shown by Evans in his figure 398 for a duck embryo of 13 somites in the ‘‘ Manual of Human Embryology” (Keibel and Mall). At the stage of 12 somites this plexus also connects with the aorta opposite the midbrain, as shown in text-figure 1. This deep primary plexus, which I have uniformly represented in a gray tone, soon gives rise to a superficial plexus opposite the region of the forebrain and the midbrain, as shown in text-figure 1. In this superficial plexus there develops a venous channel for the forebrain and the midbrain, as will be seen in plate 2, figure 2, from a chick of 16 somites, which is the stage when the blood begins to circulate. The superficial plexus opposite the forebrain and the mid- brain arises, for the most part, from the deep plexus (text-fig. 1), but I have also injected a few tiny connections between the superficial plexus and the aorta itself in early stages. These, however, disappear and the superficial plexus drains only the deep plexus. The vein which develops within the superficial plexus is characteristically placed, and is very adequately shown by Evans for the stages of 17 to 25 somites (Anatomical Record, 1909, III, figs. 3 to 6). At the stage of 29 somites this primitive cerebral vein is clearly shown in plate 6. Owing to the flexure of the midbrain, the primitive cerebral vein (v. cap. p. 1) runs directly across the thalamus; and it receives a very interesting series of branches. It is obvious that a very large number of the primitive veins opposite the cerebrum drain the eye. Beginning with the position of the Gasserian ganglion, as seen in plate 6, there is a plexus of veins which I have called the primitive maxillary veins (v. m. p.), which drain the inferior part of the eye and the most anterior border of the cere- brum. These veins have usually been called the primitive inferior ophthalmic veins, and, according to the function which they actually perform at the stage of plate 6, this would be perhaps a more logical name. However, the stage when this plexus drains mainly the eye is very transitory. Soon the capillaries of the maxillary arch develop and the plexus of veins which, at the stage of plate 6, clearly lies in the maxillary arch, drains all of the structures of that arch, the roof of the mouth, and the nose. The position of the maxillary vein and its corre- sponding artery in the maxilla is shown for the pig in plate 7. In the chick of the fourth and fifth days of incubation this group of veins clearly drains the entire maxilla and receives branches from the most anterior part of the cerebrum and a group of inferior ophthalmic veins, of which one of the most important runs in the optic stalk. Therefore I have preferred to limit the name primitive inferior ophthalmic veins to the branches of the primitive maxillary vein instead of calling 82 DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. the entire trunk the ophthalmic veins. The emphasis on the fact that this group of veins belongs in the maxilla, bringing it into line with the veins of the man- dibular arch and with the veins from the rest of the aortic arches, is interesting in connection with the origin of the middle segment of the primary head-vein. Cephalic to this plexus of maxillary veins is an extensive series of veins from the marginal vein of the optic cup. The vein in the margin of the optic cup is very characteristic. Above these is a smaller, but very important, group of veins which drain the cerebrum proper. As can be seen in plate 6, they tap the deep plexus of the cerebrum at their tips; they gradually creep dorsalward on the deep plexus until they meet with those of the opposite side in the mid-dorsal line. The anastomosis of these veins in the mid-dorsal line will ultimately give rise to the superior sagittal sinus, as has been shown by Mall and Streeter. On account of the relation of the primitive veins of the neural tube to the ultimate formation of the dural sinuses, this process of the creeping of the primitive veins toward the mid-dorsal line on the deep plexus is very important. Over the thalamus at’ this stage is the main root of the primitive cerebral vein and one large accessory root. For the midbrain the superficial branches of the primitive cerebral brain have not yet appeared, and all the blood of the midbrain drains through the deep plexus toward a characteristic deep vessel along the cerebellar ridge, which joins the primitive vessel of the hindbrain. In plate 2, figure 2, it is clearly shown that at the time when the circulation begins all the venous blood of the forebrain and midbrain must pass through the deep channel of the hindbrain and the transverse vein of the first interspace in order to reach the heart. This figure (plate 2, fig. 2) shows that the vessel of the hind- brain is mesial in position both to the primitive cerebral vein and to the anterior cardinal vein. Plate 6 shows that it is also farther dorsal than either of these veins; and also, what is well known, that the primitive vessel of the hindbrain is mesial to the Gasserian ganglion, the acoustic complex, the otic vesicle, and the ganglion of the glosso-pharyngeus. The cephalic end of the ganglion of the vagus, on the other hand, is mesial to the transverse vein of the first interspace; that is, the primitive vessel of the hindbrain runs down to the region of the cephalic end of the ganglion of the vagus. The primitive vessel of the hind-brain serves as a transitory vein for the brain of the chick during the second and third days of incubation, as is very evident in any living chick. On the other hand, it serves as the only channel for the blood to the hindbrain, and it can receive arterial blood directly from the aorta through tiny branches. These branches are so small and are so seldom fully injected that it is probable that only a small amount of blood actually passes through them in the living chick into the primary channel of the hindbrain. The permanent arteries for the hindbrain develop later, as will be shown. Plate 6 shows how the primary vascular channel of the hindbrain ceases to serve as a vein for the forebrain and midbrain, and how the true head-vein, the vena capitis prima, develops. The specimen here shown also indicates the fate of the primary vessel of the hindbrain. The deep plexus of the cerebrum, the DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 83 thalamus, and the midbrain has now made an almost complete covering for that part of the neural tube. Over the midbrain the plexus is farthest developed and has anastomosed across the mid-dorsal line with the plexus of the opposite side; over the thalamus and the cerebrum the deep plexus has almost reached the mid-line. The primary artery of the brain which supplies this extensive plexus divides into two branches—first, into a large arterial plexus which curves around the dorsal margin of the optic stalk and leads to the plexus around the eye and to the plexus of the cerebrum, and partly supplies the plexus of the thalamus; second, into an artery which curves along the ventro-lateral border of the thalamus and the midbrain, and is approaching the hindbrain. This artery will soon meet the ascending artery seen along the ventro-lateral border of the rhombencephalon. Opposite the hindbrain the development of the vessels, both the arteries and the veins, is most interesting. As is shown in plate 6, there is now a most important new vein. This is as yet a tiny, irregular vessel, hardly larger than a capillary, which connects the veins of the maxillary, the mandibular, and the second aortic arch with the anterior cardinal vein. The primitive vessel of the hindbrain is a vein for the brain only; this new capillary develops out of the capillaries of the visceral arches and by means of the relation of the maxillary veins to the primitive cerebral vein it receives the blood of the primitive cerebral vein and hence it becomes a true head-vein. We shall call this new vein, which is usually called the vena capitis lateralis, the middle segment of the vena capitis prima (v. cap. p. 2), and will say that as soon as this anastomosis between the primitive maxillary veins and the anterior cardinal veins takes place we can speak. of a primary head-vein which extends from the region of the thalamus to the duct of Cuvier and drains the structures of the head, namely, the brain and the tissues of the visceral arches. The specimen in the drawing of plate 6 is not shown from an exactly lateral aspect, but is tilted slightly to show the ventro-lateral surface of the hindbrain; but even with this tilting it is clear that the general position of the superficial vessel is such that it can become a direct line between the primitive cerebral vein and the anterior cardinal vein. This direct line is very plain in plate 2, figure 2. In other words, it is a more favorable vessel for the drainage of the large primitive cerebral vein than is the primitive vessel along the hindbrain. The exact course of this tiny chain of new capillaries is most interesting, because it conforms so closely to the structures that are present before it develops. In this connection the relation of this new capillary to the Gasserian ganglion is important to note, because it has been so little understood. As is well known, the ganglion arises from the wall of the pons at the point shown in plate 6, grows yvcntralward, and becomes adherent to the skin, making the placode of the tri- ceminus. If sections from specimens at the stage of plate 6 are studied, it will be seen that it is this attachment of the Gasserian ganglion to the skin, occurring at the stage when the tiny capillaries that give rise to this superficial vein begin, that renders it impossible for the new capillaries to pass lateral to the ganglion; hence they grow mesial to it. The primitive vessel of the hindbrain is mesial S4 DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. to the Gasserian ganglion, but lies against the hindbrain; the primary head-vein is also mesial to the ganglion, but lies ventral to the hindbrain. On the other hand, the new capillaries pass dorsal to the placode of the acoustic complex, and the slight dorsal curve of the primary head-vein (which is very evident in plate 6) indicates this adjustment of the vein. The placode of the acoustic complex is indicated in plate 6 by a film over the primitive vessel of the hindbrain opposite the root of the eighth nerves. The vena capitis prima passes ventro-lateral to the otic vesicle and again curves slightly dorsalward opposite the ganglion of the glosso-pharyngeus. In another injected specimen of this stage the superficial vein is a slender capillary plexus spanning the gap between the second aortic arch and the anterior cardinal vein, and not yet connecting with the veins of the maxillary arch. Thus this middle segment of the vena capitis prima (the so-called vena capitis lateralis) begins as an irregular capillary plexus between the aortic arches and the anterior vardinal vein. It becomes a true head-vein, in the sense that it drains the entire head, whereas the primitive vascular channel of the hindbrain (vena eapitis medialis) is a true neural vessel draining the brain only and not the entire head. Up to the stage when the capillaries of the visceral arches develop, the primi- tive channel of the hindbrain serves as the only drainage channel in the head, and this means practically for the brain alone; but as more structures in the head differentiate, a new vascular channel develops to drain these structures. This new chain of capillaries which receives the blood of the primitive cerebral vein by means of the relations of the maxillary veins is so direct and so favorable a channel for the blood of the primitive cerebral vein that the vena capitis prima develops very rapidly at the expense of the primitive vessel along the hindbrain. By far the most interesting way to follow this transformation is by watching the living chick. As is very clearly shown in plate 6, there is a stage when there are two venous channels for the head of the embryo—a large, deep channel along the hindbrain and a superficial tiny capillary chain farther ventral and farther lateral. While this more lateral channel is very tiny, it is hard to see it in the living chick, because there are few if any blood-corpuscles in it, and it is by the injection of blood, as it were, that one sees the vessels. In one chick opened toward the close of the third day and kept in a warm box, the two veins were of equal size when first observed, but in the course of about 2 hours the ventral channel had become by far the larger. This important change can be followed in the living chick either by opening a number of eggs at the close of the third day of incubation and observing the veins by the blood within them or by keeping a single chick of the right stage under observation for 4 or 5 hours. In such living specimens it can be seen that the deep vessel of the hindbrain, which remains as a single vessel for 2 days, becomes a capillary plexus as soon as the mass of venous blood from the forebrain and midbrain becomes shunted through the superficial vein. In an injection many interesting details of this process can be made out which are not so clearly seen in the living chick. In a stage still earlier than that shown in plate 6, the deep vessel of the hindbrain DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 85 begins to show very characteristic dorsal branches which conform to the surface of the hindbrain and to the roots of its nerves. In fact, the first of these branches, as can be seen in plate 6, tend to surround the root of the trigeminus, the root of the eighth nerves, and the otic vesicle. While these branches of the primitive vessel of the hindbrain are forming, the vessel itself also becomes a plexus. I have injections which show how this takes place. At first the single channel gives rise to a plexus of very large vessels which tend to run longitudinally, follow- ing the pattern of the original vessel. Gradually the vessels of this plexus become smaller and the longitudinal pattern is lost. I have not illustrated the develop- ment of the plexus on the hindbrain for the chick, but this point is well shown for the pig in plate 7, and the principles are the same in both forms. The plexus on the hindbrain ultimately covers the hindbrain as completely as the plexus on the midbrain shown in plate 6, but the pattern of the plexus is modified by the structures of the hindbrain: (1) by the roots of the nerves and their sensory ganglia; (2) by the otic vesicle, which for a time lies close to the hindbrain; (3) by the special vascular structure of the roof of the fourth ventricle. As has been said, the plexus into which the primary vessel of the hindbrain first breaks up tends to have a longitudinal pattern; the ultimate plexus over the hindbrain, on the other hand, tends, like the rest of the neural plexus, to show indistinct trans- verse lines. This is, I think, plain in plate 7, and it leads to the subject of the new arterial supply for the vessels of the hindbrain. A most important point in the history of the transformation of the primitive vessel of the hindbrain concerns its relation to the neural arteries, and this point is well brought out in plate 6. Taking into consideration the entire neural tube, it is originally supplied by a series of arteries from the aorta: (1) a group of vessels to the forebrain, that is, to the cerebrum and the thalamus, at the base of the optic vesicle from the primitive arch of the aorta; (2) a few small arteries opposite the midbrain; (3) a series of small arteries to the primitive vessel of the hindbrain; (4) a series of intersegmental arteries, of which the most cephalic is in the first interspace. In plate 6 an artery is shown on the right side from the primary arch of the aorta, which is growing along the ventro-lateral surface of the thalamus and the midbrain, and this artery is approaching a new artery, which is at the same time growing forward along the hindbrain. This new artery is very important; it starts as a longitudinal anastomosis along the neural tube between the segmental arteries. In plate 6 it connects the first, second, and third segmental arteries, which are occipital vessels, and is growing forward, making more and more new connections with the deep vessel of the hindbrain. Plate 6 shows none of the primitive arteries which connect the primitive vessel of the hindbrain directly with the aorta; but in plate 7, from a pig of a still older stage, it is very interesting to note that two of these original arteries still persist and take part in the forma- tion of this new longitudinal artery. This longitudinal artery grows rapidly forward until it joins the corresponding descending artery opposite the midbrain. It is very clear in plate 6 that the longitudinal neural artery along the hindbrain is originally along the ventro-lateral border of the hindbrain, and thus that there 86 DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. is one on each side. In plate 6 this vessel is labeled the basilar artery (a.b.), which is an illustration of the fact that the relations of the arteries of the adult may have too great an influence on the naming of the embryonic vessels. This vessel is not even a capillary which will become the basilar artery, because it is not in the mid-ventral line; it is rather a vessel which will become a part of a capillary plexus that will gradually reach the mid-ventral line, where the basilar artery will form. At the stage of plate 6 there are bilateral longitudinal arteries along the thalamus, the mid-brain, and the hindbrain, as can be proved by a direct ventral view of the specimen. The relations and the importance of this vessel would be emphasized by calling it a part of the primary longitudinal neural artery. On the other hand, the vessel shown in plate 7 from a pig embryo of an older stage is in the mid-ventral line and is thus the true basilar artery. During the fourth day of incubation the longitudinal artery seen opposite the first, second, and third somites in plate 6 grows caudalward along the ventro- lateral surface of the spinal cord on either side, to the caudal end of the neural tube. These ventro-lateral arteries develop as a longitudinal anastomosis between all the segmental arteries of the spinal cord. At the stage of the fourth day of incubation it is clear that the vascular plexus along the entire surface of the neural tube is supplied with blood by bilateral ventro-lateral arteries which extend from the groove between the cerebrum and the thalamus to the caudal tip of the tube. These two longitudinal arteries are originally in the form of a plexus on either side of the subthalamus, as is still better shown in plate 7 for the pig, and are more definitely a single channel along the rest of the course. This longitudinal neural artery receives its blood from the forerunner of the carotid arteries on either side and from the segmental arteries. It is easy to see that it is these important longitudinal arteries which will ultimately give rise to the circle of Willis, the basilar artery, and the anterior spinal artery. The development of the anterior spinal artery has been worked out in the pig by Evans (1909 and 1912). In the chick the anterior spinal artery does not form until the fifth day of incubation. During the fourth day there are two ventro-lateral arteries along the spinal cord which are placed on either side of the notochord and are not connected except by an occasional capillary across the mid-ventral line; they make a sharp ventral boundary for the lateral plexus on the spinal cord. These two longitudinal arteries are just mesial to the point where the spinal arteries meet the spinal cord, as can be seen in Evans’s figure 437¢ in the “Manual of Human Embryology” (Keibel and Mall). They give rise to the characteristic anterior arteries which penetrate the spinal cord. During the fifth day of incubation these two longitudinal arteries become connected with each other across the mid-ventral line, which is the beginning of the formation of the anterior spinal artery. The stage of the fourth day of incubation for the chick in which there are bilateral longitudinal arteries along the ventro-lateral border of the entire neural tube from the point of origin of the carotid artery to