'!':'iV i,!.i CONTRIBUTIONS TO EMBRYOLOGY Volume IV, Xos. 10, 11, 12, 13 PlBLI«HE1) by the ('AnNE(;lK InhTITITION OI' \\'.\!mbryos, and his conclusion is that magma is present in both kinds. His own words are as follows : " Bci der Ocffiuiug dcs ( 'hoiioiisackes dcr Eicr de.s or.stcii uiid zwciten Monats .sah ich, wic in dcr Einloitung onviihiit wurdc, iiiid dies oft, am bcsten nach kiirzer Erhiirtun^ in Uol)orosmiuin.'i;im(' (von }i Pioc.) odcr in Miilloischpr Losung (gowiilinlich narh dopjiolter Wrduniiuiif;) , in dcin si'lilci- niigcn Inlialt, wolchcr zwisclicn dciu Chorion tind dciii Ainnion, also ini suljclioiionischcn Raunic vorhanden war, diinnorc odor dickcrc Fiidon und Strange, die melir oder wcniger dicht von der iiusseron Fiiicho des Amnion zur innercn Fliichc do.s C'liorion liiniiherliefen, um sicii dort mit ihren Endcn an den Ix'idcn llautcn zu l)ofestigon, indcm sic sicli oft an ihnon verhreitorten und in ilirc licklcidcndc Schiclit iihcigingcn. Diosc Fiick'n und Striingo, welclip ini frisdicn Priiparatc kamn siclitl)ar waren, traten nach dvv Btiuindiuiig mit den erwahnten Fliissigkeitcn deutiich hervor. In dor Fig. 15 der Taf. XVIII babe ich ein solche.s Ei abgebildet. Das stark zottige Chorion (ch) ist geoffnet, und man sielit im subchovionisrhm Raumo di^n Aninion.'re Riuuk-l verschiedenen Calibers welche au.s dicht gedriingten Fibrilien bestehen. Es sind also fibrillar-bindegcwebige Balken, welche durch eine homogene, zahlreiche einzelnc Fibrilien enthaltende Intercellularsubstanz ziehen. Zwischen den Balken und Fibrillenziigen sieht man recht zalilreiclie Zellen, welche theils und am moisten rundlich oik-r oval, theils auch spindelformig sind und in ihrem oft reichlichten Protoplasma grossore gliinzende Korner enthalton. Die.se Zellen liegen in der (irundsubstanz ohne be.sondere anordnung zerstreut, bilden also koine Scheiden o. d. um die Fibrillenbiindel. "Man hat es hier offenbar mit einem unreifrn B'nulcgewehe zu thun, oinem embryonalen mucosen Bindegewehe, irelches indessen in der Entwicklimg zum fibrillnren liindegewebe sckon weit vorgeschrilen ist." THE MAGMA IN NORMAL DEVELOPMENT. We have now in the literature a detailed description of a number of yoimg human ova, and, according to their clinical histories, some of them, at least, must be normal. The classic s]x'cimen is that described bj- Peters, which came from a woman who had committed suicide. The specimen was hardened in situ in an alipro^■ed manner, and was worked up and described under the best possible con- ditions. Tn it the ccelom is filled with a gelatinous substance, through which radiate HUMAN MAGMA RETICULE IX 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 ])artly made up of heavier strands of tissue accompanied by nuclei. In the neigh- borhood of the embryo there are two large spaces, lined with colls, which appear to be the primitive bodj'-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 embrj-o m his plates 3 and 4. According to this authority these two body-cavities comnnmicate 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 coelom begins to form near the body of the embryo. As this cavity exj^ands subsequently, it probabh' first destroj's the more delicate strands of magma, leaving the heavier ones; thus in a short time the cavity of the ovum is lined b}' the endothelium of the ccelom, which also must cover the stronger bands of magma radiating as trabeculse throughout this cavity (Grosser, pp. 78 and 79). Keibel explains the formation of the human coelom as follows : "It is, however, not quite clear how the cavity traversed by scattered strands of niesohlast 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 extraenabryonic ca'lom; but it may also be imagined that it has arisen from an extensive loosening up of ths tissue, and not by a splitting of ths mssoderm, 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 coelom." 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 circumscriljing a space on the ventral side of the embryo. Eternod has written several papers in which he describes the formation of the exocoelom and the fate of the magma reticule. He says that it first fills the entire space between the primordium of tire 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 strikhigh' 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 exocoelom 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 RfiTICULfi 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 al)ove indicate that the space near the emliryo is the primitive e.\oca?lom and that the remainder of the so-called ca^•ity of tlie 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 general confirms the observations in the Peters ovum. A list of th(> normal speci- mens in our collection discussed in this i)aper is given in table 1. Table \.— List of normal embryos. Cat. No. Length of embryo Dimensions of chorion. Men- strual age in days. Condition of mngma. Cat. No. Length of embryo. Dimensions of chorion. Men- strual age in days. Condition of magma. mm. mm. mm. mm. 763 0.2 4X 2.2 60 Some reticular. 588 4 19X15X 8 49 Strands of magma. 391 2 16X14X12 14(?) Do. 136 4 14X11X 6 56 Keticular excessive. 779 2.75 16X14X12 40 None. 836 4 22X18X11 36(?) Delicate reiicular. 164 3.5 17X17X10 Few strands 148 4.3 17X14X10 34 Small amount of magma 463 3 9 17X12X 7 48 Much reticular. around cord. 4S6 4 22 X22 X22 44 Do. 576 17 30X30X25 Small amount of maj^tiia. 470 4 20X13 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; subsccjuently 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 undoubtedlj' 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 especiallj' favorable for defining magma fibrils. The specimen described by Herzog is also luidoubtedly normal, as it was obtained from a woman who was killed b}^ 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 ca^lom, which contains no other for- eign substance, but a jjhotograjjh (figure 24, published by Herzog) shows that the ccelom is filled bj' a very jjronounced substance, reminding one ver}' much of reticu- 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 ccelom. (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 Brj^ce and Teacher, which also shows the condi- HUMAN MAGMA RETICULE IN NORMAL AND PATHOLOGICAL DEVELOPMENT. 1 1 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 i^erfectly hardened and there is a small cleft between the chorionic wall and the mass of magma. As yet there is no exoctt'lom, 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 "exocoelom," 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 w^ith 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 bj^ Strahl and Beneke on page 18 of their monograph. Magma of uniform consistency, as seen in the Brjxe and Teacher specimen, soon arranges itself in bands, which gradually become more and more pronounced in older specimens. Between these bands are sjjaces filled with fluid, and those spaces near the embryo become hned with endothelium to form the exocoelom. There are other spaces between the exocoelom 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 exocoelom 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 fil)rils 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 IIIMAX MAGMA RftTICULfi IX NORMAL AM) I'ATHOLOGICAL DEVELOPMENT. no magma. \\'ithin 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 umbihcal vesicle attached to it. The body is of uniform diameter, measuring less than a millimeter. We are probably dealing here with a normal emliryo. In ojiening 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 uiwn 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 i)athological siiecimen with dissolution of the magma. Usually in i)athological specimens the magma is greatly increased in quantity. No. 164 is a somewhat older specimen. It came to us from an autoi)sy, with the entire uterus, and the sections of it indicate that the embryo is undoubtcdh' 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 si)ecimen, No. 463, is somewhat more achanced in development and contains a flexed embryo, 3.9 mm. in length. The ovum is covered completely on one side, and partly on the other, with villi 1.75 to 2.75 mm. long. On the partly covered side the villi leave relatively bare one area, centrally situated, measuring 8 by 4.5 mm. 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 4 mm. 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 aj^pearance 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 jellj'-like. The specimen came from a woman who was perfectly healthy and hacl gi\-cn birth to 2 children dvu'ing 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 historj' 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 fil^rils of magma, but the chorionic w^all, 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, Ijut 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 intenselj^ as do the fibrils of the chorionic wall, with which they are continuous. The contrast obtained with jVIallory '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 reticule 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 phj'sician 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 exoccelom. 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 embrj'o 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 exoccelom, 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 RftTICULft IN NORMAI, AND PATHOLOGICAL DEVELOPMENT. and the adjacent chorionic wall where the fibrils arc unusually abundant. This specimen was obtained from a liysterectoni.