the tip of the spinal cord is an important stage for understanding the blood-supply of the nervous system. DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 87 It must be made very clear indeed that the longitudinal artery seen along the hindbrain in plate 6 is a neural artery and is not the vertebral artery. This is a specimen of the third day of incubation and the artery shown in this specimen forms along the neural tube at a stage when the occipital arteries supply only the neural tube. On the fifth day of incubation, on the other hand, these same arteries also supply the corresponding myotomes with vessels, and there then forms a second longitudinal anastomosis on either side along the upper segmental arteries which is nearer the aorta than the neural vessel. These second longitudinal vessels become the vertebral arteries. These arteries form at the stage of the fifth day of incubation in the chick and are present in a pig measuring 15 mm., a very much older stage than the one shown in plate 7, which measured 6.5 mm. The vertebral arteries form as the heart is shifting farther caudalward; and indeed it is clear that the basilar and anterior spinal arteries together, as well as the vertebral arteries, provide for the arterial supply of the hindbrain when the shifting relations in the neck interfere with the direct arteries from the aorta. The fundamental relations of the neural arteries to the plexus on the surface of the neural tube has now become clear. This plexus is fed with arterial blood from bilateral longitudinal arteries which are along the ventro-lateral border of the plexus and eventually come to lie for the most part in the mid-ventral line. Over the surface of the subthalamus the vessels remain bilateral. It is now necessary to consider how the neural plexus becomes related to the veins. In the study of the development of the veins of the brain as distinct from those of the spinal cord, it is of primary importance to study how the deep plexus of vessels becomes related to branches of the primary head-vein. This point I have worked out more in detail in the pig and shall therefore take up its consider- ation later. The fundamental points are, however, (1) that the branches of the primary head-vein opposite the forebrain and midbrain are transverse veins superficial to the deep plexus which constantly tap the deep plexus at their tips and grow toward the mid-dorsal line; (2) that the transverse veins of the hind- brain are profoundly influenced by the presence of the ganglia of the hindbrain and by the otic vesicle. The sensory ganglia become as completely surrounded by a capillary plexus as the neural tube itself, and each of these plexuses gives rise to a vein or group of veins. Moreover, the same is true for the spinal ganglia. In this account of the origin of the neural vessels great stress has been laid on the development of the vessels of the hindbrain, on account of the peculiar relations of the primitive vessel of the hindbrain to the drainage of the forebrain. In the course of the development of the vessels of the hindbrain the direction of the circulation of the blood is ultimately exactly at right angles to its original course. This change takes place, (1) by the completion of the true head-vein, by which the pial vessel is relieved of a great volume of venous blood from the brain; (2) by the development of a new longitudinal arterial channel, by which it can receive a much greater arterial supply. By these changes the blood over the hindbrain soon runs from the ventral toward the dorsal border, at right angles to its original course from the cephalic to the caudal border. SS DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. In the transition from the stage in which the primitive channel of the hind- brain serves as the vein of the brain to the stage when the new lateral superficial vessel—the true primary head-vein—is complete, it is clear that the primitive transverse vein of the first interspace is cut out of the main line of drainage for the head. It does not form a part of the primary head and neck vein of the embryo. Thus the primary head-vein, from the standpoint of development, consists of three parts: an anterior division, which is the primary cerebral vein; a second portion, which is a true head-vein draining the entire brain, forebrain, midbrain, and hindbrain, as well as the visceral arches; and thirdly, the anterior cardinal vein. The transverse vein of the chick persists as a root of a character- istic vein of the hindbrain—namely, a vein which arches caudalward along the lateral surface of the medulla. This vein of the medulla will be followed farther in the pig. It was called the posterior cerebral vein by Mall. The position of the transverse vein of the chick embryo in the first interspace is also just opposite the cephalic end of the ganglia of the vagus nerve. As soon as the superficial vein—the primary head-vein—is formed, the vascular channel of the neck straightens out, and there is then no longer any way of distinguishing the exact place where the second segment of the primary head-vein joins the third segment or the anterior cardinal vein, for the two become a single, continuous channel. From now on, the place of transition can be indicated only in a general way by the root of that vein of the medulla which follows the roots of the vagus nerve along the medulla; and it is well known that veins are shifting landmarks. Stated in other words, the anterior cardinal vein extends along the entire zone of the occipital myotomes, and as the occipital muscles develop these myotomes become indistinct landmarks. The first interspace is thus a transitory landmark, and in later stages and as soon as the superficial head-vein connects directly with the anterior cardinal vein and eliminates the transverse vein of the first interspace from the direct line of drainage for the blood of the brain, the distinction between the head-vein and the anterior cardinal vein becomes less obvious. The cephalic portion of the head-vein develops to drain the forebrain and midbrain; the middle portion develops to drain the brain and the gill-arches. The vein of the anterior part of a chick of the fourth day of incubation is therefore a composite structure, so far as development is concerned. However, at the fourth and fifth day of incuba- tion there is a single long vein extending from the groove between the cerebrum and the thalamus down to the duct of Cuvier. This vein receives branches from all the various structures of the head. The neural branches come from the cere- brum and the eye from the thalamus, the midbrain, and the hindbrain. The branches from the hindbrain are especially modified by the ganglia of the hind- brain and the otie vesicle. On the ventral aspect this vein receives branches from the developing visceral arches and from the somatopleure opposite the heart. The entire vein may thus be called the embryonic head-vein, or the vena capitis prima. DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. &9 As far as the relations of the vena capitis prima to the vessels of the adult are concerned, it has been shown by Mall and Streeter that only a very small portion of the primary head-vein persists within the skull cavity—namely, the segment just mesial to the Gasserian ganglion which becomes the cavernous sinus. The neural branches of the primary head-vein ultimately give rise to the other dural sinuses. In regard to the relations of the anterior cardinal vein of the embryo to the internal jugular vein, it is interesting to note, in plate 6, that the entire anterior cardinal vein is opposite occipital myotomes; that is, it is entirely within the head. The caudal part of the anterior cardinal vein will become a vein of the neck when the duct of Cuvier shifts into the zone of the cervical myotomes. The cephalic end of the anterior cardinal vein of the embryo is opposite the upper zone of the medulla. The cardinal system of veins in general covers the entire zone of the myotomes, which includes a part of the head as well as the entire body of the embryo. In closing this account of the origin of the primary head-vein, it is important to emphasize again the relation of the new vessel, the middle portion of the vena capitis prima, to the various structures related to the hindbrain—that is, to the otic capsule and to the ganglia of the hindbrain. The middle portion of the head-vein develops after these structures are formed and must conform to their position. It grows in as straight a line as possible, and passes mesial to the placode of the trigeminus, lateral to the acoustic complex, to the otie capsule, and to the ganglion of the glosso-pharyngeus. It is entirely a new vessel, and has no remnants whatever of the preliminary vascular channel of the hindbrain which arises and runs along the neural tube. As is seen in plate 6, there are two entirely distinct vessels in the head of a chick of the early part of the fourth day— the so-called vena capitis mesialis, a neural vessel, and the so-called vena capitis lateralis, a true head-vein. After following this account of the origin of the primary head-vein of the chick, it will be of value to consider the long series of previous studies upon which it has been based. The observations which seem to me to lead to a clear under- standing of this subject are those of Salzer, Mall, Grosser, Evans, Williams, and Streeter. The view first held in regard to the development of the veins of the head was that the external jugular vein was the primary vein of this region. This view, which was incorrect, was based on the work of Rathke. In 1887 Kastschenko described a remarkable relationship between the jugular vein and the cranial nerves in the chick. He stated that up to the end of the third day the cranial nerves were lateral to the jugular vein (primitive vessel of the hindbrain), and noted that this vein was not in the form of a plexus. At the end of the third day the facial and glosso-pharyngeal nerves became mesial to the vein, and on the sixth day the vagus became mesial. He thought that the nerves cut through the veins, as it were, without the latter losing their continuity. In 1895 Salzer published an article on the development of the veins of the head in the guinea-pig, which forms the basis of the correct interpretation of this 90 DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. difficult subject. He described the head-vein, in an embryo guinea-pig 2.5 mm. long, as a vessel running from the region of the optic cup, close to the neural tube (primitive vessel of the hind-brain), to the level of the first vertebra, where it turned lateralward and lay lateral to the aorta (anterior cardinal vein), ending in the duct of Cuvier. This vein (the primitive vessel of the hindbrain), was mesial to the cranial nerves, and Salzer called it the anterior cardinal vein. It was a transitory vein, for by the time the embryo was 2.8 mm. long he found a second vein, lateral to the nerves, from the region of the acoustico-facial complex for- ward. This second vein he called the vena capitis lateralis, and concluded that, not only in the guinea-pig but in vertebrates in general, the anterior cardinal vein (deep channel of the hindbrain) is the first vein to develop in the head, and that it is replaced by a vena capitis lateralis, which as the neck develops is con- tinued into the neck as the internal jugular vein. This description of the veins of the early embryo by Salzer is nearly correct, and was a great step in advance, though more complete studies give a different interpretation and naming of the veins. The next step was made in 1907, by Dr. Mall, who studied the cerebral sinuses in the human embryo and, on the basis of this work of Salzer, demon- strated that the first drainage canal for the head (primary head-vein including the anterior cardinal) gives rise to the cerebral sinuses and the internal jugular vein. This drainage canal (the vena capitis prima) he called the anterior cardinal vein, using the term in its generally accepted sense as applying to the entire head- vein and neck-vein of the embryo. In the same year Grosser made it clear that the first vascular channel for the head (deep vessel of the hindbrain and the anterior cardinal) can be analyzed into two parts: a cephalic part which lies close to the neural tube, and a caudal part which has an entirely different position—namely, ventral to the myotomes and lateral to the aorta, in the same position as the posterior cardinal vein. He limited the term ‘“‘anterior cardinal vein” to this caudal portion, and analyzed the cerebral portion into a primary vessel (the vena capitis medialis) and a sec- ondary vein (the vena capitis lateralis). At this point Evans gave his beautiful injections of early blood-vessels, published in 1909. He showed the form of the primitive vascular plexus of the brain and also how this plexus covered the surface of the forebrain, encircling the large optic vesicle with a chain of capillaries and spreading over the surface of the thalamus and midbrain. He described how this extensive plexus became a single slender channel along the wall of the hindbrain, leading down to the transverse vein and the duct of Cuvier; and also demonstrated the connections of the plexus of the forebrain and the single vessel of the hindbrain with the aorta. Streeter has recently published a study dealing with the later stages of the vena capitis prima. It was from the branches of this vein that Dr. Mall had shown that the dural sinuses were derived. Streeter has worked out the develop- ment of the dural sinuses more in detail and has shown that the only part of the vena capitis prima to persist is the part mesial to the Gasserian ganglion which DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 91 becomes the cavernous sinus. The rest of the dural sinuses come from branches of the primary head-vein. The sinuses of the mid-dorsal line arise from the anastomoses of the veins of the two sides; the basal sinuses arise for the most part from veins which border the Gasserian ganglion and the otic capsule. In 1916 Stracher published an article on the veins of the head of the chick, in which he deals with the fate of the vena capitis medialis and the origin of the vena capitis lateralis.. In this work he uses the method of reconstruction in preference to the method of injection in a form in which it is easy to obtain abundant injected material, on the ground that with reconstructions the relations to the surrounding tissues can be better analyzed. Stracher’s own work, however, suffers from the limitations of his method. His reconstructions show the larger trunks, which are not always the most important ones, and do not show certain tiny channels which are essential to an understanding of the relations of the vessels. He shows the stage at which the primitive vessel of the hindbrain (vena capitis medialis) and the primary head-vein are both present in the same specimen and equal in size. This had not been done previously, and is an important point. He also shows in part how the middle segment of the primary head-vein arises, but misses several points that are essential to an understanding of this vein. In his text-figure 2, from a chick of 30 somites, he shows a short branch from the anterior cardinal vein and a branch from the maxillary (ophthalmic) veins, and recognizes that these two branches become connected and form the vena capitis lateralis. He speaks of the branch from the inferior orbital vein (my maxillary vein) as arising from a swelling on the vena capitis medialis, not realizing that it is a new outlet, not for the blood of the vena capitis medialis but for the blood of the primitive cerebral vein, as is plainly shown in plate 6. In discussing the origin of the vena capitis lateralis from the lower border of the Gasserian ganglion to the anterior cardinal vein he says (page 55): Kastschenko gibt keine Abbildung, die ihre Entstehung zeigen wiirde, seine Tafel stellt sie da, nachdem ihre Ausbildung vollendet ist. Nach seiner Schilderung ‘‘durch- schneiden” die Nerven die Vene. Demgegeniiber ist zu betonen, dass der eben geschil- derte Teil der Vena capitis lateralis—es folgt spiiter noch die Ausbildung weiterer caudal and cranial davon gelegener Strecken—frei im Gewebe, ziemlich entfernt von der Vena capitis medialis entsteht. Thus he realized a part of the method of origin of the vena capitis lateralis, but missed entirely its relation to the capillaries of the visceral arches. In regard to the relations of the portion of the primary head-vein in the region of the Gasserian ganglion, Stracher’s models are better than his interpretations. The essential facts are that the vena capitis medialis is a vessel on the hindbrain, the vena capitis lateralis is a more superficial vein which lies ventral to the hindbrain; both are present in the same specimen at a given stage; both are mesial to the Gasserian ganglion, one as a part of the system of vessels of the pia mater and the other as a part of the primary head-vein. _ Stracher shows both the vena capitis medialis and the vena capitis lateralis in their correct position mesial to the Gasserian ganglion, and then concludes 92 DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. that the medial vein of this area becomes transformed to make the lateral vein. His own figures do not warrant this conclusion, which was formed through not following the fate of the primitive vessel of the hindbrain. Thus his diagram (page 68), which should show the primary head-vein coming, embryologically, from three segments—namely, from (in his nomenclature) the vena cerebralis anterior, the vena capitis lateralis, and the vena cardinalis anterior, shows it coming from five: (1) the vena cerebralis anterior; (2) a short stretch of the vena capitis lateralis; (8) the vena capitis medialis; (4) the vena capitis lateralis again; (5) the vena cardinalis anterior. He observed the beginning of the breaking of the vena capitis medialis into a plexus, and then missed the plexus as it became finer, so that he lost the very important point of the fate of the primitive channel of the hindbrain. These points are covered in his summary (page 67): “Sodann entwickelt sich eme neue Venenbahn (Vena capitis lateralis), die parallel zur medialen Kopfvene, aber lateral vom Nervus acustico-facialis, glosso-pharyngeus und dem Horbliischen verliiuft. Sie verbindet das Stiick der Vena capitis medialis, das sich medial von der Trigeminusanlage findet, mit der vorderen Kardinalvene. Zur selben Zeit weiten sich Gefiisse des Venennetzes am Hinterhirn zu einer Bahn aus, die bedekt von der Trigeminusanlage beginnt, an der Seite des Hinterhirns dorsal yon Hérbliischen im Bogen verliuft und in der Gegend des Nervus glosso-pharyngeus wieder zur medialen Kopfvene zuriickkehrt (Vena capitis dorsalis). Sie tritt mit Beginn der Obliteration der Vena capitis medialis auf und verschwindet wieder, sobald die Vena capitis lateralis vollstindig ausge- bildet ist. Die Vena capitis medialis verédet zuerst im Bereich des Hérbliischen, dann caudal davon in der Gegend des Nervus glosso-pharyngeus. Weiterhin entwickelt sich dadurch um den ersten Ast des Nervus trigeminus ein Venenring, dass lateral yom Nerven eine Vene entsteht, die rostral vom Nerven aus dem Stamm austritt und sich caudal wieder mit ihm vereinigt. Der mediale Schenkel dieses Ringes verschwindet alsbald. In dhnlicher Weise entwickelt sich auch um den Nervus vagus ein Ring mit Beihilfe der dorsal ein- miindenden Zweige. Auch hier verédet die alte medial vom Nerven gelegene Bahn. Damit is die Vena capitis lateralis vollstindig ausgebildet, und die Kopfvene dndert ihren Verlauf, was ihre Lage zu den Nerven anlangt, nicht mehr, da die Nervi accessorius and hypoglossus beim Huhn auch im ausgebildeten Zustande lateral von Vena jugularis interna ziehen.”’ It is, I think, clear that the primary blood-vessels which arise in the head are neural vessels. These neural vessels form a continuous plexus of capillaries which closely invests the brain. Along the hindbrain angioblasts probably appear first, but here they form a single, characteristic long channel which serves temporarily as a vein and does not take the form of a plexus characteristic of the neural vessels until relatively late. This single, large, primitive vessel does not extend the full length of the rhombencephalon, however, but at the zone of the cephalic roots of the vagus nerve, or, in other words, opposite the first occipital myotome, becomes a plexus on the side of the medulla which gradually extends the full length of the cord and connects with every intersegmental artery and vein. The neural system of vessels becomes connected with the venous end of the heart by means of the two cardinal veins. These connections are very char- acteristic; the most cephalic, which is either in front of the first occipital myotome (as in the pig) or between the first two occipital myotomes (as in the chick), is always the largest and drains the entire brain. All the other connections are DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 93 small intersegmental veins. Thus it may be said that two organs determine the early blood-vessels, the neural tube, and the nephrotome. Soon a third set of organs (the visceral clefts) develop and give rise to capillaries, which connect on the one hand with the anterior veins of the brain and on the other with the cardinal veins; and in this manner the head-vein of the embryo is completed. The method of nomenclature of the primitive vessels of the head is certainly open to discussion. The primitive vessel of the hind-brain, of which I have shown the origin, the relations, and the fate, is the vessel seen by Kastschenko in 1887 and more clearly by Salzer in 1895, and recognized by all who have since worked on this subject as being the first long vein in the head region and as lying along the wall of the hindbrain. Grosser gave it the name of “vena capitis medialis,” a name which has been universally accepted. It may be argued that it is a mistake to attempt to change a name of this type which has been generally adopted; but on the other hand a name which would emphasize the essential point in regard to this vessel—namely, that it is a neural vessel and that it develops into neural vessels—rather than the accessory fact that it serves temporarily as a vein for the head, would, I am convinced, clear up much of the confusion in regard to the primitive veins of the head. It does serve for two days in the chick as a vein for the forebrain and the midbrain, but at the same time it is the entire capillary bed of the hindbrain and ceases to be a single long channel as soon as the cerebral blood is shunted through another channel. It then develops, as have the rest of the neural vessels, into an extensive capillary plexus in the position of the pia mater. I therefore wish to avoid the use of the term vein in connection with it and to reserve the term vein for the vena capitis lateralis, for which I shall use the term vena capitis prima, because this is the first vascular channel of the head which is purely a vein and because it is the first vessel which drains the head and not the brain alone. I have therefore called the vena capitis medialis the primitive vessel of the hindbrain. The term vessel is more indefinite than the term vein, but for that very reason it applies better to a channel which serves both as a veinand as a capillary at the same time, and ultimately becomes a capillary plexus, out of which both arteries and veins will arise. I propose to call the long vein of plate 6, extending from the region of the thalamus to the duct of Cuvier, the primary head-vein. This primary head-vein develops in three segments—a cephalic segment which is the primitive cerebral vein, a middle segment opposite the hindbrain, and a caudal segment which is the vena car- dinalis anterior. At the stage of plate 6 this vein is entirely within the head, because the duct of Cuvier is still opposite occipital myotomes. As soon as the duct of Cuvier shifts into the neck region this vein will become the primary head and neck vein. The terms vena capitis medialis and lateralis have the sanction of usage; but it seems to me that the terms primitive vessel of the hindbrain and primary head-vein better express the function of these vessels. O4 DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. DEVELOPMENT OF THE SPINAL ARTERIES. It will now be necessary to go back and follow the development of the spinal arteries. This can be done in a series of injections of the stages from 15 or 16 somites upward, or the entire process can be followed in any one chick up to the stage of about 30 somites. The process is easier to illustrate after the embryo has rotated so that the lateral instead of the dorsal surface is presented. The entire process can thus be readily followed in plate 3, figure 1, from a chick of 25 somites, with six sections from two different series of chicks of 25 and 30 somites. The general stage of development of the vessels of the head of the embryo at the stage of 25 somites can be seen in Evans’s figure 6 (Anat. Record, 1909, III, p. 505); or can be estimated from my plate 6, the stage of 25 somites being just before the superficial capillaries which make the middle segment of the primary head-vein begin. The deep vessel of the hindbrain is still the vein for the brain, and is shown in its relation to the capillary plexus on the lateral surface of the spinal cord in plate 3, figure 1. The general development of the area vasculosa at this stage is also of interest in following the vessels in sections. The roots of the omphalo-mesenteric arteries at the stage of 25 somites are opposite the twentieth and twenty-first somites. As was indicated above, in the earlier stages the entire lateral border of the aorta opposite the somites was originally connected by direct lateral (that is, ventro- lateral) branches with an arterial plexus of the area vasculosa. In this plexus, on either side of the embryo, the omphalo-mesenteric veins gradually extend caudalward from the region of the sinus venosus and thus are formed two veins, or a plexus of veins, with direct short connections with the aorta. This process explains the large veins of the splanchnopleure shown in figure 3, plate 2, and figures 2 and 3, plate 3. In the chick the spinal arteries do not arise as direct dorsal arteries from the aorta to the cord, but the direct dorsal arteries make a primary arch to the dorsal border of the nephrotome, where they give rise to the cardinal veins. The spinal arteries then arise from these arches instead of from the wall of the aorta itself. In following the development of the spinal arteries I shall begin with the more caudal segments in plate 3, figure 1, because they show the earlier stages. As has been described in connection with the origin of the cardinal veins, the first dorsal branches of the aorta are direct dorsal diverticula of the wall of the aorta into the interspaces, as is shown best in text-figure 3 for the stage of 12 somites. Figure 2 on plate 4 and figure 4 on plate 2 are both from the lower segments of a chick of 30 somites. They are both taken below the origin of the omphalo-mesenteric arteries in the zone where the arteries of the posterior limb-buds are forming. The cardinal veins are also developing in this area. Figure 2 on plate 4 passes through the twenty-fifth interspace, and plate 2, figure 4, is still lower down and passes through the twenty-seventh interspace. In plate 4, figure 2, it can be seen that even in later stages the dorsal branches start as direct diverticula of the aorta. These diverticula soon arch lateralward and, as can be seen in plate 2, figure 4, dilate slightly just dorsal to the nephrotome. These dilated portions of the arches DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 95 become connected with similar dilatations in the other interspaces and make the cardinal veins. These observations indicate that the cardinal veins begin with dilatations of the dorsal branches of the aorta—that is, that they start as an out- growth from the wall of the aorta in the different interspaces and that these inter- segmental vessels become connected along the lateral line. Very soon the vascular arches which give rise to the cardinal veins give off sprouts which extend toward the spinal cord, as is shown from the tenth to the seventeenth interspaces in plate 3, figure 1. The position of these sprouts is shown in section on plate 2, figure 3. This section passes through the twenty- first interspace of a chick of 30 somites. These neural sprouts soon reach the spinal cord, as is shown in section on plate 3, figure 4, which is to be compared with the seventh interspace of plate 3, figure 1. The section shown in plate 3, figure 4, is from a chick of 25 somites—from another series than that of all the other sections on the plates. It is from a series of nearly the same stage as that of plate 3, figure 1; it passes through the seventeenth interspace. It was selected because it shows so well the double dorsal arch to the posterior cardinal vein, the primary direct one and the secondary neural one. One has only to imagine the primary direct arch disappearing to obtain the well-known pattern of the spinal arteries shown in section on plate 3, figure 2, and in the upper interspaces of plate 3, figure 1. From this study, it is, I think, clear that the spinal arteries of the chick arise from dorsal intersegmental vessels which give rise to the cardinal veins, and not directly from the aorta. In plate 3, figure 1, it is very evident that the capillary plexus which forms along the lateral surface of the spinal cord is a direct continua- tion of the primitive vessel of the hindbrain. The original simple chain of capillaries on the lateral surface of the cord, such as is shown in plate 3, figure 1, from a chick of 25 somites, very soon becomes a plexus on the neural tube, as indicated opposite the second somite in plate 6. By the fourth day of incubation this plexus covers the entire lateral surface of the spinal cord. The relation of this plexus to the spinal arteries on the one hand and to the spinal veins on the other is very regular and characteristic. Every spinal artery, on approaching the cord, bifurcates into a short ventral branch and a longer lateral branch (plate 3, fig. 2). The ventral branch leads to a longitudinal neural artery which at the stage of the fourth day lies on the ventral surface of the cord just lateral to the notochord. In other words, there are symmetrical ventral longitudinal arteries. These arteries form a ventral border for the plexus along the lateral surface of the cord. The lateral arteries run in the plexus on the surface of the cord; they lie just cephalic to each spinal ganglion and extend nearly to the dorsal border of the cord. The veins which accompany these transverse arteries, in contrast to the arteries, are lifted off from the surface of the cord, as it were. They also correspond to the cephalic border of each ganglion, and they are more superficial in every case than the corresponding artery. I am emphasizing the fact that the arteries lie in the plexus on the surface of the cord and that the veins are more superficial, because the same is true for the primary arteries and veins of the brain, as can be seen very clearly in plate 7. 96 DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. The series of sections of injected chicks shown on plates 2, 3, and 4 allow an interesting comparison of the primitive branches of the aorta. It is clear that the branches of the aorta ean be described best according to what organs they supply rather than by regarding their exact point of origin in the wall of the aorta. The primary branches extend to the splanchnopleure and are primitively directly lateral branches, as can be seen on the left side of text-figure 3. That they come to be ventro-lateral and then ventral branches is well known and is shown in plate 2, figure 4, in which it is clear that there are three sets of arteries on the right side of the section. The first is a dorsal branch to the cardinal vein; the second is a lateral branch to the somatopleure, and the third is a ventral branch to the splanchnopleure. The branches that extend to the cardinal veins need careful attention. There are in the first place the original dorsal arches that give rise to the cardinal veins, such as are shown in figure 4 of plate 2 and figure 4 of plate 3. These branches are strictly intersegmental; moreover, the interseg- mental branches are the first arteries related to the cardinal veins because they are the only ones present at the stage of 12 somites. In later stages (for example, at the stage of 25 or 30 somites) there develop a few direct dorso-lateral arteries to the cardinal veins, and these arteries may lie opposite the somites instead of between them. Such an artery, for example, is shown on the left side of figure 3 of plate 3. This section is the next one below that of figure 2 of plate 3 in the series and indeed the edge of this artery is shown in the latter section. Similar direct dorso- lateral arteries to the cardinal veins are shown on both sides of figure 4 of plate 3. This latter figure demonstrates that these dorso-lateral arteries are new vessels and not remnants of the original dorsal arches. On the left side of figure 4 of plate 3 blood reaches the cardinal vein in three ways: from the aorta along the surface of the cord, from the aorta along the primary dorsal arch, and from the aorta through a dorso-lateral artery. It must also be brought out that these dorso- lateral arteries to the cardinal vein are not the same as the direct lateral arteries to the tubules of the pronephros and the metanephros, which develop later and are quite differently placed, as can be seen in text-figure 8 from a chick of 35 somites. These dorso-lateral arteries to the cardinal veins are of importance in connection with the extension of the cardinal veins caudalward and are very im- portant in comparing the chick with a form like the pig, where the dorso-lateral branches are more numerous. It may be well to enumerate here the different types of branches of the aorta which may be found in the embryo from the standpoint of the structures they supply: first, there are the arteries to the splanchnopleure; second, mesial branches which connect the two aorte; third, lateral arteries to the somatopleure leading to the umbilical veins; fourth, dorso-lateral arteries to the cardinal veins; fifth, lateral arteries to the limb-buds; and sixth, lateral arteries to the nephritic tubules. DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 97 THE VASCULAR SYSTEM IN YOUNG PIG EMBRYOS. In the study of the vascular system in a mammal it is not as easy to obtain young stages for injections, as in the case of the chick. The material, however, offers valuable opportunities for comparison with human embryos, and to obtain injections in much earlier stages than have ever been injected in human specimens. I shall follow the development of the vessels in the pig by the aid of six figures of injected embryos, and shall describe the specimens and follow the development of the vessels under six headings: First, the form of the heart: second, the ventral branches of the aorta, including the allantoic arteries and the subintestinal artery; third, the umbilical veins and the vessels of the thoracic body-wall; fourth, the vascular system of the nervous system and the formation of the primary head-vein; fifth, the cardinal veins; sixth, the vessels of the pronephros and the mesonephros. THE FORM OF THE HEART. ‘The youngest pig which I have injected is shown on plate 4, figure 3. This is from a specimen which measures 4 mm. in oil and which has 14 somites. It corresponds in development with a human embryo, No. 470 of the Carnegie collection, which measures 3.3 mm. and is in the fourth week of development. In this embryo pig an injection was made into the aorta opposite the origin of the omphalo-mesenteric arteries. The point of injection was obscured by extravasa- tion, so that it is not shown in the drawing. The stage of development of the specimen can be judged by the form of the brain, the otic vesicle, and the form of the heart. The extensive venous plexus covering the anterior or cephalic wall of the yolk-sac converges on either side into large right and left omphalo-mesenteric veins, which meet in a conjoined tube, the sinus venosus. The sinusoids of the liver have not yet begun to form, so that the sinus venosus stands out clearly. The sinus has a marked diverticulum, which Tandler called the horn. The dorsal wall of the sinus shows a series of sprouts, representing the duct of Cuvier, which is probably developed at this stage, as indicated by the posterior cardinal vein, but is incompletely injected. The most caudal of the sprouts form a small plexus representing the umbilical vein in the somatopleure. Above the sinus venosus is a well-marked groove between the sinus and the atrium. The atrio-ventricular canal, on the other hand, is only just indicated. The form of the heart corresponds closely with the description by Tandler (Manual of Human Embryology, Keibel and Mall, page 536), which is based on the studies of Born, in which he says that the heart becomes a horizontal loop, the two limbs of which are separated by an almost horizontal bulbo-ventricular cleft into two parts, a ventricular limb and a bulbar limb. In my specimen the bulbar limb consists of three parts: first, the bulbus cordis; second, a short constricted portion of the tube, the fretum Halleri; third, the large truncus arteriosis, which gives off the two aortze. In the use of the term fretum Halleri I am following the usage of His (Anatomie menschlichen Embryonen, pages 131 and 140). He describes this portion of the tube as the portion which ultimately gives rise to the semilunar valves. QS DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. In connection with the development of the heart, figure 1 of plate 5 and figure 1 of plate 1 are very interesting. The specimen from which the former was taken was one of a litter of five, all of which were injected. It measures 6 mm. in oil, that is, after fixation and dehydration, and has 20 somites. The specimen on plate 1, figure 1, was one of a litter of six embryos, all of which were injected. It measured 7 mm. when fresh and is 6.2 mm. long in oil. It has 238 somites. It should be noted that these embryos do not have a caudal flexure, so that these measurements must not be confused with the same measurements of older specimens after the flexure has formed. The number of somites gives more valuable data in regard to the stage of development in these stages than do measurements. If comparisons are made with human embryos at the stage of 23 somites it will be noted that at this stage the human embryo has two very marked flexures, shown, for example, in the R. Meyer embryo No. 300, represented in Felix’s figure 531, in the ‘‘ Manual of Human Embryology” (Keibel and Mall), and hence it is very much shorter. In figure 1 of plate 5 the changes in the heart from the stage shown in plate 4, figure 3, are readily followed. The direction of the ventricular arch has changed from the horizontal to an oblique position. The atrio-ventricular canal has become the characteristic long, slender channel, and there is a marked constriction between the ventricle and the large bulbus cordis. The fretum Halleri is now a long, slender tube, and both the bulbus cordis and the truncus arterlosus are shown in maximum distension. In plate 1, figure 1, the form of the sinus venosus is not clear, as it is con- cealed by the injection of the sinusoids of the liver. In all of the six specimens of this litter the sinusoids of the liver are farther developed on the left side than on the right. In all of the other specimens, however, and on the right side of this specimen, there is a marked constriction between the liver and the sinus venosus just below the upper large opening of the umbilical vein. At this stage the umbilical vein connects with the liver and with the sinus venosus by large openings, and with the duct of Cuvier by an extensive capillary plexus in the somatopleure. There is a constriction between the sinus venosus and the atrium, and a well-marked atrio-ventricular canal. The bulbo-ventricular cleft gives the effect of an hour-glass constriction of the heart. This is true of all the specimens of the litter, but in one the contraction of the bulbar portion is particularly marked. The differences in the form of the heart in figure 1, plate 