\- upon a woman, 25 years old, for a fibrous tumor of the uterus. She had been married 4 years, this being her first ])rey 20 by 20 nun., projects. It is a sac and is covered with a rather smooth moml)rane (dcM'idua reflexa), beneath whicli tortuous vessels are apparent. On one side the sac (tiie implanted chorion) is adherent to tlic uterine mucosa (decidua vera). With a sharp scalpel the entire mass was dissected away from the uterus and brouglit under a binocular microscope in warm salt solution. The middle portion of the free surface was oiK-ned carefully, beautiful \illi iicinfi foiuid, and then the delicate wall of the chorion was divided. Within, a transparent young embryo and its unibilical vesicle were seen, the embrj'o apjiearing to i)e about 5 mm. in length. Through this opening in the chorion, warm (40° C.) saturated aqueous solution of HgClo, containing 5 per cent glacial acetic acid, was gently introduced and the entire mass placed in 500 c.c. of this fi.xaf ion fluid. The main body of tlu> uterus was dissected from the myomatous nodule and fixed in 10 per cent formalin, the site of the implanted ovum being mtirked bya short wooden rod." The fixed and hardened spccimini 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 wath the decidua capsularis, measures approximately 22 by 18 by 11 mm. The adjacent decidua parietalis is thrown into large folds, which are themselves marked b}' numerous tiny elongated crack-like depressions, as well as by more circular pit-like apertures. The relatively smooth l)ut 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 and 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 bullions ones on the main stem. The villi are uniformly- distributed in the small area expo.sed. With a slender scalpel the ovum was care- fully divided under the dissecting micro.scope, the embryo and yolk-.sac being visible. The yolk-sac appears to be almost 2 cm. in diameter and the embryo is surrounded by its amnion, its head (visible from above) being about 3 cm. in length and showing the fourth ventricle covered by a transparent ependyma. Two gill-arches are visible. The yolk-sac surface presents an exqui.site picture of irregular, clear va.s- cular channels and a uniform pattern of small, opaque, white blood-islands. The preservation seems perfect. HUMAN MAGMA RETICULE IN NORMAL AND PATHOLOGICAL DEVELOPMENT. 15 After the embryo had been carefully removed, the ovum was cut into blocks which included its implantation. A block 1 mm. thick, which included the largest circumference of the embryo, was embedded in celloidin, the sections being stained in various ways. A photograph of this block is represented in plate 1, figure 4, which shows strikingly the extent of the magma. Sections which have been stained in hematoxylin and aurantia show the magma much as it appears in the other embryos that have just been considered. There is a denser magma just under the chorionic wall, and heavy strands radiate in every direction, with a fine network resembling spider-web, among the main strands. A number of loose nuclei accom- pany these strands, but they do not have the appearance of the nuclei of the main wall of the chorion. They are mostly round and are of unequal thickness, simulating very much the blood-cells. Occasionally there is a large nucleus. Sections which have been treated by the Weigert fibrin method do not show these fibrils. This confirms a previous experience which I have published elsewhere in my paper on monsters, namely, that magma fibrils do not give the reaction of fibrin, nor do these fibrils stain well in Van Gieson's mixture; however, they take on color similar to the mesenchyme of the chorion. At points it appears as though these fibrils arise directly from the chorionic wall. They stain intensely blue by the Mallory method, and in sections treated in this way the nuclei of the mesenchyme of the villi look much like the accompanying nuclei of the magma fibrils. On one side of the ovum a denser mass of the magma is directly continuous with the mesenchyme of the chorionic wall. However, just in this region the magma contains no nuclei. It, therefore, appears that the magma fibrils must be associated, at least partly, with the nuclei of the chorionic wall. Exceedingly good histological pictures were obtained from sections stained by Heidenhain's method, which show all the transition stages between magma con- taining no nuclei and magma very rich in nuclei. It would seem that there is quite a free wandering of the nuclei along the magma fibrils, and whenever they come in contact with the chorionic wall the fibrils enter it, showing direct continuity. The most instructive specimens are obtained by the Weigert elastic-tissue stain, which gives a slight blue-black tinge to the mesenchyme fibrils of the chorionic wall, as well as to those of the centers of some of the villi. The magma itself takes on a very light stain, but where it is in contact with the chorionic wall it grades over into its blue network. It appears, then, that the centers of the villi, which represent their older portion, stain somewhat with elastic-tissue stain; and, if we view the chorionic wall as the more differentiated portion of the chorion, we must conclude that the older mesenchyme fibrils behave more like elastic-tissue fibrils than do the younger. At any rate, the magma fibrils do not take on elastic-tissue stain. From all that has been said it is clear that the mesenchyme of the chorionic wall and the magma fibrils are continuous and, as I have pointed out elsewhere, they together form a common syncytium. I have already demonstrated that very young connective tissue arises directly from the mesenchyme, the earlier stages of which I have designated as the connective-tissue syncj'tium. Towards digestive reagents the connective-tissue syncytium gives somewhat the reaction of yellow elastic tissue, just as do the mesenchyme and the magma of No. 836 when treated with Weigert's 16 HUMAN MAGMA KfiTICULfi IN NORMAL AM) I'ATHOI.OCKWL DEVELOPMENT. elastic-tifssue stain. I liave also t^howii that the younger the connective-tissue syncytium is, the more difficult it is to digest it in pepsin. Frozen sections shrink but little when treated with acetic acid, while white fibers become transparent. The syncytium itself is somewhat elastic, as shown l)v pressure upon the cover- glass over a frozen section. If treated for 24 hours with pepsin, the lihrils disin- tegrate. The}' are therefore much more resistant to the action of jiepsin than are white fibrils. The action of pancreatin is, in a mea.sure, the opposite of that of jiepsin. When the main mass of syncytuim is formed by exoplasm, it digests readily in ])ancrea(in. The more the syncytium is developed, the more resistant it is towards i)ancreatin. Very young sj'ncytium fibrils, therefore, react towards pancreatin and pepsin much like elastic fibers and this is confirmed in a measure, by tinctorial methods, when applied to sections of the chorion and magma, in sjiecimen No. 830. I have discussed the denser strands of tissue within the main mass of the magma. In the fresh state it appears that these are distinct fibrils, as shown in plate 3, figure 2. They are, also, observed in plate 1, figure 3. It is not quite so clear that there are fibrils in the magma as shown on plate 1, figure 4. In fact, it apjiears as though we have compartments separated by membranes, and that at the junction of several of these membranes the fibrils become denser, and therefore often appear as distinct fibers. It would be more appropriate than to state that the exoccelum is l>roken up into compartments the walls of which are composed of meml)ranes, and that where several of the membranes come together the increased amount of tissue gives the i)oint of juncture the appearance of fibers to the naked eye and under the enlarging lens. I have taken great pains to follow the cells which mark the stronger bands of magma, and it is difficult to arrive at any conclusion, for, in a measure, thej' seem to be related to the endothelial lining of the exoccrlom. In the Peters ovum the spaces near the embryo are lined by a distinct layer of cells, but otherwi,se there is no indication of endothelial lining in any other portion of the chorionic cavity, nor is there any indication of such a lining in the figures given by Herzog, Johnstone, Jvmg. or Strahl and Beneke. It would seem that what corresponds to the exoccelom of the chorion in the later stages is represented by a diffuse mass in the si)ecimen of Bryce and Teacher where the nuclei are scattered through it. The mode of the destruction of the mesenchyme is well indicated in figures on page 18 of a monograi)h by Strahl and Beneke. These irregular cells are first of all attached to the h(\Mvi(M- strands of magma, and they must, therefore, correspond to the endothelial lining of the exoccelom. For the present, however, it appears as if the exoccelom of the human chorion is lined only in part bj' a layer of endothelium; these cells also accompany the magma fibers and line the inner side of the chorion near the emliryo. As the amnion exixuids, it naturally pushes theso strands of magma up against the chorion, and in a short time we can recognize only a few fibrils in the exoca'lom which encircle the umbilical cord. These are well seen in specimen Xo. 148, and their remnantsare shown in Xo. 576, of which I give an illustration on i)late 2, figure 2. Xo. 148 is luidoubtedly normal, for it was obtained by mechanical means, and Xo. 576 is also a normal s{)ecimen obtained from a tubal pregnancy. HUMAN MAGMA RETICUL!-; IN NORMAL AND PATHOLOGICAL DEVELOPMENT. 17 The conclusion regarding the condition of the magma of normal development is that the cavity of the ovum is filled with delicate fibrils which are interpersed with numerous nuclei and which form one continuous network, extending from the embryo to the chorionic wall, and blending with its connective-tissue network. It forms one continuous syncytium, and as the ovum grows the magma reticule differentiates somewhat. Stronger bands of membranes soon form, breaking the cavity of the chorion into compartments. This process continues until the amnion begins to expand, and then these fibrils are pushed up against the chorionic wall. The exocoelom begins as two larger spaces near the embryo, and in this portion of the ovum its cavity is lined with a layer of endothelium. It is quite certain that this sharply defined cavity does not extend to include the whole cavity of the ovum, but the cells lining it arise in common with those which accompany the magma fibrils. The exact extent and the fate of the two small spaces near the embryo in the Peters specimen is still undetermined, but Waterston's specimen indicates that they do not extend to fill the entire chorionic cavity. The examination of numerous specimens, however, indicates very definitely that the exoccelom of the ovum at 2 months does not contain a complete endothelial lining. THE MAGMA IN PATHOLOGICAL OVA. Since the publications by Giacomini it has become well known that an increased quantity of magma within the coelom indicates with certainty that the embryo is pathological. When the magma is pictured or described, it is (juite easy to deter- mine whether or not the embryos and ova published in the literature are normal or l)athological. This is demonstrated in the plates accompanying Veljieau's work. His was able to separate most of the normal from the pathological embryos, but he relied mainly upon the external form of the specimens, which he compared with other mammalian embryos. Unless an embryo appeared much like those of other mammals and was not transparent and sharply defined, he decided that it was not norrnal but pathological. The work of Hochstetter, who limited his study to embryos obtained through hysterectomy, has been used to advantage by Keibel and Else in the preparation of their Normentafel, so that now we have adequate tables and jilates which enable us to recognize with considerable certainty whether or not an embryo is normal, ^without paying much attention to the magma or the chorion. However, embryologists are well aware that they can predict whether a specimen is normal or pathological by tlie quantity of the magma which masks the embryo when the ovum is opened. By the contents of the exocoelom it is quite easy to classify i)athological ova into three chief groups. In the first group, which includes most pathological specimens, the magma is changed into an organized mass of reticular fibrils, inter- mingled more or less with granular substance. To the second group belong si)ecimens in which the exocoelom is large and contains only a fluid mass — that is, a litjuid substance which does not coagulate in either formalin or alcohol. I have pictured a number of specimens of this sort in my paper on monsters. Specimen No. 512, of which I give an illustration on plate 2, figure 1, belongs to tliis grouii. The embryo is atrophic, and it is (juestionabk" 18 UrM.VN M.\OM.V Rl'TICrLf; I\ XORM.VT. .VXD r.\TIIOI,OGIC.\L DKVKT.OPMKXT. whether or not it is encircled bj' the amnion. In these specimens the ccelom is usually oularoed and sometimes it is greatly distended. Often there is a small gran- ular jirecipitate in older specimens, but this is not of sufficient quantity or density to form a continuous mass. The histories of these specimens show that they are considerably older than their sizes indicate, and I am inclined to view them as having once had a dense mass of magma within the ccelom, which subsequently underwent dissolution, leaving a more or less flaky deposit that finally disajipeared altogether. In the third group, the ccelom is greatly distended, the amnion is usuall,v absent , and the ovum is filU^d with a gelatinous substance. This is well illustrated by speci- men No. 604, plate 1. figures 1 and 2. Table 2.— List of specimens containing pathological magma. Cat. No. Length of embryo. Dimensions of chorion. Men- strual age. Contents of ovum. Cat. No. Length of embryo. Dimensions of chorion. Men- strual age. Contents of ovum. 278 660 813 78 531 250 12 318 543 6510 244 402 122 533 545 21 560 135 mm. 1 .5 2 2.1 2.5 3 3 4 4.5 5 5 5 5.5 7 9 mm. days. 67 200 S7 45 ■41 42 75 42 65 56 53 49 Hvalino niasma. Fluid. Flaky. CJranular. Fluid. Do. Hyaline niasima. 512 636 605 104 211 1117 270 991 604 94 1189 584a 79 230 261 mm. 10 10 [10.5) 12 12 14 14 17 17 20 22 25 33 57 90 mm. 30X27X18 28X28X22 45X40X25 35X35X15 days. 56 35 73 14 91 No magma. Granular-liyalino ; also no magma. Hyaline magma. Granular; hyaline also. Hyaline magma. Granular; hyaline. Do. Do. 40X35X30 SO X50 X25 36X33X13 19X19X19 10 X 9X 8 18X18X 8 20X18X11 70X30X25 35 X30 X30 25X15X15 40X25X20 20X16X 6 35 X30 X30 12X 9X 9 12 X 9X 5 40X30X20 70 X50 X50 50X50X50 50X42X40 50X50X50 75 XOO X50 120X70X70 105X65X65 I will now review several specimens illustrating these three varieties of patho- logical magma. The specimens considered are arranged in table 2. The list could easily be increased to several hundred, but as the specimens with catalogue numl)ers less than 403 have been published in detail with illustrations in my monograi)h on monsters, I will allude only to some of them. The pathological specimens with numbers over 402 are lieing prei)ared for publication, so that a few selected speci- mens with higher numbers are illustrated. Pathological specimens from tubal preg- nancj' with numbers up to 1.000 will he found described in detail in my monograph on tubal ])regnancy. The first specimen which I shall consider i,Xo. 278) consists of an entire ovum, measuring 6 bj^ 4 mm. It was sent me by Dr. Stanton, of Albany, New York. The specimen might be viewed as normal, but it contains no embryo, and as it was obtained from a diseased uterus, it is probably pathological, the magma having undergone minor changes. This ovum was found accidentally in curettings from a woman supposed to have chronic endometritis following pregnancy. There is nothing in the history from which the age of the specimen could be estimated. Part of the specimen had l)een HUMAN MAGMA RETICULE IN NORMAL AND PATHOLOGICAL DEVELOPMENT. 19 cut into sections before it was received at the laboratory, with the statement that no embryo had been found, it having fallen out. I found that the half sent con- tained a ccelom. 3 by 2.5 mm., filled with magma, in which there was a cavity about 1.5 bj' 1 mm. Sections showed that the cavitj' was natural and not sharply defined, with nothing to indicate that it had contained an embryo. On the contrary, it was found that the magma reticule was composed of a loose network of mesoderm cells, which bound one side of the chorion with the other. These cells are directly continuous with those of the mesoderm and resemble them in every particular. At one ]ioint there is a small group of epithelial cells, which may represent what was originally the embryo. Otherwise, the chorion and its villi are normal in appear- ance, being encapsulated in decidua which has in it some uterine glands. All in all, this specimen reminds one very much of the Peters ovum. There are some leuco- cytes in the decidua, but no accumulation of them indicating inflammation of the uterus. l~^everal figures, illustrating this specimen, may be found in my monograph on monsters. Specimen No. 531 is in many respects similar to the one just described (No. 278) . It came from a Avoman who had been pregnant 6 times, her j^eriods having been 17 days overdue before this abortion. The ovum is spherical, 19 mm. in diameter, and is covered only by a mass of \illi, which appear normal. The ccelom within con- tains many magma fibrils, the meshes of which are more or less filled with dense granules, as is shown in plate 1, figure 8. Within this mass there is a detached vesicle, 1.5 mm. in diameter, which no doubt represents the umbilical vesicle. A specimen intermediate between the two just described is No. 250, of which several illustrations are published in my paper on monsters. The specimen came embedded in a mass of decidua and was obtained by scraping the uterus. When opened it was found filled with magma reticule just beneath the chorion, in which could be seen a small embryo, and farther away towards the center of the coclom was the umliilical vesicle. The whole ovum was cut into sections. The chorion and the Ailli are apparently normal in shape and structure, being also rich in blood- vessels, which are filled with embryo blood. The villi are bathed in mother's l>lood and covered with an active trophoblast. The decidua is somewhat infil- trated with leucocytes, but there are no abscesses. The front end of the amnion is absent, and its free edge and the embryo are embedded in reticular magma, indicat- ing that the amnion was destroyed before the abortion took i^lace. The general shape of the embryo and its degree of development are practically' normal. The heart is well formed and, including the blood-vessels, is filled with blood. The ali- mentary canal, brain, spinal cord, otic and eye vesicles, myotomes, and branchial arches are much like those of embryo No. 12, to be described presently. The septum transversum is well marked and the th3Toid gland is just beginning. The tissues of the embryo, however, and the cavity of the front end of the brain are filled with numerous small round cells with fragmented nuclei. All stages of frag- mentation are seen, just as may be observed in the leucocytes in small abscesses. Most of the red blood-cells are within the blood-vessels, l)ut those within the ti.ssues appear perfectly normal. On account of the diminished number of mesoderm cells, which, in fact, diminish in pro])ortion as the fragnienled cells increa.se, the con- 20 HUMAN MAGMA UKTICn.K IX NORMAL AM) I'A TIK H.OCICAI, DKVELOPMENT. elusion must be drawn that the fragmented cells arise from the mesoderm cells. The epidermis covers tlie whole embiyo. The primary change in this specimen is no doubt in tiie mesoderm, for all the rest of the embryo appears normal. That the equilibrium was overthrown is indicated by the necrotic amnion and the great amount of reticular magma in the exoccelom. ^yhat is especially interesting in this specimen is the partial destruction of the anmion, which l:)rings the embryo directly in contact with the pathological magma of the coelom. Embryo No. 12, which has been just referred to, may also be discussed in this connection. It was questionable for a long time whether or not the embryo was normal, as the villi and contents of the coelom and embryo are beautifully preserved and show no pathological change. However, more careful consideration of the specimen shows that there are a few fibrinous masses between the villi, w^th every indication of uterine inflammation and infection. The extent of the reticular magma is more pronounced than usual, aiul it was necessary to dissect it away before the embryo could be isolated sufficiently so that it could be well seen. The head is no doubt atrophic, and I am fully convinced that this part of the embryo must have undergone pathological changes a short time before the abortion. Specimen No. 318 is much like No. 250. The ovum, measuring 20 by 18 by 11 mm., is covered with villi which ajijiear to be };)erf(Ttly normal. Ui)on ojiening, it was found to be filled with stringy magma, on one side of which was eml:)edded an embryo 2.5 mm. in length. The head is sharply outlined, but th(> embryo seems to continue directly with the umbilical vesicle, leaving an atrophic tail. Sections show' that the amnion over the head has dissolved, leaving a picture very much like that shown in No. 250. ^^'e have here a small embryo with a very large coelom, the ovum being moderately filled with reticular magma and a small embryo only partly covered with the amnion. No. 543 is another embryo of the same type. The magma is a little denser than in No. 318. The chorionic villi are developed, but markedly pathological, as the photograph shows. The embryo within is 3 mm. long, lying (juite free within the mass of magma. It is covered bj' a ragged amnion; that is, the amnion is partly destroyed. An interesting specimen in this connection is No. 402, which is partly described in my paper on monsters, since the issue of which the embryo and chorion have been cut into serial sections. The villi of the ovum are not well developed, and they are distributed irregularly over the surface. The coelom is filled with reticular magma. The embryo is club-shaped, the head being much too far advanced for the body. The umbilical vesicle is normal in size; the heart is well outlined, and the extremities are just appearing. Sections show that the amnion is greatly distended. Sections of the chorion were stained with cochineal and \'an Ciieson, and show beautifully llic fibrillated structure of the chorionic membrane. These fibers take on red stain, as do those of the reticular magma. The two are continuous, as shown by the illustration on i^late 3. figure 3. In fact, this continuity is much more i)ronounced in pathological than in normal specimens. Specimen No. 533 (plate 2, figure 3) shows a more ad\ anced stage of an extensive development of reticular magma. The vilh of the ovum appear to be normal and the reticular magma is verj- dense. Between the meshes there are a number of HUMAN MAGMA RETICULE IN NORMAL AND PATHOLOGICAL DE^•ELOPMENT. 