5, and figure 1, plate 1, are partly due to the fact that the hearts in these specimens were fixed while beating and were caught at different phases of the beat. For example, in plate 5, figure 1, and bulbus cordis and the truncus arteriosus show a maximum distension, while in plate 1, figure 1, the bulbus cordis and the truncus arteriosus are con- tracted and there is a general distension of the cephalic aorta. On the other hand, in plate 1, figure 1, is shown the beginning of a torsion of the ventricular loop, by means of which the beginning of the fretum Halleri will come to be opposite the ventricular end of the atrio-ventricular canal. DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 99 This torsion is more clearly seen on figure 2 of plate 5 and figure 1 of plate 4. These two specimens are from the same litter. They measure 7.1 mm. in oil, and have 27 somites. Figure 1 of plate 4 is given because of an extravasation in the vessels of the head in the specimen of figure 2 of plate 5. In this latter figure the sinusoids of the liver have markedly developed. The sinusoids of the left side anastomose across the ventral line with those of the right side. The opening of the left umbilical vein into the liver is directly mesial to the umbilical vein itself and is hidden by it, while the opening into the duct of Cuvier is plainly visible. There is also a plexus from the umbilical vein in the somatopleure connecting it with the posterior cardinal vein and with the duct of Cuvier; but this is omitted in the drawing. There is a well-marked constriction between the sinus venosus and the atrium. The change in the heart is due to the twisting of the obliquely placed ventricular arch, whereby the point which marks the beginning of the fretum Halleri comes to le exactly opposite the opening of the atrio-ventricular canal into the ventricle. The bulbus cordis lies far to the right and its connection with the fretum Halleri is hidden by the ventricle, while the opening of the auricular canal is far to the left. These relations as seen from the other side are shown in plate 4, figure 1. From these two figures it is obvious that a still further twisting of the heart must take place before the arterial orifice comes to lie directly anterior. VENTRAL BRANCHES OF THE AORTA, INCLUDING THE ALLANTOIC ARTERIES AND THE SUBINTESTINAL ARTERY. One of the most interesting subjects in connection with these injections has been the study of the ventral branches of the aorta, or the branches to the yolk- sac, the gut, and its derivatives. The study of the early vessels of the embryo emphasizes the fact that the vessels should be considered in relation to the organs which they supply. The fundamental relations of the ventral branches of the aorta to the yolk-sae and to the allantois are shown in two total preparations of injected pig embryos (plate 5, fig. 1, and plate 1, fig. 1) and in two sections (text-figs. 5 and 6). Plate 5, figure 1, is from a specimen of approximately the same stage as in Evans's figure 394 in the “Manual of Human Embryology,’ which shows the state of develop- ment of vessels of the brain at this stage. The position of the embryo should be carefully noted. The caudal half of the specimen is seen from the direct ventral aspect, while the cephalic half is from a direct lateral view. The place of rotation is around the ninth somite. Extending from the level of the eleventh somite to the caudal end of the embryo there is a series of tiny ventral arteries from the two aorte. These are of uniform size and are placed at regular intervals, approximately one opposite an interspace and one opposite a somite. In this particular embryo only a few of these ventral branches are injected; but other specimens show that the entire length of both aorte gives rise to branches like those shown opposite the twelfth, thirteenth, and fourteenth somites. From the region of the eleventh to the four- 100 DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. teenth or fifteenth somite these tiny branches from the two aortz unite in a plexus of large arteries on either side of the stalk of the yolk-sac, which join and vive rise to the omphalo-mesenterie arteries on the yolk-sac. The large arteries are seen only on one side in plate 5, figure 1, and plate 1, figure 1, but are shown on both sides in figure 2 of plate 5. From the fourteenth somite caudalward the ventral branches of the aorta are uninjected in this specimen (plate 5, fig. 1), but show in other specimens leading to a single artery which arises in the caudal end of the embryo. Opposite the caudal end of the embryo the ventral branches of the aorte form a sheet of capillaries on either side of the alimentary canal, which deserves careful consideration. These two sheets of capillaries form a plexus which completely surrounds the entire caudal end of the gut cephalic to the allantois, the stalk of the allantois, and the blind end of the gut, caudal to the allantois. This capillary plexus gives rise to two arteries, the paired allantoic arteries and the single subintestinal artery. Thus, we have here examples of arteries in the embryo which arise in a capillary plexus and end in a capillary plexus. The primitive allantoic arteries arise in a plexus around the stalk of the allantois and pass to the capillaries of the body of the allantois; the subintestinal artery arises in a capillary plexus around the gut and runs to the capillaries of the yolk-sac. The allantoic arteries, as seen in plate 1, figure 1, extend into a plexus on the ventral or cephalie surface of the allantois; this plexus arches around the dome of the allantois, though not completely shown in the drawing, and reaches the veins on the caudal surface. The two allantoic veins join the umbilical veins at the point where the stalk of the allantois is fused with the body-wall. A section through the allantoic arteries from an injected embryo of the same litter as the specimen of plate 1, figure 1, is shown in text-figure 6, and shows the allantoic arteries following the wall of the gut into the allantois. In the series from which text-figure 6 is taken there are a few tiny capillaries extending dorsalward from the allantoic arteries just at the point where these arteries pass ventral to the ccelom. These capillaries grow lateral to the ccelom, and when the posterior limb-buds begin they will anastomose with the iliae arteries. These capillaries will become the umbilical arteries in the somatopleure. These observations on the pig agree with the findings of Hochstetter in the rabbit (1890) and show that in these forms the primary allantoic arteries are vitel- line vessels, while the central ends of the umbilical arteries are vessels of the soma- topleure, which appear later and anastomose with the primitive allantoic arteries. In the study of the R. Meyer human embryo 300, Felix (1910) gives an exceedingly interesting reconstruction of the vascular system of a human embryo which is of the same stage as my figure 1 of plate 1. This reconstruction (fig. 7, Morph. Jahrb, 1910, XLI, p. 590) shows that the primitive artery of the fetal mem- branes at the caudal end of the embryo arises in a capillary plexus around the gut, Just as is shown in my figure 1 of plate 5 and figure 1 of plate 1. The posi- tion of this plexus in the wall of the gut is shown in section in Felix’s figure 9, which is to be compared with my text-figure 6. The same relations are shown for the chick in Duval’s Atlas, plate xxi, figure 372. In the human embryo this artery DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 101 has been traced back as a vitelline vessel to the stage of 5 somites by Felix (1910), and to the stage of 8 somites by Dandy (1910). This artery in the wall of the gut, which is the primitive allantoic artery in the pig, has been called the um- bilical artery in the human embryo on account of the insignificance of the allan- tois and the earlier vascularization of the chorion. The relations of these two vessels in connection with the human embryo were summed up by Evans (1912, page 595) in the phrase that the umbilical artery is merely a modified vitelline vessel. The entire question of the relation of the arteries for the fetal membranes at the caudal end of the embryo has centered around the position of the central end of the arteries with reference to the ccelom, as can be seen in text-figure 6; that is to say, whether the artery is mesial or lateral to the ceelom. In general, both in birds and in mammals there is a primitive artery mesial to the ccelom; that is to say, a splanchnic vessel, and a secondary vessel, the umbilical artery, lateral to the ecelom running in the somatopleure. Thus the vessels develop in the same manner in the different forms, for there is a primitive splanchnic artery followed later by an artery in the somatopleure, but there are variations in the relative importance of the allantois itself. Besides the two arteries of the allantois, the two sheets of capillaries of the wall of the caudal end of the gué give rise to another artery. Extending forward from the stalk of the allantois, as seen in plate 5, figure 1, the two plexuses meet in a capillary plexus ventral to the caudal root of the yolk-sac. This plexus is continued as a single, ventral, subintestinal artery which joins the omphalo- mesenteric plexus opposite the fourteenth or fifteenth somite. The point where the subintestinal artery joins the omphalo-mesenteric plexus is the well-known intestinal landmark where the stalk of the yolk-sac joins the gut. A figure which gives a very clear understanding of these relations is Tandler’s figure 1 in the Anatomische Hefte, 1904, 1%, page 192. This subintestinal artery in the pig is the more interesting in view of the corresponding subintestinal vein in the chick, discovered by Hochstetter in 1888 and accurately described by him. He described its relations not only to the omphalo-mesenteric veins, but also to the intestinal and the allantoic vessels, and noted that it disappeared and that the left vein was larger than the right. A complete understanding of the development of this vein in the chick can be gained from the figures of Popoff (1894). As was mentioned in connection with the chick, during the early hours of the third day of incubation the entire capillary plexus of the area vasculosa caudal to the omphalo-mesenteric arteries must be regarded as an arterial capillary plexus down to the marginal vein, as shown in Popofi’s plate v. During the last hours of the third day, as seen in Popoff’s plate v1, branches of the omphalo-mesenteric vein gradually extend caudalward on either side of the embryo in the wall of the yolk-sac, and arch around the posterior end of the embryo; the left vessel starts ahead of the right and is always larger than the right. As these veins gradually extend backward into the terri- tory of the pre-existing arterial plexus, forming more and more new connections with the plexus, they change the direction of the current of the blood in the 102 DEVELOPMENT OF ‘PRIMITIVE BLOOD-VESSELS. plexus (which has been away from the heart) to a direction towards the heart. The vein on the left side quickly extends to the marginal vein, making the single posterior vein of the yolk-sae of Popoff, which lies a little to the left of the mid-line, as shown in Popoff’s plate vim. The two lateral veins form an arch around the posterior end of the embryo; this arch is just cephalic to the point where the stalk of the allantois will develop. On the third day of incubation there is a very extensive capillary plexus on either side of the posterior end of the gut, and beginning at the very caudal tip of the gut on either side are symmetrical ventral veins, which unite in a loop just cephalic to the base of the allantois and then run forward, at first as two veins and then as a single ventral vein in the ventral wall of the yolk-sac. The sub- intestinal vein is thus the primitive vein for the entire posterior end of the gut, for the caudal tip of the gut, the allantois, and the entire rectum and intestine up to the margin of the yolk-sac. Caudal to the allantois these vitelline veins receive the most caudal branches of the posterior limb-bud. This relation has been described by Evans (Anat. Record, 1909, ii). On the third day the um- bilieal artery develops around the somatopleure in connection with the posterior limb-bud and anastomoses with the primitive allantoic capillary plexus in the wall of the allantoic stalk. By the beginning of the fourth day the vessels in the stalk of the allantois show an exceedingly interesting relation. On either side there is one large artery coming from the aorta and now running in the somato- pleure instead of in the splanchnopleure; but this artery is fed also from a capillary plexus in the wall of the splanchnopleure, which completely surrounds the stalk of the allantois and the caudal tip of the gut, and by a few capillaries of the somatopleure from the tail of the embryo, which capillaries, however, tend to drain more and more into the posterior cardinal veins. These relations are clear in the light of the development of these vessels. There is at first a plexus of capillaries arising from the aorta and running in the stalk of the allantois, in which arise the primitive allantoic arteries; and secondarily, a capillary plexus in the somatopleure of the caudal end of the embryo, in which an umbilical artery develops. The umbilical artery joins the original allantoic artery in the fused area of allantois, somatopleure, and amnion (see text-fig. 6), and then the primitive allantoic arteries from the aorta become reduced again to a capillary plexus. Thus the allantois has a double arterial supply and a double venous drainage, the former in the wall of the gut and the latter in the somatopleure. The primitive allantoic arteries arise in a plexus of the splanchnopleure, and the corresponding venous return is through the sub- intestinal vein; the subintestinal vein anastomoses with the allantoic veins, but the direct continuation of the allantoic veins is into the umbilical veins, which develop in the somatopleure. Finally the umbilical arteries develop in the somatopleure, connect with the allantoic arteries, and soon bring most of the blood to the allantois. DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 103 The fate of the subintestinal vein in the chick is very interesting. If an injected chick of the fourth and fifth days be dissected so as to expose the caudal end of the gut and the straight posterior segment of the gut which leads up to the open bell of the yolk-sac, it will be seen that the entire wall of the gut is surrounded by a capillary plexus. At the caudal end of the gut and just cephalic to the stalk of the allantois the ventral vein has entirely disappeared in this capillary plexus, while far- ther forward it is still clear in the ventral wall of the gut, though freely connected with the plexus. It is clear also that this posterior segment of the gut is receiving new arterial and venous connections which grow in along the dorsal border at the cephalic end of the segment. The new artery is a branch of the omphalo-mesen- teric artery given off just at the root of the yolk-sac; it extends caudalward along the dorsal wall of the gut and anastomoses with the aortic branches which are the forerunners of the inferior mesenteric arteries. The new veins are branches of the omphalo-mesenteric veins within the mesentery, the forerunners of the portal system. The entire subintestinal vein gradually disappears as a single channel by developing into the plexus of the wall of the gut. In this plexus it is clear that the direction of the flow of the blood in the wall of the gut is from the ventral toward the dorsal border, at right angles to the direction of the stream in the subintestinal vein. It may seem curious that the pig should have a subintestinal artery in place of the well-established subintestinal vein of the chick. As has been shown, the subintestinal vein in the chick develops as a part of the process by which the primitive circulation of the yolk-sac, with arteries and veins as far apart as possible, becomes changed so that every zone of the area vasculosa is invaded by veins. The pig of plate 5, figure 1, represents the more primitive condition for compar- ison with Popoff’s plate tv, in which the caudal part of the yolk-sac is still arterial. The subintestinal artery of the pig can be seen in section in text-figure 5 from a pig of the same litter as the one shown in figure 1, plate 1; it receives numerous ventral arteries from the aorta, as does the corresponding vein in the chick; but it joins the omphalo-mesenteric arteries at the point of loop of the mesenteric arteries instead of the veins. This same artery is still present in the pig measuring 9 or 10 mm. after the caudal flexure has formed, at which stage it is breaking up into the capillary plexus within the wall of the gut. By the time the pig is 15 to 17 mm. long there is a new longitudinal artery in the dorsal wall of the gut, extending from the superior mesenteric artery caudalward and anas- tomosing with all the ventral aortic branches which represent the interior mesen- teric artery. At the same time the accompanying venous plexus from the omphalo- mesenteric vein extends along the dorsal border of the gut. As this new blood- supply for the lower half of the intestine develops, the ventral vein of the earlier stages of the chick, or the ventral artery of the pig, becomes reduced to a part of the capillary plexus in the wall of the gut. It is interesting to note that in a pig of 9.5 mm. the ventral artery of the gut is also accompanied by a plexus of ventral veins, which correspond to the single ventral vein in the chick. Thus the differ- ence in the two forms becomes readily understandable, for the invasion of that LO4 DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. part of the gut by the veins is merely relatively later in the pig, and the veins are thus much more transitory. Branches of the omphalo-mesenteric veins growing down the mesentery begin early inthe pig. These are shown in plate 4, figure 3. They are not seen in plate 1, figure 1, because uninjected in the specimen. In other specimens from the same litter there is a vein in the mesentery underneath the umbilical vein, as seen from the side, and joining the main omphalo-mesenteric vein at the lower margin of the liver. These branches are shown in plate 5, figure 2. The veins in the root of the mesentery anastomose with the mesial cardinal (subeardinal) vein as soon as it develops. This anastomosis was described by Hochstetter. The ventral subintestinal artery here described was discovered by Ravn in 1894 in the rat and mouse. The vessel was also described by Evans in the pig (Manual of Human Embryology, Keibel and Mall, foot-note 56 on page 656). Ravn’s description can be readily followed in my plate 5, figure 1, as he described the main omphalo-mesenteric artery arising in the caudal end of the embryo. Both Ravn and Evans describe this subintestinal artery as arising from the um- bilieal artery. My specimens, however, are from still earlier stages, and prove that this vessel arises, as does all the rest of the omphalo-mesenteric system, in the wall of the yolk-sac or gut; that it is a true vitelline vessel. Its anastomosis with the umbilical arteries in the somatopleure occurs later. Thus the subintes- tinal artery in the pig and the subintestinal vein in the chick are vitelline vessels. They disappear as single channels and help in the formation of the primitive plexus in the wall of the gut in connection with the changes by which the gut receives its permanent blood-supply and in connection with the gradual reduction of the yolk-sac. The study of the ventral branches of the aorta in the human embryo is based on the work of Mall, who in 1891 published an account of a human embryo 7 mm. long, in which he described two main ventral branches, a cceliac axis and an omphalo-mesenteric artery, and a series of small ventral branches in the lumbar region, making a capillary network in the mesentery. He noted that the position of both the cceliac axis and the omphalo-mesenterie artery was farther forward than in the adult, and analyzed all the available material in a study of the shifting of the arteries caudalward