21 opaque nodules about 0.5 mm. in diameter. With much difficulty the embryo was teased out, but it was practically impossible to clear it entirely of the magma fibrils. The embryo is long and slender, looking more like that of a dog than a human speci- men, the head being unusuallj' small and thin for a human embryo of 0.5 mm. long. The fibers are irregularly stuck together by small granules, and there is a gap in the center which represents the i)lace in which the embryo was located. The illustration shows this condition beautifully. The specimen was sent me bj' Dr. Fewsmith, of Trenton, New Jersey, who obtained it from a woman whose menstrual period had been a month overdue. An extremely interesting specimen is No. 545, well illustrated in figure 1, plate 3. The magma is not extensive, but it is pronounced. The embryo is atrophic, and the chorion is only partly covered with villi. The specimen was sent me by Dr. Rand, of New Haven, Connecticut. It was obtained from a woman who is the mother of one healthy child. The last menstrual period began on September 2. Bleeding began on October 22 and ended with the abortion on October 25. The ovum was found embedded in the clots of blood attached to the cervix of the uterus. An extreme case of degeneration of the magma is shown in No. 660, also well illustrated in figures 4 and 5, plate 3. There is a tendency towards membrane formation, tough strands of fibrils, spaces, and clumps of granules. The chorionic wall is hemorrhagic and degenerated; within there is a collapsed amnion containing a cheesy granular mass. I shall use two more specimens to illustrate the nature of granular mass in more advanced stages. The first is No. 605 and the second is No. 584a. No. 605 is a white transparent specimen, covered with a uniform layer of villi which branch two or three times. The entire specimen measures 45 by 40 by 25 mm.; a small patch of decidua adheres to the outside. The interior is partly filled with coarse strands of reticular magma, having numerous granules attached. On one side of the specimen the umbilical cord is seen, surrounded by a ragged amnion. The tip of the cord has a piece of intestine and stomach hanging from it. The larger masses of tissue which are intermingled with the reticular magma must be the remnants of the embrj'o, parts of which appear to be normal, and judging from the form and size of the arms and legs the embrj^o is about 10.5 mm. long. The second sjiecimen is unusually interesting because it contains a normal embryo with hernia of the liver. The exocoelom is unusually large and is filled with a more extensive layer of reticular magma than should be found in an ovum containing a normal embryo of this size. The remaining three specimens are given because they well illustrate various degrees of reticular magma within the ovum. No. 560 (plate 1, figure 6) shows very pronounced reticular magma inter- mingled with much granular. Two stages of somewhat later development are given in Nos. 636 and 991. In the former (plate 1, figure 10) the magma is more pro- nounced than in normal development, and in the latter fjilatc^ 1, figure 7) it is in an extreme amount. Finally, a unique specimen (No. 1189) throws some light upon the formation of the reticular magma. The ovum came to us within the uterus, having been re- moved b}^ an operation. At first it seemed to be normal, but on opening it the 22 IIIMAN .M.V(;.\1\ HKTlCTl-r; IX XOUMAl, AM) I'A1II()I,( HIK'AI. 1)K\ Kl.Ol'MENT. embryo was found I'licircli'd hy a larjro mass of traiisi)aront, tough, stringy reticular magma, whicli was remo^•e(l only with groat dithculty. It behaved nmch like the vitreous humor of the eye. On account of its great ciuantity we at once suspected that the specimen was pathological, and after the embryo was removed it proved to be so. Although ciuite advanced in development, its head was found to be smaller than normal, the tissues of the face were dissociated, and the borders of the eye were not sharj) but ragged. Xo doubt the specimen had continued to develop normally until shortly before the operation, and the magma increased in (juantity and became tough and fibrous. It is an interesting specimen, showing changes in the magma late in development. Sections of the implanted ovum have not yet been made The specimen is from a negress, 45 years of age, who had had 9 previous i)regnaii- cies. Her last menstrual period was 67 days before the operation. Pregnancy was suspected before the removal of the uterus, but a hysterectomy was performed because her i)(n-io(ls had Ix-come very severe, lasting 8 days and causing faintness and weakness. The two types of degeneration which the reticular magma undergoes have been considered above. The magma becomes granular and denser as it lessens and be- comes liquid. The liciuid again either coagulates or remains fluid when the speci- men is fixed in formalin. The two fluid tyjics may be r(>lat(^d to the destruction of the amnion, but as yet I have been unable to reach a conclusion regarding this pcjint. The lieginning of the formation of granular magma is shown in specimens No. 560 and 991 (plate 1, figures 6 and 7) as well as in Nos. 533 (plate 2, figure 3) and 660 (plate 3, figure 5). It appears to extend into the cavity of the amnion, and often forms great crusts, which surround the embryo, as shown in several specimens pictured in my monograph on monsters (e. g., Nos. 79, 94, 104, 230, and 261). An extreme specimen of granular magma within the exoccelom is sho\\-n in specimen No. 651<7 (plate 1, figure 9). It is extremely difficult to determine with certainty the structure of the granular uKigma, but in studying pathological ova (especially those obtained from tubal preg- nancy) I have frequently observed that there are large masses of granular magma which take on hematoxylin stain. These granules are mixed with a slimy mass which also takes on hematoxylin stain. ]\Iy attention was called to these granules because they have a characteristic circular stratification and contain within their centers small granules which also stain intensely. I am by no means certain whether all granular magma stains in this way with hematoxylin, and what I have just stated may apply only to a portion of the granular magma. Si)ecimen No. 531 (shown in figure 8, plate 1) has its coolom filled with a liciuid mass m which there is a granular deposit that surrounds the eniljryo anlage. Such specimens are numerous and, without opening them, they may fre([uently be recog- nized by the transparency of the chorionic wall, which is covered with but few atrophic villi. A more advanced embrj'o, showing the sam(> condition, is .shown in specimen No. 512. In it the embryo is atrophic and macerated, without the jiresence of an amnion. The chorion is thin and is fully covered with delicate degen- erated villi. Other specimens which come within this group are Nos. 21, 78, 122, and 244a. These are all illustrated in my monograph on monsters. HUMAN MAGMA RKTICULK IN NORMAL AND I'ATIK tl.(H IICAL DEVELOPMENT. 23 l^ometimcs the entire specimen is filled with a gelatinous mass, which becomes firmer when fixed in formalin and separates into a more solid mass, and into a licjuid when preserved in formalin. This mass appears to lie within the amnion in most specimens, as in cases where it fills the whole ovum the amnion is missing. Speci- men No. 604 (plate 1, figures 1 and 2) is quite typical, as is also Xo. 135. In both the embrj'os are atrophic and necrotic, and the jelly-like fluid fell out with ease when the ovum was cut open. The chorion is atrophic in both of them and is covered only with a few atrophic villi. Specimen No. 604 came to mc without a history, and measures 70 by 50 by 50 mm. It is full}- covered with fibrinous clots, between which there are few large villi, as the picture shows. The chorionic wall is 3 to 4 mm. in thickness, and its interior is entirely filled with jelly-like magma of uniform consistency. On one side of the specimen, lying free within the hyaline magma, is a straight embryo, 17 nnn. in length, with atrophic head, arms, and legs. The same descrij^tion aj^plics eciually well to No. 135. Specimens like these are ciuite numerous in our collection of human ova, but usualh' the jelly is lost when the speci- men is opened. Figures illustrating embryos of this sort may be seen in my pai:)er on monsters, under the description of embryos Nos. 79, 94, 230, 261, and 270. No. 1117 (plate 1, figure 5) contains an embryo well packed in the jelly-like magma. The cavity of the ovum is small and its wall is xevy hemorrhagic. The specimen came from a woman, age 26 3'ears, who was married at 15. She had two births at term and one i)revious abortion. She believed she became pregnant about 3 months before the operation, although she had not missed her regular periods. Another specimen belonging to this category is No. 813. It consists of a fleshy mole, well filled with tough, jelly-like magma. All the villi are destroyed and its surface shows ulceration. Further study of this magma is necessary before it can be related to the granular magma which forms with the reticular magma in the exocoelom. I am inclined to t)elieve that the hyaline substance which is so often found within the amnion of pathological specimens arises from the amniotic liquid, which has become richer in albumen, and therefore congeals into a jelly-like mass when preserved in formalin. CONCLUSION. The fibrils forming reticular magma are always in direct continuity with those of the mesenchyme of the chorionic wall. This can easily be demonstrated by means of Van Gieson stain, and reticular magma must therefore be viewed as embrj'onic connective tissue extending into the cavity of the ovum. The stronger strands are accompanied more or less by mesenchyme nuclei, showing that the magma itself must be viewed as independent connective tissue identical with the mesenchyme of the chorion. As the amnion extends these strands are pushed aside, their final remnants being seen in that portion of the exocoelom which encircles the umbilical cord. In pathological specimens the reticular magma increases in quantity in the earlier stages of development, continuing for a number of months of pregnancy. Fre- (luently the meshes between the reticular fibrils are filled with peculiar stratified granules which take on an extensive hematoxylin stain. Often the amnion is 24 HIM.W MACMA HKTICrLK IX XOHMAI. AND I'ATIini.ofUf AT. ni;VKT,(M'MK\T. destroyed early in development, in which case the magma may dissolve, but some- times it increases fj;reatly in (luantity, forming a gelatinous mass. Freciuently pathological ova are encountered in which the devel()])nient of the embryo is re- tarded, and the amnion is often found filled with a flaky deposit that, as time goes on, increases greatly in quantity and finally forms large crusts which invest the embryo. In other cases there is marked hydramnios, and in certain instances, where \hv anniion is destroyed, the magma dis.solves, leaving only the eml)ryo floathig in tlu- fluid encircled by the chorionic wall. Specimens are also found in which the cavity of the amnion is greatly enlarged and is filled with a jelly-like sub- stance, which in later stages may form crusts encircUng the embryo. The tiuc relation between the pathological changes of the contents of the exoccelom and of the cavitv of the anmion remains to be determined. lilMAX MAU-MA lU-niCULE IN NOUMAL AND PATHOLOGICAL OKNELOPMEXT. 25 BIBLIOGRAPHY. Bryce and Teachkr: ( 'uiilrilnitiuns Id the study of the early doveloijineiit and injlxildiiig of the human ovum. Cdasgow, 19()s. Kternod, .\. {'. !■".: La ga.stnile duns la seric animale ot phis speeialement ehez I'honune et les mammiferes. Tirage i part du Hull. .Soe. V'aud. Se. Nat., 1906, xi.il, L56, Lausanne, 1900. . Des premiers stades de I'tt-uf humain et de son im- planation dans I'uterus. Mfmoire presente au premier Congrcs feder.atif international d'anatomie (Geneve, 0-10 aout 1905), Nancy, 1900. . L'ccuf humain. Implantation I't gestation tropho- derme et placenta. Memtnre public k Tocoasion du Jubile de rUniversilC", l.'w9-1909, Geneve, 1909. . Inegalites de croissancc du chorion ovulaire humain et localisationa consccutives en chorion Iteve et chorion frondosum. C. R. de la Rf'union de I'Association des Anatomistes (Nancy, 5-7 avril 1909), Lille, 1909. FRA.'^sr, L. : Ueber ein junges menschlielics Ei in situ. Archiv fiir mikroskopische .Anatomic untl Kntwicklung.s- gcschichte, Bd. 70, 1907. ( liAcoMiN'i, C.: Probleme aus Entwickclungsanonialien d. Menschl. Embryo. Merkel and Bonnet "Ergeb- nis-se," iv. 1S94. Grosser, O.: Eihiiute und der Placenta. Wien und Leipzig, 1909. . The development of the egg membranes and the placenta; menstruation. Keibel and Mall, Human Embryology, i, 1911. Herzog, M.: A contribution to our knowledge of the earliest- known stages of plaeentation and embryonic devel- opment in man. American .Tourna! of .\natomy, ix, 1909. His, W. Anatomic menschlicher Embrvonen. Leipzig, lSSO-1885. HocHSTETTER, F. ! Bildcr der iiusseren Kcirperform einiger menschlicher Embryonen aus den beiden ersten Mon.aten der Entwicklung. Miinchen, 1907. Ing.\lls, N. W.: Beschn-ibung cinc.s nienschliehen Embryos \'fni 4 : 9 mm. .\rchiv. fiir mikroskopisclie Anatomie mid Eiitwickhingsgeschichtc, Bd. 70. 1907. ,J(jH-Nstii.\e, H. \V.; Contribution to the study of the early liuinan ovum. .Journal of Obstetrics and Gyne- cology of the Briti.sh Empire, 1914. Juxo, P.: Ei-Einbetlung beim nienschliehen Weil)e. Berlin, 1908. Keibel, F. : Die iiussere Ktirperform von Affcnembryonen. Selcnka, Entwickelungsgeschichte, xiv, Wiesbaden, 1900. . The formation of the germ layers and the gastrula- tion problem. Keibel and Mall, Human Embry- ology, I, chapter v, 1911. Keibel und Elsa: Normentafel zur Entwicklungsges- chichte des Menschen. Normentafel zur Entwick- lung.sge.schichte der Wirbeltiere. Achtes Heft. .Jena. 190S. Lewis, F. T.: The development of the intestinal tract and respiratory organs. Keibel and Mall, Human Embryology, ii, 1912. M.\LL, F. P.: Origin of human monsters. Journal of Mor- phology, XIX. Published also as a monograph by the Wistar Institute of .\natflmy, Philadelphia, 190S. . Develo|)mcnt from the connective tissue of the syncytium, .\mcrican .Journal of Anatomy, i, 1901 . . On the fate of the human embryo in tubal pregnancy. Publication No. 221, f'arnegic Institution of Wash- ington, 191."). Peters, H.: Einbettung des nienschliehen Eics. Leipzig und Wien, 1899. Retziis, G.: Ueber das Magma reticul6 des nienschliehen Eics. Biologische Unter.suchungen. I, Stockholm, 1890. Strahl and Beneke: Ein junger menschlicher Embrxo. Wiesbaden, 1910. Velpeau, a. L. M.: Embryologie ou Ovologic Humaine. Paris, 1833. Waterston. a young embryo with 27 somites. .Journal of .\natoniy and Physiology, xlix, 1914. Fic. 6. Fi(i. ( . Fig. 8. Fici. 9. Fig. 10, EXPLANATION OF PLATES. I'l.ATi; 1 . Figs. 1,2. Pliotograplis of the two lialvcs of ovum \o. 004. Natural sizo. Tho cavity of the ovum is lillcil witli a jelly-like substance in which a i)athological embryo is embedded. Fig. 3. Section through a normal ovum Xo. 836, encajwulated in the decidua. X'-i\. Drawn by Mr. Ditlusch. The embryo lies witliin the ccrlom, and hands of mapma fibrils radiate from the amnion to the chorionic wall. The head of the embryo shines through the more transparent portion of the amnion. Fig. 4. Photosra|)h of a block of the ovum, Xo. 830, in »ilu after the embryo had been removed. X22. The supporting strands of magma are strikingly shown. Fig. 5. Pathological embryo Xo. 1117, embedded in hyaline magma. X4. From a tubal pregnancy following gonorrhea (?). Pathological ovum X'o. 560, containing a great quantity of reticular magma. X2j. The embryo is normal in form. From a case of retroversion of the uterus. Pathological ovum Xo. 991, with the cavity completely filled with reticular magma. Xatural size. The embryo is normal in form. From a negro woman. Sect ions of the embryo indicate that it is macerated. Pathological ovum Xo. .")31, containing a granular deposit around a nodular embryo. Xlj. Pathological o\um with a nodular embryo (G.^lg). X2. The exoco-lom is gorged with p;ranular magma. Specimen Xo. 630. X21. The embryo and chorion are normal in form, but the reticular magma is markedly increased in quantity. Pl.vte 2. Fig. 1. Pathological embryo Xo. 512, lying free within the ovum. XO. The villi arc thin and scattered and the embryo is atrophic. There is no formed magma. An o\-um, Xo. 570, obtained from tubal i^regnancy, showing a delicate layer of magma fibrils around the attachment of the umbilical cord to the chorion. X3. Ovum Xo. 533, showing very extensi\c magma. X6. Pl.ate 3. Ovum Xo. 545. X7. There is a delicate network of fibrils below the amnion and the chorion. Embryo Xo. 588. XS. Delicate strands are shown radiating from the umbilictil cord and yolk-sac. This figure is given to show the appearance of magma in vesicle de\-elopment. From a woman who has had numerous mechanical abortions performed upon herself. Uterus badly inflamed. Section through the chorion and magma of Xo. 402. X2S0. The specimen was stained with Van Gieson stain and shows that the fibrils of the magma are continuous witli those of the mesenchyme of the chori- onic wall. It came from a case with subinvolution and symptoms of endometritis. Outline of the ovum of Xo. 000. Xatural size. The diagram indicates the part of the specimen shown enlarged in figure 5. Xo. 000, showing very extensive changes in the magma. XO. The upper tip of the amnion is shown. The magma is fibrillar and granular, and at places the fibrils seem to form membranes. The chorionic wall is very hemorrhagic. Fig. 2. Fig. 3. Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. o. Kig. 3 (836) Fig:. 6 (560) Fij;. 8 (531) Fig. 9 (65I-G) Fig. 10 (636) Fia;. 1 (512) Fig. 2 (57G) Didusch fee. Fig. 3 (.ooo) Fig. 1 (.->4.-,) WMr Fifi. 4 (mi)) i^^."?^ *^;^ ■J Fifj. 3 (402) Didusch fee. Fig. 2 (o.->s) Collupsi'il amnion. Fig. 5 (CdO) CONTRIBUTIONS TO EMBRYOLOGY, No. 11. THE STRUCTURE OF UHROMOPHILE CELLS OF THE NERTOUS SYSTEM. By E. \. CowDRY. Anatomical Laboratory, Johns Hopkins I'niversity. With one plato. 27 THE STKUCTUllE OF CIIRO.MOPIIILE CELLS UF THE .NERVOUS SYSTEM. By E. V. CowDRv. INTRODUCTION. It has long been known that certain pecuhar nerve-cells, well characterized by their structural appearance, occur in the normal human brain, and indeed in the brains of all the vertebrates which have been examined. In fixed preparations they are slightly shrunken, they stain deeply with both acid and basic dyes, and their nuclei are obscure and hard to define. Flesch (1887, p. 196) called them "chro- mophile" cells. Nissl (1896, p. 1154) thought at first that they were artefacts of some sort, but Cajal (1909, p. 210) and others brought forward strong evidence against this view. Cajal (1909, p. 211) concluded that they were resting cells. On the other hand, in the light of DoUey's (1910, p. 333) work, they would seem to be in the initial stages of fatigue, as evidenced by the increase in the amount of NissI substance in them and by their obscure, deeply-staining nuclei. Our knowl- edge of their structure is incomplete so far as the mitochondria and the canalicular apparatus are concerned. Busacca Archimede (1913, p. 332), alone, has observed that the mitochondria in certain cells in the brain of Testudo grceca stain particu- larly intensely with iron hematoxylin, and in some cases seem to lose their definite outlines and to form homogeneous masses. Rina Monti (1915, p. 39) has made a comprehensive study of the canalicular apparatus ("apparati di Golgi") in nerve-cells, but she does not mention cells in the chromophilic condition. I shall consequently venture to present in this paper my observations on these two structures in the chromophile cells in the brain of the white mouse. MATERIAL AND METHODS. White mice were employed because they are the smallest mammals which can be conveniently used in the laboratory for experimental purposes. The small size of their nervous system permits the study of the distribution and the arrangement of chromojihile cells in serial sections. All the mice were of known age and care was taken that they were perfectly normal. A modification of the methods of Altmann (1890, p. 27), Galeotti (1895, p. 466), Regaud (1910, p. 296), Bensley (1911, p. 309), and Shirokogoroff (1913, p. 523) was devised for the study of mitochondria. The method has many advantages. In the first jjlace, the use of a mixture of formalin and potassium bichromate as a fixative (Regaud) gives a much more uniform preservation of mitochondria than the osmic acid containing fixatives in general use. The application of the fixative by 'The work was aided by the Departmoiit of Embryology of the Carnegie Institution of Washington, and part of it was done at the Marine Biological Lalx)ratory. Woods Hole, Massachusetts, where, throujih the kindni'ss of the Director. Dr. T.illie. a room was placed at mv disposal. 