along the aorta. In this study he recorded human embryos with the cceliac axis opposite the first, second, fourth, and sixth dorsal nerves, as compared with the position in the adult opposite the twelfth nerve. In 1897 he made a further study of the ventral arteries, especially in a human embryo 2.1 mm. long. In this specimen he showed a series of ventral branches extending from the seventh somite to the caudal end of the aorta. These vessels he grouped together as the omphalo-mesenterie arteries. In the reconstruction he showed that the upper arteries tended to be opposite the middle of the somites rather than between the somites, as are the dorsal intersegmental vessels. In a second analysis of the ventral aortic branches he showed that there is a constant shifting of the coeliac axis and omphalo-mesenterie arteries caudalward. A double origin of the omphalo-mesenteric¢ arteries in one embryo suggested the method of the wandering of the vessels. DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 105 The same idea of the shifting of the arteries caudalward was further developed by Tandler in two papers in 1904 and by Broman in 1908. These workers ex- tended their observations over a long series of embryos, Broman giving a study of 41 specimens. In one of the youngest specimens in his series, an embryo measuring 3.4 mm., the upper ventral branch was between the sixth and seventh interspaces. He described the branches as tending to occur between the inter- spaces, there being two or three to a somite. He found that the cceliae axis and the superior mesenteric artery are not segmental vessels (that is, opposite the interspaces), while the inferior mesenteric artery is sometimes opposite and some- times between the somites. Broman gives an analysis of the literature and an extensive discussion of the methods by which the shifting of the ventral arteries may take place. In the human embryo ventral branches of the aorta have been described from about the seventh segment caudalward. In the pig these ventral branches are very numerous—approximately one to a segment and one to an interspace. They are originally of uniform size and about equidistant apart. They unite into an extensive plexus of larger vessels in the more cephalic region and into a long artery in the caudal region. It is easy to follow the method of the shifting of arteries from such a primitive pattern; that is, any of the vessels of the original system could easily enlarge and the blood-stream be increased or decreased accord- ing to the development of the region of the organ supplied. The entire wandering of the arteries can be understood without presupposing the development of any new vessels, but rather through the shunting of the blood through different channels already present in response to the varying development of the parts supplied by these arteries. Moreover, it is plain that the point brought out by Evans is of importance—namely, that the so-called wandering of arteries takes place while the vessels have the structure of capillaries; that is, while their wall consists of endothelium alone. From the position of the primitive ventral arteries it is also easily seen that there might be variations as to whether the ultimate ventral arteries of the older embryo came opposite an interspace on the same level as the dorsal arteries or opposite a somite. THE UMBILICAL VESSELS. My series is not very complete in regard to the umbilical veins, but it shows a few interesting points. In plate 4, figure 3, the relation of the somatopleure to the fold of the amnion is very plain. Jn the somatopleure is the beginning of a capillary plexus representing the umbilical veins. In plate 5, figure 1, the umbilical veins are not injected, but they are well shown in plate 1, figure 1, in which it is clear that the return flow of the blood from the caudal end of the embryo is in part through the subintestinal artery in the splanchnopleure and in part through the umbilical veins in the somatopleure. At the stage of plate 1, figure 1, the umbilical veins have established their connections with the liver, though they still connect with the sinus venosus. In figure 1 of plate 1 and figure 2 of plate 5 it is clear that cephalic to the duet of Cuvier there is also a capillary plexus in 106 DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. exactly the same position as the umbilical veins; that is, in the somatopleure. In the specimen of plate 1, figure 1, this venous capillary plexus connects with a tiny lateral aortic branch shown Just opposite the zone of the second aortic arch. This lateral artery is not the second aortic arch, which arises from the ventral rather than from the lateral surface of the aorta. From this tiny lateral artery a straggling chain of capillaries is injected within the somatopleure, out over the heart, and down to the duct of Cuvier; they are omitted in the drawing. It is clear that they are vessels for the body-wall analogous to the vessels which drain into the umbilical veins; but they are cephalic to the duct of Cuvier. The venous end of the plexus is injected in plate 5, figure 2. It was shown in the chick that the corresponding vessel of the somatopleure over the heart develops very early. In later stages these vessels in the somatopleure over the heart anastomose freely with a plexus of capillaries lateral to the occipital myotomes, as shown in text- figure 5 in my article on the Origin and Development of the Lymphatic System, 1913. NEURAL BRANCHES OF THE AORTA AND THE PRIMARY HEAD-VEIN. In connection with the neural vessels, I have no specimens of embryo pigs corresponding to the chicks of 6 somites in which to trace their beginning. I have one litter of very young pigs, measuring 3 mm., in which the heart and aorta are present; the neural folds are open at the cephalic end, and I can find no angio- blasts along the closed hindbrain. At the stage of figure 3, plate 4, the vessels to the forebrain can be injected; and the vessel of the hindbrain must be present, for it is seen in a human embryo of the same stage of development. Opposite the third and fourth somites the lateral plexus of the neural tube has been injected from the aorta. I found only one specimen of the litter of figure 3, plate 4; but the fact that the posterior cardinal vein is almost completely injected indicates that the anterior cardinal vein is present and that it connects with the deep vein of the hindbrain. At the stage of plate 5, figure 1, the vessels of the head are in about the stage of development of those of plate 1, figure 1, as is proved by the injections of the same litter. In one specimen of the same litter as plate 5, figure 1, the anastomosis of the capillaries around the optic stalk is complete, just as was shown by Evans for the later stage of three aortic arches in his figure 395 (Keibel and Mall, Manual of Human Embryology, II, p. 579). The best view of the early neural vessels in my series is given in plate 1, figure 1. In order to analyze the relations of the vessels of the head, I have used gray to indicate all of the capillaries which are true neural vessels, in the sense of lying close to the wall of the neural tube and giving rise to the vessels of the sub- sequent pia mater. As can be seen in plate 1, figure 1, the deep capillary plexus of the forebrain and midbrain is covering the wall of the brain, and the form of this plexus indi- cates the form of the brain. The vascular arch which surrounds the large peduncle of the optic vesicle (see Evans’s figure 395) is incompletely injected in DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 107 plate 1, figure 1. It shows the relative size of the optic vesicle and the forebrain at this stage. The side of the thalamus and the midbrain is nearly covered by a plexus extending toward the dorsal wall of the neural tube. Along the cephalic part of the hindbrain is a wide vessel connected with the aorta by two arteries. It already shows sprouts along its dorsal border, two of which bound the otie vesicle. This deep single channel becomes a plexus along the side of the neural tube at a point just in front of the first myotome. This is the point where the cephalic end of the anterior cardinal vein joins the neural vessels, and, in terms of the neural tube, it is at the cephalic end of the origin of the roots of the vagus nerve. The transverse vessel of the first interspace which is so prominent in the chick is but a small vein in the pig like the other intersegmental veins, and does not become an important vessel, as in the chick. As is well known, the upper myotomes are occipital myotomes, so that it is clear that the point of transition between the deep vessel of the hindbrain and the primitive plexus, as shown in plate 1, figure 1, is not between the hindbrain and cord, but is near the upper part of the medulla. The lateral plexus along the cord is injected in the specimen of plate 1, figure 1, down to the fourteenth somite, which is opposite the lowest transverse artery injected, and the spinal arteries are injected down to the twentieth interspace. These lower vessels are omitted in the drawing. In plate 1, figure 1, can be traced very clearly the origin of the cephalic part of the primary head-vein; that is, the primitive cerebral vein. Extending from the groove between the telencephalon and the diencephalon (as Evans showed in his figure 395 in 1912), is a superficial capillary plexus, indicated in blue, which receives its blood from the deep plexus of the forebrain and midbrain and drains into the deep vessel of the hindbrain. In this plexus will develop the primitive cerebral vein; at this stage it is entirely a plexus without any definite longitudinal channels. The specimen is just at the stage of the second vascular arch, which is probably present and uninjected, as shown in Evans’s figure 394 from an earlier stage. Opposite the lower end of the primitive vessel of the hindbrain is a plexus of exceedingly tiny vessels spanning the gap between the deep vessel of the hind- brain and the anterior cardinal vein on the one hand, and reaching toward the second aortic arch on the other. These tiny capillaries form the origin of the lateral vein of the region, that is, the middle segment of the primary head-vein, just as has been shown for the chick. This plexus will span the gap between the second and third aortic arches as they form, and the cephalic end of the primary head-vein, until there is a double vascular channel from the head, as shown on plate 4, figure 1. Figure 1 of plate 4 is from a specimen of the same litter as that of figure 2 of plate 5, and is given because of the extravasation in the head region in the latter figure. At the stage of three aortic arches the primary head-vein is complete. The primitive veins which pass ventral to the eye are not injected in the specimen of plate 4, figure 1, except just where they join the primary head-vein in front of the ganglion of the trigeminus. The primary head-vein starts opposite the thalamus and extends in a double curve down to the anterior cardinal vein. It 108 DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. lies mesial to the Gasserian ganglion and lateral to the otic vesicle. The pattern of the deep and the superficial vessels in plate 4, figure 1, deserves careful study. The place of origin of the roots of the Gasserian ganglion is marked by a plexus of the deep vessels which are growing around it, leaving a non-vascular area where the nerves emerge. The deep plexus is also forming a dorsal arch around the otic vesicle which now lies between the deep and the superficial vessels. The pattern of the vessels also indicates the position of the acoustic complex of ganglia and the glosso-pharyngeal ganglion, both of which lie between the deep capillaries and the superficial veins, one cephalic to the otic vesicle and the other just caudal to it. It is clear that the relations of the primary head-vein to the Gasserian ganglion and to the acoustic complex are the same in the pig as in the chick, and are due to the fact that this vessel forms while the ganglia are attached to the skin in their respective placodes. The primary head-vein develops mesial to the placode of the Gasserian ganglion, but curves dorsalward opposite the acoustic ganglia and opposite the ganglion of the glosso-pharyngeus. Sections of an embryo slightly older than that of plate 4, figure 1, cut so that a long stretch of the primitive head-vein is included in one section, show that the lateral border of the acoustic ganglion is in a straight line with the mesial border of the Gasserian gang- lion, so that the superficial vein takes the shortest course in passing mesial to the ganglion of the trigeminus and lateral to the ganglia of the acoustic complex. The relations of the branches of the vena capitis prima are very important at the stage of plate 4, figure 1. The branches from the aortic arches are not injected, nor are the primitive maxillary veins. The lateral veins from the cere- brum have hardly begun. The superficial veins opposite the midbrain have a very characteristic pattern; they are, as it were, creeping along on the deep plexus toward the mid-dorsal line. It will be noted that the deep plexus itself has not yet reached the mid-dorsal line at this stage, but it is in advance of the superficial veins. This gradual extension of the branches of the vena capitis prima to the mid-dorsal line characterizes the branches of this vein over the entire brain. When the superficial veins meet in the mid-dorsal line they will give rise to all of the sinuses and veins of this line, as has been shown by Mall and Streeter. Opposite the hindbrain the branches of the vena capitis prima have the same fundamental relation to the deep plexus. It is true that caudal to the otic capsule there are a few veins from the deep plexus draining into the ventral border of the vena capitis prima, which may be forerunners of the small ventral veins of the medulla in the adult, but almost all of the veins of the hindbrain drain into the dorsal border of the primary head-vein. These veins have the same characteristics as the rest of the neural veins; that is, they gradually creep dor- salward on the deep plexus. Over the hindbrain, however, the pattern of the veins is not as simple as over the midbrain, because here they are profoundly affeeted by the ganglia of the hindbrain and by the otic capsule. It has been shown that the deep plexus makes an arch of capillaries around the roots of the nerves, as seen around the root of the trigeminus in plate 4, figure 1. The superficial veins also curve around the roots of the nerves. Their DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 109 beginning is shown in plate 4, figure 1. Here it is clear that branches of the primary head-vein are tapping the deep neural plexus around the root of the trigeminus. Lateral to the acoustic complex and to the ganglion of the glosso-pharyngeus, the branches of the primary head-vein make a very extensive plexus. The super- ficial venous arch around the otic vesicle is just beginning in plate 4, figure 1. The veins around the trigeminus and around the otic capsule are exceedingly important, because of their ultimate relations to the basal sinuses of the dura. These vessels are shown in plate 4, figure 1, at the stage when they are scarcely more than capillary sprouts. They will be traced farther in the next figure. The specimen of plate 7, from a pig which measures 6.5 mm. in oil, is given to emphasize the fate of the primitive vein of the hindbrain, to bring out the ventral artery that now extends the full length of the nervous system from the base of the optic cup to the tip of the tail, and to show the characteristic relations of the veins to the neural tube and its ganglia. The injection of the specimen of plate 4, figure 1, did not bring out the ascending neural arteries as did a corresponding injection of the chick (plate 6), but the specimen of plate 7 shows that there is now a longitudinal artery which extends from the primary aortic branch to the brain opposite the subthalamus, along the ventral or ventro-lateral border of the neural tube to its caudal tip. This artery is an anastomosis between all of the neural arteries, both cerebral and spinal. As can be seen in plate 7, the carotid artery leads to an arterial plexus which covers the lateral surface of the subthalamus and gives rise to a cerebral artery passing dorsal to the eye. The plexus on the subthalamus anastomoses with the plexus of the opposite side in the mid-ventral line; it is tapped by a vein leading to the primary head-vein just cephalic to the maxillary vein. Opposite the groove between the thalamus and the midbrain the two plexuses on either side of the subthalamus gives rise to a single ventral artery which curves along the ventral border of the neural tube down to the level of the third occipital inter- space, where the single median artery becomes an arterial plexus. From this point to the caudal end of the spinal cord there is a double line of capillaries, such as was shown by Evans in his figure 440 (1912). As Evans showed, this double capillary chain will give rise to the anterior spinal artery. The importance of this longitudinal neural artery, which gives rise to the circle of Willis, the basilar artery, and the anterior spinal artery, is obvious. The anastomosis of the arterial plexus of the subthalamus and the ventral surface of the cerebrum with the corresponding plexus of the other side across the mid-ventral line accounts for the anterior com- municating artery of the circle of Willis. At the stage of plate 7 the longitudinal neural artery is supplied by the two carotid arteries, by direct arteries opposite the hindbrain, of which two are shown on the right side of plate 7, and by all the intersegmental arteries on either side. This artery is not supplied as yet by the vertebral arteries, which form later as an anastomosis between the upper intersegmental arteries. 