29 30 rilK STIUCTtUK OK CHHOMOl'HILE CELLS OF TlIF, Xl'-inoiS SVSTK.^L injection throufili I he hlood-vcssels ( Shiroko^'oroff) eliniinalrs many very ol).i('('t ion- able artefacts due to faulty penetration. The use of permanganate and oxalic acid (Bcnsley) facilitates the staining of the mitochondria with th(> anilin fuchsin (Altmann), and the counterstaining with methyl green (Gah'ottii permits of the demonstration of the Xissl substance in the same cell with the mitochondria. The fact that \hv method gives good results in the case of other ti.'^sues where the mix- tiu'es of Altmann, Flemming, and others are useless on account of their poor powers of jjenetration, justifies the following detailed statement: Fixation: Chlorofoiin the aiiiinaL Inject wanned ().8.t ])er eent NaC'l solution into the aorta thi()ujj;li tlic ventricle. If the brain alone i.s to ho studied clamp the descending aoria. If the entire nervous .system is to he fixed, clanip the cd'liac, the renals, the sujierior and inferior mesenteries, the iliacs, and the brachials. Continue the injection until the .salt solution is returned uncoloreil through the juRulars. DuriuK this time lay bare the arch of the aorta and the carotids from connective tissue, so that they mas- expand easily and carry more fluid to the brain, (iravity jiressure of not more tiian (i feet may be employeil. Cut the infeiior vena cava and tlie jufiulars so that the salt solution may run through easily. Follow the salt solution with the formalin and bichromate mixture: 3 i)er cent pota.ssium bichromate, 4 parts; neutral foi-malin, 1 part. The potassium bichromate acts l)cst when freshly prei)ared. Neutral formalin is made fi'om the commercial variety by the addition of magnesimn carbonate, a dejMJsit of which sliouUI always remain at the bottom of the formalin bottle. It is imjiortant that the jjressure should be at the maximum when the mixture is first injected, so that the blood-vessels may be fixed in a state of dilation. If the pressure is low when the fixative comes in contact with the vessel-walls they will be fixed in a condition of collapse. It will then l)e difficult, or even iinpo.ssible, to obtain a complete injection. The injection sliould lie continued foi' .-iboul an hour. The brain is then dissected out and immersed in the fluid. In the case of the mouse's brain it is sufficient to divide it longitudinally into halves. The fixative must l)e changed every day for 4 or 5 days, otherwise il undeigoes a cliange evidenced by a darkening in coloi-. This change is accelerated by light and by h<>at , so that the tissue should be kept in the dark and in a cool place. Fixation may al.so be effected by simple immeision of the tissue in the fixative, instead of by injection, but this procedure is not lecomnunded. After this jirolonged fixation th(> tissue is mordanted in a fresh 3 per cent solution of pot;issium bichromate, in which it remains for cS or fl days, changing every second day. Wash in running water for 24 hours. The object of this careful washing is to lemovi' most of the formalin and bichromate, for otherwise the tissue will be extremely brittle and hard to cut. Dehydration and embeddimj: 50 per cent alcohol 12 hours; 70 per cent and 9,5 per cent alcohol 24 hours each; absolute alcohol (5 to 12 hours: h;df absolute and xylol (i hours: xylol .3 hours; paraffin ()()° C. ,3 hours; cut in An s(>rial .sections. S^lainimj: (1) Pa.ss the .sections, mountecl on sli(l(>s. down through toluol, absolute. '.)."). 70. and .")(! per I'cnl idcohol to distille(l watei', (2) 1 per cent acpieous >oluliiin of potassium permanganate M) seconds: Imt this tinii' niu>t be iletermined exijerimeiitally. (3) 5 per cent aqueous solution of oxalic acid also about 30 seconds, (4) Rinse in several changes of distilled water about a minute. lnroiii|ilele washing |irevents the staining with fuchsin. (5) Stain in .Vltmann's anilin fuchsin. which is to be made up as follows: .Make a saturated solution of anilin (jil in distilled water by shakingthetwo together (anilin oil goes into solution in water in aliout 1 per cent). Filter and add 20 grams of acid fuch.sin to 100 c.c. of the filtrate. The stain shoulil he ready to use in about 24 hours. It goes bad in about a month. To stain, dry t he slide wil h a towel, excej)! the small area to which the sections are attached. Cover the sections on the slide with a small amount of the st.ain and heat over a sjiirit lamj) until fumes, smelling strongly of anilin oil. THE STRUCTURE OF CHROMOPHILE CELLS OF THE NERVOUS SYSTEM. 31 come off. Allow to cool. I.ct the stain remain on the sections for about (i minutes. Return tiie stain to the bottle. (6) Dry off most of the stain with a towel and rinse in distilled water, so that the only stain remaining is in the sections. If a large amount of the free stain remains it will form a troublesome precipitate with the methyl green; on the other hand, if too much stain is removed the coloration of the mitochondi'ia will be impaired. (7) Again dry the slide with a towel, except for the area covered by .sections. Allow a little 1 per cent methyl green, added with a pipette, to flow over the sections, holding the slide over a piece of white paper so that the colors may be seen. Apply the methyl green for about .o seconds at first and then modify the time to suit the needs of the ti.ssue. (8) Drain off excess of stain and plunge the slide into 9.") per cent alcohol for a second or twn, I lien rinse in absolute for the same time, clear in toluol, and mount in balsam. Several difficulties may be met with: (1) The methyl green may remove all the fuchsin, even when it is only a]ipli(>d for a short time. This is due to incomplete mordanting of the mitochondria by the chrome salts in the fixative. It may often be avoitled, either by omitting the ti-eatment with permanganate and oxalic acid, or by treating the sections with a 2 per cent solution of potassium bichromate for a few minutes immediately before staining (as advised by Bensley). The action of the permanganate and oxalic is to remove the excess of bichromate. (2) Or the fuchsin may stain so intensely that the methyl green removes it very slowly or not at all. This, on the other hand, is due to too much mordanting. It may be corrected by i)rolonging the action of the permanganate and oxalic. (3) Sometimes, after obtaining a good differentiation, the methyl green is washed out before the slide is placed in toluol. This may be avoided by omitting the 95 per cent alcohol, by passing from the methyl green to the absolute direct. (4) Unfortunately the stain is not very perma- nent. Under favoral)le conditions it will last for 3 or 4 .years. The fading in color is hastened by light and by heat, and it proceeds very rapidly in a damp atmosphere. Cajal's (1912, p. 211) uranium-nitrate method was employed for the canaUcuhir apparatus in its original form, except for the substitution of methyl green in tli(> place of carmalum as a counterstain. Control ]ireparations were fixed in a variety of fluids and were .stainc^d in many ways, as will ai^joear later. The figures have been made from specimens prepared by the ab()\e-iuentioncd fuchsin-methyl green method, by which th(> mitochondria are stained red, the Nissl substance green, while the canalicular api)aratus nmiains uncolored; and also from specimens made by the uranium-nitrate method, which black(>ns the canalicular apparatus and colors the Nissl substance green. OBSERVATIONS. Chromophile cells, as the name implies, po.sse.ss an unusual affinity lor stains, which may be either acid or basic. Their structure is variable. A glance at the figures is sufficient to show this. The variations may represent stages in a i>rocess, which, when i)tislK>d to an extreme, results in a cell in an advanced stage of chromo- philia, but of this we have no conclusive proof. Neither can we assert that the jn-ocess proceeds in this direction, for the changes observed may en lose their discrete outlines and seem to merge into the surrounding cytoplasm. Mito- chondria may not be very numerous in cells of this kind. The nucleus stains intensely and a few clear canals maj' be seen in its vicinity. The cell has ai)pai-entl_\- shrinkage spaces on either side of it. Prej^arations, made by fixing in alcohol and staining with toluidin l)lue, contain cells in which the Nissl .substance is in this con- dition and Cajal preparations show that the canals are unaltered. Figure 4 illustrates a cell in a rather more advanced stage of chromophilia. In this cell there is an unusually large amount of Nissl substance and there are further evidences of tlie disappearance of formed mitochondria, especially in the cell process. The outlines of the nucleus can barely be made out. The canalicular apparatus shows no modifications either by this method or by the Cajal technicjue. A very interesting condition is shown in figure o. Here, with this degree of differentiation, only a few typical mitochondria persist near the origin of the cell process. The Nissl substance is overshadowed by a cloud of material staining the same way as the formed mitochondria do in adjacent cells. Figure 8 illustrates a similar cell in a ( 'ajal preparation. The Nissl substance in it is increased and there is no modification in the blackened canalicular apparatus. Cells in this condition are often shrunken. It is difficult to determine whether the shrinkage is the expres- sion of an actual diminution in the .size of the cells during life, or whether it is simply the result of a difference in the reaction of chromojihih' cells to the fixation and sub- sequent treatment. The presence of what appear to be shrinkage spaces around the cells .seems to indicate that it is in reality due to the technifiue employed, because if, on the other hand, it was due to a decrease in the size of the cell during life, one would expect the space to be filled uj) by a shifting of neighboring structures. It may be emphasized that the fact that other cells, in actual contact with chromoi)hile cells, show no signs whatever of .shrinkage must be regarded as one of the distinctive properties of cells in the chronioi)hilic condition. Then^ is, of course, still another interpretation, namely, that the spaces in (juestion are uiuisually large i)erineuronal spaces, the enlargement l^eing in some way connected with the difference in the physiological condition of chromophile cells as contrasted with other cells. THE STRUCTURE OF CHROMOPHILE CELLS OF THE NERVOUS SYSTEM. 