110 DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. The arterial plexus over the subthalamus leads into a finely meshed plexus which covers the entire cerebrum except a small area in the mid-dorsal line near the thalamus. This plexus is not shown in the drawing, but it has the same character as the plexus over the midbrain. The cerebral plexus completely surrounds the optic stalk; in this plexus the only vessel larger than the rest is the cerebral artery, which is seen dorsal to the eye in plate 7. The longitudinal neural artery along the ventral border of the midbrain and the hindbrain gives off a series of nearly equal, regular, small arteries which lead into the capillary plexus on either side of the neural tube. The capillary plexus on the neural tube is very characteristic. As has been said, it is finely meshed over the cerebrum, the thalamus, and the midbrain; it is more coarsely meshed over the hindbrain, where the plexus has developed later, especially around the roof of the fourth ventricle, which has not yet been invaded by the vessels. The plexus on the hindbrain in plate 7 demonstrates the fate of the primitive vessel of the hindbrain, the beginning of this plexus as coming from the primitive vessel of the hindbrain having been seen in the living chick. The primitive vessel of the hindbrain disappears only in giving rise to the capillary plexus of the hindbrain. If the pattern of the neural plexus in plate 7 is observed carefully it will be seen that there is Just a suggestion of trans- verse lines in the plexus, indicating that the direction of the flow of the blood is from the ventral to the dorsal border of the neural tube. In this plexus will ultimately come transverse arteries. Opposite the first somite will be noticed the beginning of three layers of vessels, a deep layer of very fine capillaries, a second layer of larger vessels also shown in gray, and a third layer of more superficial veins. This is the very beginning of the next stage in the development of the neural vessels. The most important point about the form of the deep plexus on the neural tube is the way it conforms exactly to the neural tube and its nerves. Over the midbrain the plexus is very uniform, but over the hindbrain the character of the plexus indicates very clearly the position of the nerves. At the stage of plate 7 there are bare spots, that is, places with no blood-vessels, on the hindbrain corresponding to each nerve root; in later stages the vessels penetrate between the small bundles of the fibers of each root and then an injection of the deep plexus does not show the position of the nerves so clearly. As seen in plate 7, the posi- tions of the roots of the trigeminus nerve and of the acoustic group of nerves are very clear. The otic capsule now les just lateral to the deep capillary plexus, and thus its position is indicated only by the superficial veins. Opposite the ganglion of the glosso-pharyngeus is a bare spot in the deep plexus, which is nearly hidden by a very extensive group of superficial veins. The position of the roots of the vagus and the spinal accessory roots along the line of the posterior cerebral vein is very important. It is clear that the deep plexus outlines this long line of nerve roots, and the same is true along the more ventral line of the medulla, where the pattern of the vessels indicates the position of the roots of the hypoglossal nerves. DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. ie Along the spinal cord the pattern of the capillary plexus shows the position of the ventral nerve roots in the same manner. The veins which form the branches of the vena capitis prima must now be followed. The veins from the visceral arches, still largely in the form of capillaries, are completely injected in the specimen, but are indicated in the drawing only at the point where they join the middle segment of the primitive head-vein. In plate 7 the cerebral and the cardinal segments of the vena capitis prima are shown in plastic form, but the middle segment is shown merely in outline in order to make more plain the relations of the neural artery beneath. Beginning with the maxillary vein, the entire maxilla is filled with a capillary plexus which leads to the maxillary vein. This capillary plexus anastomoses with the plexus of the mandibular arch. Besides these capillaries the vein receives a large group of tiny superficial veins which arise in the deep plexus that covers the entire olfactory area of the cerebrum, together with primitive ophthalmic veins which arise in the marginal vein of the optic cup, as in plate 6. One of these subophthalmic veins runs in the groove of the optic stalk. These cerebral veins from the rhinenceph- alon and from the inferior part of the eye are very important in the early drainage of the brain, but it is well known that the main permanent ophthalmic veins develop dorsal to the eye. In the zone dorsal to the eye at the stage of plate 7 is a group of tiny super- ficial veins opposite the cerebrum which are like the small veins over the mid- brain. They were omitted in plate 7, but are adequately shown for the chick in plate 6, and they are alike in both forms. These are the primitive cerebral veins. Over the midbrain the veins are characteristic. It is plain that they are lifted off from the surface of the neural tube, that they are all superficial to the deep plexus; they spread out like a fan from the primary head-vein and clearly extend along the deep plexus, which they tap at their tips, and approach the mid-dorsal line. Opposite the hindbrain the veins are exceedingly interesting; they follow exactly the same general course of development as the rest of the neural veins; that is, they lie superficial to the deep plexus, are transverse to the long axis of the neural tube, and gradually extend toward the mid-dorsal line, constantly tapping the deep plexus at their tips. On the other hand, they are profoundly modified in their development of the ganglia of the hindbrain and by the otie vesicle, so that their pattern is much more complex than the pattern of those opposite the midbrain. The vessels around the ganglion of the trigeminus deserve careful study. At this stage the entire lateral surface of the Gasserian ganglion is covered by a capillary plexus which was omitted in the drawing. This capillary plexus extends along the second and third divisions of the nerve and becomes continuous with the capillary plexus of the maxillary and mandibular processes. Besides this sheet of capillaries which covers the lateral surface of the ganglion, there are two transverse veins above and below the ganglion which outline the root of the trigeminus nerve. These veins are very characteristic, and mark the position 112 DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. of the Gasserian ganglion in any injected specimen up to the stage measuring 20 mm., when the transformation of the veins into the dural sinuses is well advanced, as can be seen in Streeter’s figure 3 (Amer. Journ. of Anat., 1915, XVIII, page 156). The superficial vessels around the ganglia of the eighth nerve are still in the form of capillaries in plate 7. Opposite the otic vesicle the deep plexus has completely covered the surface of the hindbrain; there are a few super- ficial veins across the lateral surface of the vesicle, which are shown cut off in the drawing close to the primary head-vein. Two of these transverse veins make a border for the otie vesicle exactly as do those above and below the Gasserian ganglion. In other words, the veins of the hindbrain can be most simply de- scribed as a series of transverse vessels, some of which are forced to curve by the Gasserian ganglion and the otic vesicle. Opposite the ganglion of the glosso- pharyngeal nerve is a series of transverse veins draining into the primary head-vein. The veins opposite the vagus nerve are also very interesting. It is clear that the largest vein of the medulla at this stage is one which in a general way follows the roots of the vagus nerve. This vein was called the posterior cerebral vein by Mall. In general, the place where it joins the vena capitis prima marks the cephalic end of the anterior cardinal vein; it may be a single vein at its roots or a group of veins. In the pig the vagus nerve curves around its cephalic border, passing in the angle between this vein and the primary head-vein. Some of the injections show the nerve passing through a venous loop in this angle. Stracher describes the vagus nerve just caudal to the vein in the chick. The relations of the vagus nerve to the primary head-vein formed the basis of Kastchenko’s original study of the primitive veins of the head. As will be seen in plate 7, the main vein of the medulla primarily follows the course of the roots of the vagus nerve. It arches caudalward along the dorso- lateral surface of the medulla in the line of the spinal accessory nerve and roots of the vagus. The line of the vein on the medulla can be well seen by following the vagus roots in Streeter’s plate 11 (Amer. Jour. of Anat., 1905, IV). While it is clear that this vein and its tributaries originally follow the path of the vagus nerve, if its development is followed it will be seen that it becomes a very important vein of the embryo, not even limited to the drainage of the neural tube. At the stage of plate 7 it anastomoses with the lateral venous plexus of the lower medulla, and the first and second occipital veins are correspond- ingly small. Subsequently it gives rise to an extensive group of dorsal branches that grow over the caudal part of the roof of the fourth ventricle and largely drain the developing choroid plexus. The posterior cerebral vein next develops an exceedingly interesting relation to the vascular system of the occipital myotomes. This relation was illustrated in two figures from injected embryo pigs in my article on the origin and development of the lymphatic system (1913, figs. 4 and 5). Opposite the entire zone of the myotomes a plexus of capillaries develops, forming the third vascular sheet of this region. Primarily there is a plexus of capillaries on the surface of the neural tube; secondly, a more lateral plexus of capillaries and veins especially related to the ganglia; thirdly, this sheet of capillaries lateral to DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 113 the myotomes. Opposite the occipital myotomes the capillary plexus drains, by a series of veins on the one hand into the main vein of the medulla, on the other hand into the anterior cardinal vein. The history of the neural branches of this vein of the medulla involves the entire subject of the circulation of the medulla. The relation of the branches from the occipital myotomes involves the subject of the development of the external jugular vein and its branches. The main stem of the vein was shown by Mall, in 1905, to become a part of the great transverse sinus. For this vein I am using the term primitive posterior cerebral vein. It might also be termed the primitive vein of the medulla. The stage of plate 7 shows the beginning of the veins of the hindbrain. It will be seen that the primitive branches of the primary head-vein draining the hindbrain are greatly modified by the ganglia of the hindbrain and the otie capsule. Opposite the midbrain these veins are regular and nearly equidistant; opposite the hindbrain they are grouped according to the ganglia. Of these veins of the hindbrain, the group caudal to the Gasserian ganglion and the stem of the posterior cerebral vein bear the most important relations to the future cerebral sinuses at the base of the brain. In this account of the early blood-vessels of the neural tube three facts have been brought out which are essential to an understanding of the development of the neural vessels. First, there forms a ventral neural artery, originally paired, which extends along the ventral surface of the entire neural tube from the base of the optic cup to the caudal end of the spinal cord, which is an anastomosis of all the direct neural arteries from the aorta; second, this artery leads to a capillary plexus which completely invests the neural tube and all its ganglia; third, the primary veins of the neural tube are all transverse vessels superficial to this primary plexus, and they gradually extend toward the mid-dorsal line and are profoundly modified by the ganglia, both cerebral and spinal. All of the veins of the brain drain into the primary head-vein. As has been shown by Mall and Streeter, the only segment of the vena capitis prima which remains as a part of the dural sinuses becomes the cavernous sinus, which is that portion of the primary head-vein medial to the Gasserian ganglion. All other dural sinuses develop from the branches of the vena capitis prima. It has been shown that the middle segment of the vena capitis prima develops in the pig, as in the chick, as a chain of capillaries between the aortic arches and the anterior cardinal vein; it becomes very large, because it makes a more direct outlet for the primitive cerebral vein. The vena capitis prima develops from three seg- ments and is the first true vein for the head; the primitive vessel of the hindbrain serves temporarily as a vein for the brain and then gives rise to the capillary plexus of the upper part of the hindbrain. 114 DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. CARDINAL VEINS IN THE PIG. It was shown in the chick that the cardinal veins begin from dorsal diver- ticula of the aorta which project into the interspaces and dilate just opposite the dorsal border of the nephrotome. In the line of the nephrotome these separate dilatations become connected, making a common cardinal vein which, at the stage of 12 somites, is opposite every interspace. I have not the corresponding early stages of the cardinal veins in the pig. In my earliest stage in the pig, the eardinal veins are related to the aorta and to the spinal veins, as is shown for the chick in the section on plate 3, figure 2; that is, there are direct spinal arteries from the aorta to the cord and spinal veins leading to the cardinal vein. At the stage of plate 4, figure 3, the posterior cardinal vein is injected, extending from the zone of the ninth intersegmental artery almost to the duct of Cuvier. The anterior cardinal vein is not injected, but must be present in the specimen. The pig embryo shown in figure 1, plate 1, gives the best view of the cardinal veins in my series. In this specimen it is clear that the anterior cardinal vein joins the neural plexus cephalic to the first somite, so that the vein of the first interspace which was so important in the chick is like all of the rest of the intersegmental veins in the pig. Opposite the first nine somites in the pig, as shown in plate 1, figure 1, the cardinal veins appear to be an accompanying vein to the aorta. Just below the ninth intersegmental artery in the pig there are the lateral arteries to the nephrotomes, and over all of the rest of the course of the posterior cardinal veins the lateral cardinal vein must also be considered. Opposite the first nine somites I have not been able to find any direct connections between the cardinal veins and the aorta, such as were shown for the earlier stages in the chick. In other words, the cardinal veins are well formed rather than just beginning in all of my specimens. One embryo, of the same litter as the one in plate 1, figure 1, showed some tiny sprouts of the anterior cardinal vein opposite the second somite extend- ing toward the aorta; sections, however, did not demonstrate any connections, and I could not prove that they were not the beginning of tiny veins that soon drain the pharynx. The series of the pig embryos also does not show the origin of the duct of Cuvier, but the fact that it is made up of an extensive plexus is well shown in plate 1, figure 1, as well as its relation to the umbilical veins. Below the zone of the ninth somite the cardinal veins will be considered with relation to the vessels of the pronephros. NEPHRITIC VESSELS IN THE PIG. The nephritic tubules in the pig receive an early and characteristic blood- supply. For the limit for the chick between the pronephros and the mesonephros I have followed Lilly, who regards the tubules as belonging to the pronephros down to the fifteenth or sixteenth somite (page 190). For the pig I have arbi- trarily followed Felix’s estimation for the human embryo (1912, page 762). He places the limit of the pronephros at the fourteenth somite. It will be seen in figure 3, plate 4, that just below the ninth intersegmental artery a series of lateral DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 115 arteries gives rise to a plexus which is ventral to the posterior cardinal vein, but which connects with it. The Wolffian duct intervenes between this plexus and the posterior cardinal vein. In figure 1 of plate 5, and figure 1 of plate 1, are given very characteristic views of these lateral arteries to the pronephros, and in figure 5, plate 3, is shown a ventral view of the arteries of the pronephros from a specimen of the same litter as that of plate 5, figure 1, but a little farther developed. Figures 1 of plate 5 and 1 of plate 1 show a series of tiny lateral arteries beginning just below the ninth dorsal segmental artery. These arteries are about four to a somite, corresponding to the number of the nephritic tubules, and are connected by a tiny longi- tudinal artery close to the aorta and by a tiny lateral vein. In text- figure 4 is shown a section from a specimen of the litter of plate 1, figure 1, passing through about the fourteenth somite, showing an injec- tion of one of these lateral arteries. Its exact position with reference to the developing tubule is, I think, important. This is most clearly recognized from the diagram given by Felix of the development of the nephritic tubules (fig. 561, Keibel and Mall, Manual of Human Em- = Fic. 4.—Transverse section of an embryo pig of 23 somites, passing bryology, page 804). Thestagecor- ~ through one of the lateral arteries of the pronephros. ‘The section responds with diagram d of Felix’s = {om svedinen of the same liter asthe one shown, on plate 1 figure, and the artery passes directly the section is shown by a line on plate 1, figure 1. The section is 20 uw thick and is stained with hematoxylin and counterstained with across the curved bowl which makes orange G, eosin, and aurantia. X115. A. om., a. omphalo-mesen- - 5 terica; A. pr., a. of the pronephros which was injected from the the neck, of the future Malpighian aoria; 7. om., v. omphalo-mesenterica; V.c., v. cardinalis lateralis: corpuscle. This is the earlieststage "©? v- cardinalis posterior; W..d.. Wolffian duct. of the vessels of the nephritic tubules I have injected. As is seen in text-figure 4, the lateral vein, the vena cardinalis lateralis, lies ventral to the Wolffian duct, while the vena cardinalis posterior lies directly dorsal to the duct. The posterior cardinal vein is plainly shown in text-figure 4, but was not injected so far caudal- ward in any of my series. Text-figure 5 gives a very interesting section from the same series as text- figure 4. The level of the section is shown in plate 1, figure 1; it is about halfway between the level of the lowest transverse artery injected and the allantoic arteries. At this level the nephritie tubule is in the stage of Felix’s figure 5615, consisting of a Wolffian duct and a mass of nephrogenic epithelium. Here, instead of an artery which can be injected, the section shows a chain of angioblasts running ventral to the nephritic tissue to the lateral cardinal vein, and other sections show similar chains of angioblasts connecting the aorta and the posterior cardinal vein. 116 DEVELOPMENT OF These angioblasts are mesial to the nephritic tissue. PRIMITIVE BLOOD-VESSELS. This section is, I think, similar to the section in Evans’s figure 416 from a human embryo of the same Fic. 5.—Transverse section of an embryo pig of 23 somites, passing stage, namely, with 23 somites, which shows the posterior cardinal vein dorsal to the Wolffian duct and the lateral cardinal vein ven- tral to the duct. A comparison of text-figures 4 and 5 seems to me to indicate that the primary arteries of the nephro- genic tissue are ventral to the ne- phrotome, but when the tubules through one of the lower myotomes and the mesonephros, to show the position of the subintestinal artery and a chain of angioblasts which will form an artery of the mesonephros. The section is from a specimen of the same litter as the one shown on plate 1, figure 1, and from the same series as figures 4 and 6. The level of the section is shown by a line on plate 1, figure 1. The section is 20 » thick and is stained with hematoxylin and counterstained with orange G, eosin, and aurantia. X115. A. mes., a chain of angioblasts which connect the aorta with the vy. cardinalis lateralis and which will form an artery of the mesencephalon but were not injected because they are still solid; A. si., a. subintestinalis; V. c. l., v. car- are farther developed the artery crosses the neck of the tubule; in other words, the tubules grow ven- tral to the arteries. The study of the embryo pig at the stage of 23 somites (as shown in dinalis lateralis; V. c. p., v. cardinalis posterior. plate 1, figure 1, and in the sections of text-figures 4 and 5), seems to me to indicate’ that the posterior and lateral -ardinal vessels extend caudalward in connection with chains of angioblasts from the aorta which pass dorsal and ventral to the nephritic tubules in lines which are very plain in figure 5. In figure 5 of plate 3 is shown a ventral view of the pronephritic vessels in a pig of 20 somites, in which it is clear that there is a tend- ency toward a grouping of the transverse arteries of the prone- phritic tubules around segmental lateral arteries. For example, be- tween the ninth and tenth spinal arteries there is one lateral artery giving off four branches; between the tenth and eleventh spinal ar- teries are two lateral arteries from the aorta, with three transverse branches. The longitudinal artery shown in plate 3, figure 5, persists for some ~— A.al Fic. 6.—Transverse section of an embryo pig of 23 somites, passing through the allantoic arteries to show that the primitive allantoic arteries are in the splanchnopleure. The section is from a specimen of the same litter as the one shown on plate 1, figure 1, and from the same series as figures 4 and 5. The level of the figure is shown by a line on plate 1, figure 1. The section is 20 » thick and is stained with hematoxylin and counterstained with orange G, eosin, and aurantia. 53. A.al., artery of the allantois; C., coelom; V. al., vein of the allantois in the zone where the splanchnopleure, the somatopleure, and the amnion are fused, time in the pig and connects the glomerular arteries even after the arterial tufts of the glomeruli are well formed. As seen in plate 3, figure 5, the transverse arteries lead directly to a lateral vein, which in turn connects with the posterior cardinal vein. \oreover, as is shown opposite the tenth somite, the posterior cardinal vein has many direct connections with the aorta. DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 117 The next stage in the development of the circulation of the Wolffian bodies is the formation of the mesial cardinal vein. I have illustrated the position of this vein in two sections, one from an injected pig embryo of 30 somites, measuring 7 mm. before the caudal flexure has formed, and the other from an injected chick of 69 hours’ incubation (text-figs. 7 and 8). The mesial cardinal vein lies ventral to the nephritice arteries, close to the aorta, in the angle between the root of the mesentery and the Wolffian ridge. The course of the mesial cardinal vein can be readily imagined in plate 3, figure 5, wherein it is noted that opposite the tenth and eleventh somites the posterior cardinal vein is in the form of a plexus, dorsal Fic. 7.—Transverse section of an injected embryo pig of 30 somites, to show a typical cross-section of the vessels of the pronephros of the pig after the v. cardinalis mesialis has formed—that is, to show the pronephros with a cen- tral artery and three peripheral veins. The embryo measured 7 mm. after fixation and dehydration; it had no caudal flexure and was a little farther developed than the one on plate 5, figure 2. All the vessels shown were in- jected. The arteries are represented in black, the veins in white. The section is 50m thick and is unstained. X53. A. pr., artery of the pronephros which gives off capillaries to the tubules and extends to the v. cardinalis lateralis; V. c. l., v. cardinalis lateralis; V. c. m., v. cardi- nalis mesialis; V. c. p., v. cardinalis posterior; W. d., Wolf- fian duct. Fic. 8.—Transverse section of an injected chick of 35 somites, after 69 hours of incubation, passing through the fifteenth somite. The section shows a typical cross- section of the vessels of the pronephros in the chick after the v. cardinalis mesialis has formed—that is, it shows the pronephros with a central artery and three peripheral veins. All the vessels were injected. The aorta is shown with a black rim, the artery is black, and the veins are white. The section is 50 u thick and is unstained. X53. A. p., artery of the pronephros; V. c. L, v. cardinalis lateralis; V. c. m., v. cardinalis mesialis; V. c. p., v. car- dinalis posterior; W. d., Wolffian duct. to the nephritic tubules (text-fig. 7). At the stage of 30 somites a vein from this plexus passes ventral to the nephritic artery opposite the eleventh somite and grows caudalward just ventral to the nephritic arteries, between the aorta and the longitudinal artery of plate 3, figure 5. This is the medial cardinal vein, the subeardinal vein of F. T. Lewis. There is thus formed the primitive pattern of the circulation of the Wolffian body, as shown in text-figures 7 and 8, consisting of a central artery and three longitudinal superficial veins—the posterior cardinal vein dorsal to the Wolffian duct, the lateral cardinal vein just ventral to the duct, and finally the mesial cardinal vein near the root of the mesentery. The mesial cardinal forms the connection with the vessels of the liver and (as shown by Hoch- stetter) also anastomoses with branches of the omphalo-mesenteric vein along the mesentery. I emphasize the lateral cardinal vein because it has not been adequately recognized in the literature. In the pig it is very obvious in total preparations, 11S DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. such as are shown in plate 3, figure 5. It develops early and is very straight. In the chick it is not straight and therefore is much less striking in total prepara- tions. Its primary connections with the posterior cardinal vein are lateral to the Wolffian duct, as seen in text-figure 5 for the pig. That this is also true for the chick is shown by Graefe’s figure 6 (1906), which shows the pronephros of the chick at the stage of 2 days and 15 hours. Later, in both the pig and chick, these two veins are connected by branches which are mesial to the duct, as shown in text-figures 7 and 8. The failure to take into account the lateral cardinal veins has led to some confusion in the literature; for example, in the study of the pronephros, Graefe (in his figure 11) has labeled the lateral vein close to the Wolffian duct the sub- cardinal, while in figure 13 he has labeled the true subcardinal vein ventral to the nephritie artery the subeardinal, but has not labeled the lateral vein at all, though it is shown in the section. In Keibel and Mall’s Embryology, Felix gives some extremely interesting sections from the R. Meyer human embryo No. 300. This embryo had 23 somites and was 2.5 mm. long. It is to be compared with my plate 1, figure 1. In figure 532 Felix shows solid angioblasts, both dorsal and ventral to the Wolffian duct; he does not label the dorsal angioblasts which represent the posterior cardinal vein, but on the other hand calls the ventral angioblasts the posterior cardinal vein. Again, in figure 559 he calls angioblasts which are ventral to the duct the posterior cardinal vein. These sections show that in the human embryo there are angio- blasts both dorsal and ventral to the duct and bring out the value of the two names for the veins, the posterior and lateral cardinal veins. They also show that the posterior and lateral cardinal veins extend as solid angioblasts and so bring up the question as to whether these veins may not differentiate as chains of angioblasts connected with the aorta by chains of angioblasts. CONCLUSION. In this study it seems clear to me that the chick affords very valuable material for the study of the most fundamental point in connection with the vascular system that is still at issue, namely, how long in the life of the embryo do new angioblasts continue to differentiate from mesenchyme and join the blood-vessels? The answer to this question involves more extensive observations on the living blastoderm than I have yet made. It has been shown that blood-vessels first arise not only in the membranes but also in the embryo by a differentiation of cells into angioblasts, by the process which His had described, and not from a dilatation of spaces in the mesenchyme and a flattening-out of cells to form their border. It has been proved that the aorta at least in part differentiates in situ. [vi- dence has been given that a part at least of the neural vessels and their connec- tions with the aorta differentiate in situ. On the other hand, the cardinal veins begin as a growth from the wall of the aorta. They are a longitudinal anastomosis between direct branches of the aorta. A more detailed study of the later stages of the cardinal veins is necessary to determine if any part of them differentiates in situ. DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 119 I think that it is important to emphasize the extent of the development of the blood-vessels both of the membranes and of the embryo at the time when the circulation begins. This has been done for the chick, and it would be of great value to obtain the same observations for the mammal. This study gives a more complete account of the primitive vessel of the hind- brain than is to be found in the literature. I have followed its origin, its rela- tions, and its fate. The fate of this vessel is a very important point. This primitive vessel of the hindbrain differentiates early, opposite the first part of the neural tube to develop. It has been shown why it remains so long a single channel, namely, because it serves temporarily as a vein for the forebrain and midbrain before it takes the characteristic form of a plexus like the other early vessels on the surface of the neural tube. As the vena capitis prima becomes complete, so that the blood of the forebrain and midbrain is shunted out of the primitive channel of the hindbrain, this channel receives new arterial connections and breaks down into the very important capillary plexus of the rhombencephalon. It has been shown that the first true vein of the head, the vena capitis prima, as contrasted with veins which drain only the brain, develops in three segments. The anterior segment is a purely cerebral vein which drains the forebrain and midbrain and originally empties into the primitive vessel of the hindbrain; the posterior segment is the anterior cardinal vein; the middle segment develops last, as a capillary chain between the capillaries of the maxillary, the mandibular and the other visceral arches, and the anterior cardinal vein. This middle segment anastomoses with the primitive cerebral vein from the forebrain and midbrain and forms,a much more direct and favorable channel for draining the brain, and so rapidly supplants the more indirect channel along the hindbrain. It drains the other structures of the head in addition to the neural tube. The embryonic vein extending from the region of the thalamus to the duct of Cuvier is the first true vein of the head, in the sense of draining the entire head, that is, the brain and the visceral arches, and may thus be termed the vena capitis prima. In connection with the vascular system of the nervous system, it has been shown that the early pattern of the blood-vessels is very uniform for the entire tube. There is a capillary plexus which completely invests the tube and all of its ganglia. It is fed by bilateral longitudinal arteries, which form as an anasto- mosis between all of the neural arteries from the aorta and extends from the carotid arteries at the base of the optic stalk to the tip of the spinal cord. The bilateral character of these arteries persists only around the subthalamus, where the circle of Willis is formed; elsewhere the two arteries become a single ventral artery—the basilar artery and its primary continuation, the anterior spinal artery. I have thus brought out the origin and the significance of the basilar and anterior spinal arteries and have shown that they precede the vertebral arteries. The first neural veins are all transverse superficial vessels, which tap the deep plexus and gradually extend dorsalward on the deep plexus. They are profoundly modified by the eye, the ear, and by all the sensory ganglia. Opposite the brain they all drain into the primary head-vein; all the rest of the neural veins 120 DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. are intersegmental branches of the cardinal veins. It is thus clear that the general direction of the blood to the neural tube is from the ventral to the dorsal border and that the direction of the flow of blood from the neural tube is the reverse. In connection with the pig it has been shown that the large branches of the aorta near the caudal end of the embryo are primary allantoic arteries which run in the splanchnopleure, and that the umbilical arteries in the somatopleure develop later and anastomose with the primary allantoic arteries, exactly as in the chick. I have also given an analysis of the subintestinal vein of the chick and of the corresponding artery in the pig, and have shown that the fact that the vessel is an artery in the pig means that the primitive type of circulation of the yolk-sae persists longer in that form than in the chick. It has been shown that both the primitive allantoic arteries and the subintestinal arteries arise in a capil- lary plexus and end in a capillary plexus, so that in the case of these two vessels the blood must pass through two capillary plexuses in its return to the heart. The study of the circulation of early embryos by means of injecting living embryos and watching the flow of the ink in them or by watching the circulation of the blood in the living specimen brings out some remarkable changes in the direction of the circulation; for example, the change in the direction of the cir- culation in the vessels of the area vasculosa in the chick when the veins invade a plexus which had been arterial. Again, in connection with the development of the primitive vessel of the hindbrain into a capillary plexus, the direction of the ~ circulation is entirely changed. In the original vessel the blood flowed from the cephalic to the caudal border of the hindbrain, while when the new arterial connec- tions bring blood to the entire ventral border of the vein the blood begins to flow from the ventral to the dorsal border of the hindbrain. In the case of the subin- testinal artery is a third example of a profound change in the direction of the circulation. The blood originally runs through this artery out to the yolk-sac, but when the vessel becomes a capillary plexus in the wall of the gut, the blood flows toward the heart within the embryo in the new mesenteric veins. From these studies it is clear that it is important to consider each vessel of the embryo from the standpoint of the function it performs throughout its develop- ment and that the effort toward a precise usage of the terms artery, capillary plexus, and especially of the term vein, is an effort to understand the circulation of the embryo. BIBLIOGRAPHY. Von Barer, K. E.: Ueber Entwicklungsgeschichte der Thiere, Beobachtung und Reflexion. Kénigsberg, Gebr. Borntrager. 1 Theil, 1828; 2 Theil, 1837. Bracuet, A.: Recherches sur le déterminisme héréditaire de l’ceuf des mammiféres. Développement ‘‘in vitro”’ de jeunes vésicules blastodermiques du lapin. Arch. de Biol., Liége, 1913-14, XXVIII, 447-503, 2 pl. Broan, I. Ueber die Entwicklung und ‘‘Wanderung” der Zweige der Aorta abdominalis beim Menschen nebst Bemerkungen iiber Gefiisswurzelwanderungen im allge- meinen. Anat. Hefte, 1 Abt., Wiesb., 1908. XXXVI, 405-550, 5 pl. Danpy, W.E. A human embryo with seven pairs of somites, measuring about 2 mm. in length. Amer. Jour. Anat., Phila., 1910, X, 85-108, 6 pl. Duvat, M. Atlas d’Embryologie. Paris, G.Masson, 1889. Evans, H. M. On the earliest blood-vessels in the anterior limb buds of birds and their relation to the primary subclavian artery. Amer. Jour. Anat., Phila., 1909, IX, 281-319, 9 pl. - On the development of the aorts, cardinal and umbilical veins and other blood-vessels of vertebrate embryos from capillaries. Anat. Record, Phila., 1909, III, 498-518. Die Entwicklung des Blutgefisssystems. Hand- buch d. Entwcklngsgesch. d. Menschen, (Keibel and Mall). Leipzig, 1911, II, 551-688. Also, Manual of Human Embryol. (Keibel and Mall), Phila. and Lond., 1912, II, 570-709. Feirx, W. Zur Entwicklungsgeschichte der Rumpfarterien des menschlichen Embryo. Morphol. Jahrb., Leipz., 1910, XLI, 577-614. Die Entwicklung der Harn- und Geschlechtsor- gane. Handbuch d. Entwcklngsgesch. d. Menschen, (Keibel and Mall), Leipz. 1911, II, 732-955. Also, Manual of Human Embryol. (Keibel and Mall), Phila. and Lond., 1912, II, 752-979. Grare, E. Beitrage zur Entwicklung der Urniere und ihrer Gefasse beim Hiihnchen. Arch. f. mikr. Anat., Bonn, 1906, LXVII, 143-230, 5 pl. Griprer, L. Untersuchungen iiber die Herzbildung der Vogel. Arch. f. Entwcklngsmechn. d. Organ., Liepz., 1907, XXIV, 375-410, 4 pi. Grosser, O. Zur Anatomie und Entwicklungsgeschichte des Gefiisssystemes der Chiropteren. Anat. Hefte, 1 Abt., Wiesb., 1901, XVII, 203-424, 13 pl. Die Elemente des Kopfvenensystem der Wirbeltiere. Anat. Anz., Verhandl. d. anat. Gesellsch., Jena, 1907, XXX. Haun, H. Experimentelle Studien tiber die Entstehung des Blutes und der ersten Gefasse beim Hithnchen. Arch. f. Entweklngsmechn. d. Organ., Leipz., 1909, XX VII, 337-433, 3 pl. His, W. Untersuchungen iiber die erste Anlage des Wir- belthierleibes. Leipz., F. C. W. Vogel, 1868. . Anatomie menschlicher Embryonen. JI. Embryo- nen des ersten Monats. Leipz., F. C. W. Vogel, 1880. Lecithoblast und Angioblast der Wirbelthiere. Abhandl. d. math.-phys. Cl. d. k. sachs. Gesellsch. d. Wissensch., Leipz., 1900, XXVI, 171-328. Hocustetter, F. Beitrage zur Entwicklungsgeschichte des Venensystems der Amnioten. Morphol. Jahrb., Leipz., 1888, XIII, 575-585, 1 pl. Ueber die urspriingliche Hauptschlagader der hinteren Gliedmasse des Menschen und der Saugethiere, nebst Bemerkungen iiber die Entwicklung der Endaste der Aorta abdominralis. Morphol. Jahrb., Leipz., 1890, XVI, 300-318, 1 pl. Horrmann, C. K. Zur Entwicklungsgeschichte des Venen- systems bei den Selachiern. Morphol. Jahrb., Leipz., 1893, XX, 289-304, 1 pl. KasrscHenko, N. Das Schlundspaltengebiet des Hithn- chens. Arch. f. Anat. u. Entweklngsgesch., Leipz., 1887, 258-300, 3 pl. Lewis, F. T. The development of the vena cava inferior. Amer. Jour. Anat., Balt., 1901-1902, I, 229-244, 2 pl. Lewis, M. R., and Lewis, W. H. The cultivation of tissues from chick embryos in solutions of NaCl, CaClz, KCl, and NaHCO;. Anat. Record. Phila., 1911, V, 277-293. Litute, F. R. The development of the chick. New York, Henry Holt & Co., 1908. Matt, F. P. A human embryo twenty-six days old. Morphol., Bost., 1891, V, 459-480, 2 pl. Development of the human ceelom. Jour. Morphol., Bost., 1897, XII, 395-453. On the development of the blood-vessels of the brain in the human embryo. Amer. Jour. Anat., Balt., 1904, IV, 1-18, 3 pl. McWoorter, J. E., and Wuterte, A.O. The development of the blastoderm of the chick in vitro. Anat. Record, Phila., 1912, VI, 121-139. Pororr, D. Die Dottersack-Gefisse des Huhnes. Wiesb., C. W. Kreidel, 1894. Rast, C. Ueber die Entwicklung des Venensystems der Selachier. Festschr. z. 70. Geburtstage Rud. Leuckarts, Leipz., W. Engelmann, 1892. Jour. Ravn, E. Ueber die Arteria omphalo-mesenterica der Ratten und Mause. Anat. Anz., Jena, 1894, IX, 420-424. Reacan, F. P. YVascularization phenomena in fragments of embryonic bodies completely isolated from yolk-sac blastoderm. Anat. Record, Phila., 1915, IX, 329-341. Remax, R. Untersuchungen iiber die Entwicklung der Wirbelthiere. Berlin, G. Riemer, 1851 [1855]. Saprn, F. R. Der Ursprung und die Entwicklung des Lymphgefisssystems Anat. Hefte, 2 Abt., Wiesb., 1913, XXI, 1-98. Also transl. The origin and development of the lymphatic systems. Johns Hopkins Hosp. Rep., Monographs, New Series, No. 5, Baltimore, 1913, 1-94. On the fate of the posterior cardinal veins and their relation to the development of the vena cava and azygos in the embryo pig. Contributions to Embry- ology, No. 7, Carnegie Inst. Wash., Pub. No. 223, 1915. Sauzer, H. Ueber die Entwicklung der Kopfvenen des Meerschweinchens. Morphol. Jahrb., Leipz., 1895, XXITI, 232-255, 1 pl. SpaLTenotz, W. Ueber das Durchsichtigmachen von menschlichen und tierischen Praparaten und seine theoretischen Bedingungen. Leipz., S. Hirzel, 1911; 2 Aufl., 1914. Srreeter, G. L. The development of the venous sinuses of the dura mater in the human embryo. Amer. Jour. Anat., Phila., 1915, XVIII, 145-178. Srracker, O. Entwicklung der Kopfvenen beim Huhn bis zur Ausbildung der Vena capitis lateralis. Morphol. Jahrb., Leipz., 1916, L, 49-71, 2 pl. TANDLER, J. Zur Entwicklungsgeschichte der Kopfarterien bei den Mammalia. Morphol. Jahrb., Leipz., 1902, XXX, 275-373, 3 pl. Zur Entwicklungsgeschichte der menschlichen Darmarterien. Anat. Hefte, 1 Abt., Wiesb., 1903, XXIII, 187-210. Ueber die Varietaten der Arteria cceliaca und deren Entwicklung. Anat. Hefte, 1 Abt., Wiesb., 1904, XXV, 473-500. Die Entwicklungsgeschichte des Herzens. Hand- buch d. Entweklngsgesch. d. Menschen, (Keibel and Mall). Leipz., 1911, II, 517-551. Also, Manual of Human Embryology (Keibel and Mall), Phila. and Lond., 1912, II, 534-570. Witurams, L. W. The somites of the .chick. Amer. Jour. Anat., Phila., 1910-11, XI, 55-100. 121 to to EXPLANATION OF PLATES. PuLaTe 1. . Injection of the vascular system of an embryo pig of 23 somites, which measured 7 mm. when fresh and 6.2 mm. after fixation and dehydration. The injection is nearly complete and shows especially the primitive relations of the vasa primitiva rhombencephali at the stage when it serves as a vein for the forebrain and midbrain and as a capillary plexus for the hindbrain. 