33 The mitochondria may apparently disappear more or less completely in cer- tain cells, and their place be taken by a mass of amorphous material with the same staining properties (figure 6). The nucleus may or may not be visible. Cajal preparations of cells in the same condition (figure 9) show that the canals are unal- tered. The nucleus is obscured bj' the cloud of Xissl substance. The appearance of these cells, in advanced stages of chromophilia, would perhaps lead one to sup- pose that they are degenerating and that their nuclei have disappeared. That this is not the case may be seen if one of the mitochondrial preparations is stained with hematoxylin and eosin. The hematoxylin and eosin does not color either the amorphous deposit or the Xissl substance, which, in the mitochondrial and in the Cajal preparations, hides the nuclei. The nuclei have in reality distinct and defi- nite outlines and appear to be quite unaltered, except that they contain rather more than the usual amount of chromatin. In fact, the change in the mitochondria and the increase in the amount of the Nissl substance would never have been suspected if hematoxylin and eosin had alone been used. The distribution of chromophile cells is important. They often occur singly. They may be surrounded on all sides by cells which show no tendency toward an assumption of the chromoi)hilic condition. They may, on the other hand, occur in clumi)s. The clumps vary greatly in size. They contain cells in all stages of chro- mophilia in addition to a varialjle number of unaltered cells, which are always pres- ent, scattered among them. The neuropil in which the chromophile cells are embedded differs in no way from the neuropil elsewhere. It seems, by all the mitochondrial methods, to be studded with mitochondria. ]kit it must not be thought that the mitochondria occur in anything like equal numbers in the neuropil of different regions, because there is a remarkable variation in this respect. The mitochondria appear to be intercellular, l)ut unhaj^pily a source of error is introduced by the fact that the unmedullated, and to a lesser extent the meduUated, processes stain in much the same way as the mitochondria, so that in some cases it is impossible to distinguish between them. Undoubtedly a large number of the mitochondria in the neuropil are contained in nerve-cell processes cut in section, but there is no y side with the chromophile cells, is hard to explain on the basis of mechanical injury, because whatever pressure had been brought to bear upon the tissue must necessarily THE STRUCTURE OF CHROMOPHILE CELLS OF THE XER\OUS SYSTEAL 35 have acted upon both; but one shows the condition and tlie other does not (as is shown in all the figures). Furthermore, if mechanical injury is the cause of the condition, it is difficult to understand why chromophile cells are so rare in the spinal cord and in the ganglia of the cranial nerves, which are bound down by membranes and which in removal are consequently subjected to greater mechanical injury than the cortex of the brain. In order to settle the (luestion the results of intentional mechanical injury brought about by bruising the cerebrum and the spinal ganglia with a blunt instru- ment were studied. It was found that the lesion produced was characterized bj^ the flattening or comj^ression of many cells in the same direction, at right angles to the direction in which the pressure had been exerted. All the cells in the area were uniformly affected. Normal cells were not scattered among them. The injured cells stained intensely, but they did not simulate the chromophiL- cells. The neuropil between them showed marked changes and could readily be distinguished from the neuropil elsewhere in the same section. Chromophile cells are not the result of differences in the time or in the degree of fixation. The whole brain is uniformly fixed by the methods of technique emploj^ed. The distribution of chromophile cells is not related to the arrangement of the blood- vessels, which are the avenues of approach of the fixative. Neither do the mito- chondria varj' in number with the vascularity of the region. The condition is not due to irregular mordanting with the j^otassium bichro- mate, because complete extraction of the bichromate by prolonged treatment with l)ermanganate and oxalic acid does not essentially modify the appearance of the chromophile cells when the sections are stained. Another possibility is that the intense staining of the chromophile cells results from incomplete differentiation. Even if this were the case the differences in the rate of decolorization must be the visible expression of real differences in the cells themselves. I have found, however, that the same differences obtain in undiffer- entiated specimens stained lightly with fuchsin, crystal violet, and iron hematoxylin. I have made a number of experiments to determine whether more complete differ- entiation would bring to light formed mitochondria in cells in which they appear to have been replaced by the amorphous deposit which stains in the same way. Specimens were stained in the usual fashion with fuchsin and methyl green and were mounted in balsam. Drawings were then made of chromophile cells which had been stained intensely with the fuchsin and in which no formed mitochondria could be seen. The cover-glass was then dissolved off and the slide was passed down through toluol and graded alcohols to water. It was then r(\stained with fuchsin, differentiated more strongly with the methyl green, mounted in balsam, and examined. The same condition was apparent, except that the homogeneous deposit had a distinctlj' greenish color. The same process was repeated as many as five times with the same cell, increasing each time the extent of differentiation, until the cell stained intensely with methyl green and very little trace of the fuchsin was left; still no formed mitochondria were observed; this was repeated with other cells with the result that in some of them formed mitochondria were brought to light, while in othei-s thev were not. 36 TITK STRrCTlRE OF CHHOMOPHILE CELLS OF THE XERVOl'S SYSTEM. Similar experiments were performed with individual cells stained a homogejieous black color with iron hematoxylin. The results obtained are easier to interpret because the differentiator, iron alum, does not itself color the tissue like the methyl green. This advantage is counterbalanced by the fact that both the mitochondria and the Nissl substance stain in the same way and it is often difficult to distinguish between them. In many cases, particularly in slightly undifferentiated specimens, the extraction of the stain from chromophile cells l\v further differentiation brought to light a varial)le number of formed mitochondria. Moreover, it is worthy of note that the chromophile cells in the cerebral cortex are the last to become decolorized and that the differentiation occurs with unequal rapidity in different parts of the cell, thus indicating that the homogeneous deposit is not present in the same con- centration in all jxirts of the cell. The end-result of this experimentation is that chromophile cells, particularly those in advanced stages of the condition, contain a diffuse deposit, which stains in a tj'pical way with all mitochondrial dyes, and which is probably formed by the solution of some of the mitochondria in the cell. The condition is not due to technique and it is not associated with a visible pathological change on the part of the animal. All the mice emjjloyed were apparently normal. They ate well and showed no signs of sickness. They were killed with chloroform, and it may at once be said that the changes are not due to acute chloroform poi.soning, because animals killed in other ways, bj^ decapitation, for example, showed the same condition. The mice were not excited, or disturbed or exercised in any unusual way before they were killed. A careful autopsy of each mouse was made to make sure that it was quite normal. Fome were found to contain a parasite, present in the cysticercus stage in the liver; these were invariably discarded. The chromophile cells were found in mice of both sexes in almost all seasons of the year. They were found in mice varying in age from 25 days to adults, so that thej^ can not be regarded as an expres- sion of senility. It was thought that they might occur in consequence of abnormal conditions due to ca{)tivity. In order to settle this point a wild field-mouse was captured alive and in good condition and its brain was prepared in the usual way. It, also, showed chromophile cells. An apparently analogous partial solution of mitochondria was observed in liver-cells ])oisoned with phosi)horus by ]\Iayer, Rathery, and Schaeffer (1914, 1). 609). Accordingly, ^^'. J. M. Scott tried the effect of ex])erimental i)hosphorus poisoning on the nervous system of white mice. The chromophile cells were apparently entirely unaffected and a solution of mitochondria was not brought about. Dr. Bensley made the interesting suggestion to me that this jiartial solu- tion of mitochondria in chromoi)hile cells might be due to a swing of the reaction in them toward the acid side, with the liberation of free fatt}' acids. I therefore made some preliminary experiments on acidosis in mice ])ro(luced by the sub- cutaneous injection of dilute hydrochloric acid, all of which yielded negative results as far as the chromophile c(>lls were concerned. I have, further, f(jund that slight exercise does not alter the api)earance of the chromophile cells in the brains of white mice to any noticeable extent. THE STRUCTURE OF CHROMOPHILE CELLS OF THE NERVOUS SYSTEM. 