38. A. 1., artery of the first interspace; al., allantois; a. 8t., a, subintestinalis; b. c., bulbus cordis; L., liver; pl., plexus on the spinal cord; s. v., sinus venosus; 1. a., truncus arteriosus; v. ¢. a., v. cardinalis anterior; v. c. l., v. cardinalis lateralis; v. c. p., y. cardinalis poste- rior; v. u., v. umbilicalis; va. p. r., vasa primitiva rhombencephali; ven. c., ventriculus cordis. . Partial injection of the vessels of a chick of 12 somites. The needle was introduced into one of the omphalo- mesenteric veins near the heart. The transverse lines show the position of the interspaces. The sections shown in text-figures 1, 2, and 3 are from a chick of the same stage which was completely injected. X54. Me., mesencephalon at the level of the section shown in figure 1; v. om., v. omphalo-mesenterica. . Injection of the heart and the cephalic aortz, both dorsal and ventral, in a chick of 9 somites. The needle was introduced into the dorsal aorta opposite the somites. 94. Ao. d. c., aorta dorsalis cephalica; ao. v. c., aorta ventralis cephalica ; h., heart; me., mesencephalon; v. om., v. omphalo-mesenterica, PLATE 2. . Partial injection of the vessels of a chick of 14 somites. The needle was introduced into the dorsal aorta opposite the somites. The vascular plexus on the mesencephalon is not injected, though it is present at this stage, as is shown in text-figure 1. 100. A. so., artery of the somatopleure; d. C., duct of Cuvier just before it has connected with the omphalo-mesenteric vein; me., mesencephalon; v. c., v. cardinalis communis, that is, before it has an anterior and a posterior division; v. so., vein of the somatopleure; v. t., v. transversa of the first interspace; va. p. r., vasa primitiva rhombencephali. . Injection of the blood-vessels of a chick of 16 somites, to show the relation of the primitive vessel of the rhomben- cephalon to the primitive cerebral vein, on the one hand, and to the anterior cardinal vein, on the other. This is the stage before the vena capitis prima is completed. X58. D. C., ductus Cuvieri; v. c. a., y. cardinalis anterior; v. c. p., v. cardinalis posterior; v. ce. p., v. cerebralis primitiva, which will become the cephalic division of the y. capitis prima; v. 4, v. transversa of the first interspace; va. p. r., vasa primitiva rhombencephali. . Transverse section of an injected chick of 30 somites, after 52} hours of incubation, passing through the twenty- first interspace, The section is from the same series as figure 4 on the same plate, figures 2 and 3 on plate 3, and figure 2 on plate 4. Figure 4 on plate 3 is from another series. This section is to show the beginning of the spinal arteries as they show in the tenth to the seventeenth interspaces on plate 3, figure 1. The section is below the level of the omphalo-mesenteric arteries. It is 50 « thick and is unstained. 140. Ao., aorta; v. ¢. p., VY. cardinalis posterior; v. om. p., v. omphalo-mesenterica posterior; W. d., Wolffian duct. . Transverse section of an injected chick of 30 somites, after 52} hours of incubation, passing through the twenty- seventh interspace. The section is to show the relative position of the direct arteries to the posterior cardinal vein and the arteries of the somatopleure. The section is below the level of the omphalo-mesenteric erteries and is in the region of the posterior limb-bud. It is 504 thick and is unstained. 140. Ao., aorta; v. ¢. p., y. cardinalis posterior; ». om. p., v. omphalo-mesenterica posterior; W. d., Wolffian duct. PLATE 3. . The cardinal veins from an injected chick of 25 somites, to show the method of origin of the spinal arteries. 106. A. 3 and a. 18, arteries of the third and eighteenth interspaces; d. C., ductus Cuvieri; pl., plexus on the spinal cord; v. c. a., vy. cardinalis anterior; v. c. p., v. cardinalis posterior; v. ¢., v. transversa of the first inter- space; va. p. r., vasa primitiva rhombencephali. Transverse section of an injected chick of 30 somites, after 52} hours of incubation, passing through the fifteenth interspace. The section is to show a spinal artery like that of the seventh interspace of plate 3, figure 1; it is above the level of the omphalo-mesenteric arteries and shows the posterior omphalo-mesenteric veins on either side. The section is 50 « thick and is unstained. 140. Ao., aorta; v. c. p., Vv. cardinalis posterior; vom. p., ¥. omphalo-mesenterica posterior; W.d., Wolffian duct. . Transverse section of an injected chick of 30 somites, after 524 hours of incubation, passing through the sixteenth somite, This is the next section in the series below that of plate 3, figure 2. It shows a direct dorso-lateral artery to the posterior cardinal vein, The section is 50, thick and is unstained. X140. Ao., aorta; v. ¢. p., V. eardinalis posterior; v, om, p., v. omphalo-mesenterica posterior; W. d., Wolffian duct. 122 DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. 123 Piate 3—Continued. 4. Transverse section of an injected chick of 25 somites after 52 hours of incubation, passing through the seventeenth interspace. The section is to show the transition between the stage of figure 3 of plate 2 and figure 2 of plate 3, in the formation of a spinal artery. It is 50 » thick and is unstained. 140. Ao., aorta; v. c. p., v. eardi- nalis posterior; W. d., Wolffian duct. 5. Injection of the aorta, the arteries of the pronephros, and the lateral and posterior cardinal veins in an embryo pig of 20 somites, from a specimen of the same litter as the one on plate 5, figure 1. The specimen is shown for the ventral aspect. 140. A.9to A. 12, arteries to the spinal cord in the ninth to the twelfth interspaces; ao., aorta; v.c. l., v. cardinalis lateralis; v. c. p., v. cardinalis posterior. PLATE 4. 1. Injection of the vessels of the head of an embryo pig of 27 somites, measuring 7.1 mm. after fixation and dehydration The specimen is from the same litter as the one on plate 5, figure 2, and is to show the completion of the vena capitis prima and its relation to the vasa primitiva rhombencephali. It shows that the primitive vessel of the hindbrain does not atrophy when the vena capitis prima is completed, but rather develops into a plexus on the hindbrain. X94. Al., atrium; b. c., bulbus cordis; f. H., fretum Halleri; ¢. a., truncus arteriosus; v. cap. p. 1, V. capitis prima, first or cerebral segment, which drains the forebrain and midbrain; v. cap. p. 2, vy. capitis prima, second segment, which drains the forebrain, the midbrain, and the visceral arches; v. cap. p. 3, V. capitis prima, third segment, which is the anterior cardinal vein; va. p. r., vasa primitiva rhombencephali; ven. c., ventriculus cordis; ves. a., vesicula auditiva; V, position of the root of the n. trigeminus; VJJII, position of the roots of the nn. cochlearis et vestibularis; ].X, position of the root of the n. glossopharyngeus. 2. Transverse section of an injected chick of 30 somites, after 52} hours of incubation, passing through the twenty- fifth interspace. The section is to show the diverticula of the dorsal aorta like those of the first and second interspaces in plate 1, figure 2, which give rise to the cardinal veins. The section is 50y thick and is unstained. 140. Ao., aorta; W.., Wolffian duct. 3. Partial injection of the vessels of an embryo pig of 14 somites, measuring 4 mm. after fixation and dehydration. The specimen was injected through the dorsal aorta opposite the somites. 56. A. 9, artery to the spinal cord in the ninth interspace; at., atrium; 6. c., bulbus cordis; f. H., fretum Halleri; s. 1, first somite; s. v., sinus venosus; ¢. a., truncus arteriosus; v. c. p., v. cardinalis posterior; v. om., Vv. omphalo-mesenterica ; ven. c., ventriculus cordis; ves. a., vesicula auditiva. Puate 5. -_ . Partial injection of the vessels of an embryo pig of 20 somites, measuring 6 mm. after fixation and dehydration. It shows the omphalo-mesenteric arteries, the subintestinal artery and the arteries of the pronephros; X41. A.9, artery to the spinal cord in the ninth interspace; a. om., a. omphalo-mesenterica; a. si., a. subintestinalis; al., allantois; . c., bulbus cordis; s. al., stalk of the allantois; ¢. a., truncus arteriosus; v. c. l., cardinalis lateralis; ven. c., ventriculus cordis. 2. Partial injection of the vessels of an embryo pig of 27 somites, measuring 7.1 mm. after fixation and dehydration. It shows the general development of the vascular system at the stage when the vena capitis prima is completed. The vessels opposite the hindbrain, both deep and superficial, are extravasated in this embryo and hence they are shown on plate 4, figure 1, from an embryo of the same litter. 51. A. om. d., a. omphalo-mesenterica dextra, the other two omphalo-mesenteric arteries in the figure being on the left side; at., atrium; 5. c., bulbus cordis; d. C., ductus Cuvieri; l., liver; t. a., truncus arteriosus; v. cap. p. 1, v. capitis prima, first or cerebral segment, which drains the forebrain and midbrain; v. cap. p. 3, v. capitis prima, third segment, which is the anterior cardinal vein; v. om., v. omphalo-mesenterica; v. u., v. umbilicalis; ven. c., ventriculus cordis; x., extravasation involving both the vasa primitiva rhombencephali and the vena capitis prima, as can be seen on plate 4, figure 1. PLATE 6. Injection of the vessels of the head of a chick of 29 somites, to show the origin of the vena capitis prima. The vein extends from the region of the diencephalon to the duct of Cuvier. The injection shows that the vein arises in three segments; the first segment is a true primitive cerebral vein, which drains the forebrain and will soon drain the midbrain; the second segment is an anastomosis between the maxillary, the mandibular, and the other vis- ceral arches and the anterior cardinal vein, and it drains the forebrain, the midbrain, and the visceral arches; the third segment is the anterior cardinal vein, which drains the brain and the visceral arches. X128. A. 6., artery on the rhombencephalon, which at this stage is bilateral and is part of a plexus which will give rise to the basilar artery; a. 3, artery to the medulla in the third interspace; d. C., ductus Cuvieri; v. c. p., y. cardinalis posterior; v. cap. p. 1, v. capitis prima, first or cerebral segment which drain the forebrain and midbrain; v. cep. p.2, ¥. capitis prima, second segment which drains the forebrain, the midbrain, and the visceral arches; v. cap. p. 3, Vv. capitis prima, third segment, which is the anterior cardinal vein; v. m. p., y. maxillaris primitiva; v.om., v. omphalo-mesenterica; v. t., v. tramsversa of the first interspace; v. u., plexus in which the v. umbilicalis will arise; va. p. r., vasa primitiva rhombencephali; ves. a., vesicula auditiva; V, position of the root of the n. trigeminus; V/JJ, position of the roots of the nn. cochlearis et vestibularis. 124 DEVELOPMENT OF PRIMITIVE BLOOD-VESSELS. PLATE 7. Injection of the vessels of the brain of an embryo pig measuring 6.5 mm. in length after fixation and dehydration. 7 The injection is a complete one, but the vessels of the visceral arches and most of the vessels of the cerebrum have been omitted in the drawing. The figure shows, first, the longitudinal artery of the central nervous system, which extends from the tip of the carotid artery to the caudal tip of the spinal cord; this artery is a plexus opposite the subthalamus, a single vessel down to the lower part of the medulla, and again a plexus on the cord; second, a part of the capillary plexus which invests the entire neural tube; third, the relation of the primitive veins of the forebrain, the midbrain, and the hindbrain to the vena capitis prima. 73. A. b., a. basilaris; a. c. 1,4. carotis interna; a. 1, artery to the medulla in the first interspace; a. m. p., a. maxillaris primitiva; v. cap. p. 1, v. capitis prima, first or cerebral segment, which drains the forebrain and midbrain; v. cap. p. 2, ¥. capitis prima, second segment, which is shown only in outline, and which drains the forebrain, the midbrain, the hindbrain, and the visceral arches; v. cap. p. 3, v. capitis prima, third segment, which is the anterior cardinal vein and which drains the brain and the visceral arches; v. m. p., y. maxillaris primitiva; ves. a., vesicula auditiva; 3, 4, and 6, third, fourth, and sixth aortic arches, which are coming from the heart and are leading to the descending aorta, which is concealed by the cardinal segment of the vena capitis prima; V, position of the root of the n. trigeminus; VJJ/J, position of the roots of the nn. cochlearis et vestibularis; JX, position of the root of the n. glosso-pharyngeus; X, position of the root of the n. vagus; XJ/J, position of the root of the n. hypoglossus. J. F. Didusch fecit AHoen & Co Lith. § nm ve 24 PLATE 2 SABIN \ WARMER GA remand ghee a. Fig. 2 Fig. 1 AHoen & Co Lith. J. F. Didusch fecit SABIN J. F. Didusch fecit ey ie ke we a a ah x PLATE 3 S7tooodw Fig. 5 Fig. 4 Fig. 1 PLATE 4 SABIN AHoen & Co Lith J. F. Didusch fecit | o 2 £ G ra 3 i (=) w 2 = PLATE 6 SABIN e — wits @ .°) Cid - wy: AHoen & Co Lith. J. F. Didusch fecit % * , os SABIN PLATE J. F. Didusch fecit Afoen &Col wed A HUMAN EMBRYO OF TWENTY-FOUR PAIRS OF SOMITES, By FRANKLIN PARADISE JOHNSON, Of the University of Missouri. Wight plates, nine text-figures. 125 TABLE OF CONTENTS. Introduction. ... LO Groth Uy GR AMMeRmRAGn S cre To crg ena Sao roc Integument. . Nervous system....... Braman a. ule es Medullary tube. . Neuromeres. . . . Cerebral nerves. . . a: vate Cae a a Ganvlonicvcrestsecssemtee se ee ee ee Digestive system. iciteraetiis sists cee te eee Mouth... Foregut....... Pharynx: 3 6432 t a at. «tee een races aes Pulmonary diverticulum.................. 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Tel PLATE 5 ‘quinee'n ~p'quin'e ~s'quun'e ~~ “S'O'PseBo'A JOHNSON é 'S'B'PseO'A ‘}| pue ‘ue f~ (a¥ ‘s'd'pavora- “Y= *\- co) Ashes an Buv3 4 tee \ QUIN'AA'N s'quuin‘a ~ re ata ~s'quun’A a Se 'S'O'PUBO'A _-'y-Buiy'a 'S'R'PIBO'A™ Won | oy’ oe dues Bue (giB'ovn A foen & Co. Lith. JOHNSON PLATE G. T. Kline del. JOHNSON Sees G3 2 Q = bo = = bs fom oO A = = — & 33 3 z ary Se a —— = . 2 =) 3 < : =f ors = = i z = ——| = } =e = = e © = X ne) co x ee t oL 2 ; T a Ee IY 5 oO 5 a Syed! nT wu “i a o> 2 oS SSS Seo = = «ao £ = = y = re) ro) 3 = oC a > a a > = Oo oO = = > JOHNSON inl ae a neur.9 PLATE # am._f 4 v.card.a.d, } r f } va2 i phr. | a, v.a.l_ =F Ot gen v.card.a.s. Ieee neil ai > —# 20.al CPN _{mes. \ —— aS ty Acari. pros. H CUE . / v.card.p._/ of 4s J + vl Re) Bok a « « x < » * x p t i * is" a er : a) - 3 i “4 < $= —) > ~ 7 . ¥ 4 7 : é , As 4 i ¢ ‘ . e ‘ e 7 . : 3% 4 7 - : < ‘ . : -* - umb., . umb. d., - umb. s., aa. umb., all., a a a a. seg. v., a. a a b. w., bul. cor., cap., cap. pl., ec. int., ch. d., ch. can., ch. p., cloa., cl. men., c.m., coe., coe. cloa. d., coe. cloa. s., coe. d., coe. f. g., cop., > by ect., end. ht., end. p., end., ex. coe., f 2:, gang. ac. fe., Zang. c., gang. gl. ph., gang. tri., gang. va., gas. r., £265 ht. sw., hi gi; inf., int., 163; ire ea tzg liv. d. (p. ¢.), liv. d. (p. h.), liv. tr., lg. d., ABBREVIATIONS. . basilaris. . cerebri (?). . carotis interna. . segmentalis dorsalis. . segmentalis lateralis. . segmentalis ventralis. . umbilicalis. . umbilicalis dextra. . umbilicalis sinistra. aa. umbilicales. allantois. amnion. amnionie cavity. aorta dorsalis. aorta dorsalis dextra. aorta dorsalis sinistra. aorta ventralis. aortic arch. aortic process of sclerotome. arterial plexus of tail. atrial projection. atrium. auditory pit. epererres es blood-corpuscles. blood-vessels. body-stalk. body-wall. bulbus cordis. capillary. capillary plexus. caudal intestine. chorda dorsalis. chordal canal. chordal process of sclerotome. cloaca. cloacal membrane. cuticular membrane. ceelom. celom on right side of cloaca and caudal intestine. eceelom on left side of cloaca and caudal intestine. coelomic depressions. ccelom surrounding fore-gut. copula. dorsal branch of medial limb of a. seg. d. ectoderm. endothelial heart. endothelial process. endothelium. exoccelomic cavity. fore-gut. ganglion acustico-facialis. ganglion crest. ganglion n. glossopharyngei. ganglion n. trigemini. ganglion n. vagi. gastric region. gill-cleft. heart swelling (Grosser). hind-gut. infundibulum. integument. intersegmental enlargement of v. card. p. lateral branch of a. seg. d. lateral tributary of v. card. p. liver diverticulum (pars eystica). liver diverticulum (pars hepatica). liver trabeculae. lung diverticulum. md. a., m. b., m. and I. t., m. ., mes., mes. ves., meso., mit., m., m. €., mye., myt., nep., n. t., neur., op. V., ot., or. m., p. ¢., p. d. mes., pr. ch., proc. inv., pros., p. Vv. mes., u. aa. umb., li. VV. umb., v. ¢ard. v. ecard. v. card. vy. card. v. ¢ard. v. card. p. s WiC: as, (2) Vv. ct. i, v Vv gees ” ist L Foe Das5 y. ling-f., v. oph., v. umb. d., v. umb. s., v. vit. d., Vv. vit. s., VV. &., vy. umb., v. pl. b. w., v. pL h. g., v. pl. y. st., Vv. b:; p. gr., v: p. gr. 1 (p.1.), vent., vil., Vv. a, i < n mandibular arch. medial branch of a. seg. d. ; medial and lateral tributaries of v. card. p. mesocardium. mesencephalon. mesonephric vesicles. mesothelium. mitotic figure. mouth. myoepicardium. myocoele. myotome. nephrostome. medullary tube. neuromere (1—21). optie vesicle. otocyst. oral membrane remnants. pericardial cavity. position of dorsal mesentery. pleuro-pericardial passage. pharyngeal pouch. pharynx. primary excretory duct. principal collecting tubule. position of lung diverticulum. pronephric chamber. proctodeal invagination. prosencephalon. position of ventral mesentery. position of yolk-sac. position of yolk-stalk. sclerotome. septum transversum. sinus sagittalis superior. sinus venosus. somite (1-22). tail. tear in tissue. thyroid diverticulum. truncus arteriosus. tuberculum impar. termination of primary excretory duct. union of aa. umbilicales. union of vv. umbilicales. vy. cardinalis anterior dextra. y. cardinalis anterior sinistra. v. cardinalis communis dextra. v. cardinalis communis sinistra. . eatdinalis posterior dextra. ardinalis posterior sinistra. . cerebri anterior (?)- . cerebri media. . eerebri posterior. . linguo-facialis. . ophthalamicus. . umbilicalis dextra. v. umbilicalis sinistra. v. vitellina dextra. y. vitellina sinistra. veins of visceral arches (I-IV). ven umbilicales. venous plexus of body-wall. venous plexus of hind-gut. venous plexus of yolk-stalk. ventral branch of medial limb of a. seg. d. ventral pharyngeal groove (1-3). ventral pharyngeal groove l-posterior limb. ventricular portion of heart. villus-like projections of body-wall. visceral arch (1-3). adqddddad yolk-sac. yolk-stalk. 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On the development of the super- ficial veins of the body-wall in the pig. Amer. Jour. Anat., Phila., rx, 439-462. . Srreetrpr, G. L., 1912. Development of the nervous system. Jn Manual of Human Embryology (Kei- bel and Mall), Phila. and Lond., m, 1-156. Also, Handbuch d. Entweklngsgesch. d. Menschen (Kei- bel and Mall), Leipzig, 1911, 1m, 1-156. 3. TanpLeER, J., 1907. Ueber einen menschlichen Embryo vom 38. Tage. Anat. Anz., Jena, xxx1, 49-56. 4. Tanpver, J, 1912. The development of the heart. Manual of Human Embryology (Keibel and Mall), Phila. and Lond., 1, 534-570. Also, Handbuch d. Entweklngsgesch. d. Menschen (Keibel and Mall), Leipzig, 1911, 1, 517-551. . THomeson, Perer, 1907. Description of a Human Embryo of twenty-three paired somites. Jour. Anat. and Physiol., Lond., xu, 159-171, 3 pl. ———, 1908. A note on the development of the sep- tum transversum and the liver. Jour. Anat and Physiol., Lond., xi, 170-175. » VAN DEN Brorck, A. J. P., 1911. Zur Kasuistik junger menschlicher Embryonen. Anat. Hefte, 1. Abt., Wiesb., xurv, 275-304, 5 pl. . Warr, J. C., 1915. Deseription of two young twin human embryos with 17-19 paired somites. Con- tributions to Embryology No. 2, Carnegie Inst. Wash. Pub. No. 222. . Witrrams, L. W., 1910. The somites of the chick. Amer, Jour. Anat., Phila., x1, 55-100. : iy $ dy ie a ses EY Te ENT * = . ‘ San t4 ‘ ‘ ‘ ede ANE ORM hin dye Ba Ah ocd ¥ ots Fh Nae aan é —h . "7 PS Me pe 6 os * } * eee aa os, Tm heer ) ut : ee ’ or zs 4& a A at | ; . + = - ~ 5 , + ae ‘, f . 4 x ; . « ’ ~ 4 - . QM Carnegie Institution of 601 Washington AlC3 Contributions to embryo-~ v.4-6 logy Biological & Medical Serials PLEASE DO NOT REMOVE CARDS OR SLIPS FROM THIS POCKET UNIVERSITY OF TORONTO LIBRARY STORAGE ~~ eae 6 a ee ee eeerree. eb we ROR ee eS eee ee ee tia ed md errr bd ba So) rere GSTS SBT hae elaoacad Ws tv aR ge ae <= es wpe cay res ~ citer es Seseeneaics