37 It seems highly i)robable, therefore, that chromophile cells occur normally in the brain of the white mouse and that we have to reckon with a partial solution of mito- chondria just as we have for many j'ears recognized a chromatolysis, or solution of the Nissl substance. DISCUSSION. This work on chromophile cells has, I believe, an important bearing upon (1) the question of differential nerve-cell activity; (2) the phenomena of chondriolysis and hyperchromatism; (3) the functional independence of the -mitochondria and the canalicular apparatus; and (4) our conception of the structure of living nerve-cells. (1) The distribution of chromophile cells in the different parts of the brain is interesting. The fact that they occur most abundantly in the cerebral cortex and in the cerebellum, and that they are rarely foimd in the lower centers like the spinal cord, would seem to indicate that the central neurones differ in some way from the more peripheral ones. The difference may be one of lability, for Dolley (1914, p. 56) has found that more highly specialized cells are more prone than less special- ized ones to respond with structural changes to physiological experimentation. Moreover, the occurrence of these cells in groups, which vary in size and in position in different brains, is in accordance with our conception of the alternation of rest and activity in the higher centers and may well have some bearing upon the vexed problem of cortical localization, for as yet neither the mitochondria nor the canalicu- lar apparatus have been considered in this connection. (2) We must recognize a "chondriolysis, " or a partial solution of mitochondria, in nerve-cells as well as a "chromatolysis." The word "chondriolysis" was first employed by Romeis (1912, p. 139) to describe the disintegration of certain mitochon- dria which escaped from the cells into the uterine fluid of Ascaris. It is, to my mind, more appropriate than the term "chromatolysis," which is frequently applied to the so-called solution of Nissl bodies, for the simple reason that I am of the opinion (1914, p. 20) that the Nissl substance is usually in solution in the living nerve-cell, whereas the mitochondria are assuredly present as definite formed bodies (except of course in the chromophilic condition). Chemical changes are undoubtedly involved in the phenomena of conduction (Tashiro and Adams, 1914, p. 329) and, in view of the distinct differences in the chemical constitution of the mitochondria and of the Nissl su])stancc, the one being of a li])oid albumin nature (Faure-Fremiet, Mayer and Schaeffer 1910, p. 95) and the other being apparently a complex nucleoprotein containing iron (Scott, 1905, p. 507), it seems probable that the study of mitochondria and the changes which they undergo may bring to light variations in the activity of the nerve-cell which could never be detected by the study of the Nissl substance alone. Quite ai)art from the role of the nucleus in the elaboration of the Nissl substance and the purely cytoplasmic nature of mitochondria, there is further evidence of a functional diver- sity between the two structures. I have found that in the nerve-cells of the mouse the mitochondria vary directly with the volume of the cytoplasm and that the Nissl substance varies inversely with the nucleus cytoplasmic ratio; also that the mito- chondria are of more general occurrence in nerve-cells than the Nissl substance. 38 THE STHLCTIKE OF CHKO.MOI'IJILE CELLS OF THE NEH\OUS SYSTE.^^ They are i)resent in the granule-cells of the cerebellum, as is also evident from the earlier work of Altmann (1890, plate xiii. figure 1) and Xageotte (1909, p. 826), and in the granule-cells of the olfactory bulb of mice and rats, which are well known to be devoid of Xissl substance. IVIoreover, in certain cell-groups, under normal con- ditions, there is often a variation in the mitochondria, as between different cells, without any corresjxniding change in the Xissl substance. Mitochondria occur abundantly throughout the length of the axone, where no Xissl substance has ever been seen. They also occur in certain dendritic processes which do not contain any Xissl substance. Evidence of this sort may be multiplied. Just how the mitochondria are concerned with the activity of the nervous system is unknown. I have presented evidence elsewhere (1914, p. 18) that they l)lay a part in the basic processes of metabolism which are comn^on to all cells, but this is unfortunately a very broad statement and we naturally desire to learn some- thing rather more specific about them. Coghill's (1915, p. 350) behef that the mitochondria are concerned in the constructive (analx)lic) side of metabolism is of interest in this connection, i)articularly since it falls so well in line with the well- known " eclectosome " theory of Regaud (1911, p. 699), which, in turn, is an exten- sion of the "side chain" theory of Ehrlich. M. R. and W. H. Lewis (1915, p. 393) make the interesting suggestion that the mitochondria take jiart in cellular respira- tion, which is also a fundamental i)roce.ss common to all cells. We may confidently exjiect that this new avenue of approach to the study of the activity of the nervous system will yield results of importance, not onlj' because our histological methods of technique are now sufficiently accurate to permit of the actual enumeration of the mitochondria, a thing which can not be accomplished in the case of the Xissl substance, but also because W'aldemar and Mathikle Koch (1913, p. 427) have recently succeeded in devising chemical methods for the quali- tative and (luantitative estimation of substances, very closely related, perhaps identical with mitochondria, in the nervous system. These substances are phos- pholijMns. Iloppe f^eyler long ago jwinted out that lecithin (a t>-i)ical phospholipin) and cholesterol are to be foimd almost everywhere that life phenomena e.xist. In fact, a great wave of revived interest is manifested in recent chemical and patho- logical literature in these complex compounds of fatty acid, phosphorus, and nitro- gen. Mathews (1915, p. 88) very aptly remarks that the phospholipins are the most important substances in living matt(>r: "For they are fouiul in all cell.s, and it i.s uiKloubtedly their function to produce, with chole.sterol, the peculiar semifluid, seiuisolid state of protoplasm. The latter holds much water in it, but it does not dissolve. Indeed it may Ix' said that the phospholipins with cholesterol make the essential sub- stratum of living matter. This physical substratum of phospholipin differs in different cells and probably in the same type of cell in different animals, but everywhere, from the lowest plants to the highly diflferentiatcd brain cells of mammals and of man himself, it possesses certain fundamental chemical and physical properties. In all cases the phospholiiiin substratum is soluble in .-dcohol containing some water," etc. In view of these considerations it is interesting to inquire whether the distribu- tion of mitochondria in cells corresponds with that of the phospholipins. It is cer- tainly true that mitochondria are more widelv distributed than anv other kind THE STRUCTURE OF CHROMOPHILE CELLS OF THE NERVOUS SYSTEM. 39 of cell granulation now known to us. They occur in almo.st all cells. Yet certain cells, like the fully differentiated non-nucleated red blood-cell, unquestionabl}' con- tain a large amount of phospholipin, though no formed mitochondria can be seen. The mitochondrial substance is probably present in solution, just as it appears to be in chromophile cells, for it would obviou.sly be absurd to state that it must always occur in that state of condensation which makes it visible with the aid of certain powers of the microscope. The recent investigations of Levene (1915, p. 41) on cephalin are of interest. A new field of investigation is evidently opened up. It may thus be possible to pursue this line of w'ork with chemical as well as with his- tological and ]ihysiological methods, a combination which has been but rarely effected. Work along these lines seems the more desirable since, as will be seen, it may throw new light upon certain problems in the pathological anatomy of the nervous system as well. Wells (1907, p. 460), in his discussion of mental fatigue, writes: ".Since the lecithin forms .so important a part of the nervous system, it is tempting to imagine that in fatigue exce.ssive (luantities of its toxic decomposition product, cholin, and the still more toxic derivative of cholin, nenrin, are formed in considerable amounts and cause part, at least, of the intoxication." Now we have seen that, in the opinion of certain investigators, mitochondria are largely composed of lecithin. It is possible, therefore, if Wells's reasoning is correct, that the symptoms of mental fatigue are the result of their decomposition. Moreover, Halliburton (1907, p. 74) and others are convinced that organic diseases of the nervous system may be distinguished from functional neuroses on account of the formation of cholin in the one and not in the other. This opens up the possibility of a differentiation between these two great groups of diseases on the basis of cell structure, as to whether or not there is a change in the mitochondria. (3) The persistence of the canalicular apparatus in chromophile cells is of interest in general cytology. In chromophile cells, in which there arc marked structural changes, the canalicular apparatus remains without anj' great modifica- tion. This is rather surprising, since investigators have gradually come to regard the canalicular apparatus as the most labile cell organ; but it is in conformity with Key's as yet unpublished observations on degenerative changes in spinal ganglion cells. Key finds that the canalicular apparatus persists without much modification for from 12 to 24 hours after death in spinal-ganglion cells left in the animal. I have shown (1912, p. 494) that a canaHcular apparatus, in the form of a system of clear, uncolonnl canals, occtu's in the same cell with tyjiical mitochondria and that consequently the canalicular apparatus and the mitochondria are structu- rally distinct. This conclusion is strongly supported by my observation that they may hkewise be seen together in chromophile cells, the difference being that while the mitochondria are greatly changed, the canalicidar apparatus remains with little or no modification, so that they are functionally as well as structurally different. My positive impregnations of the canalicular apparatus by the uranium-nitrate method of Cajal confirm this observation. Now, Cajal (1908, p. 123) is so certain of the identity of the clear canals (described originally by Holmgren) and the "Apparato roticolare interno" of Golgi 40 Tin; siiucrrKic ok ciiHo.Moi'iiii.p; ciii.i.s oi' iiii-: xkuvous system. that he refers to them as "conduits de Clolf^i-Hohngren."' Hut Rina Monti (1915, p. 40) has made th(> statement that the hirge internal reticular aj^paratus corre- sponds to the chondriome (/. c, to mitochondria) in the nerve-cells of mammals; to (luote her own words: "II grande apparato r('tic;)huv inttM'no dal (Jolgi nelle CL'llule nervose di mammiferi corrisponde aduiuiue al condrioma, conune il grande ai)parato descritto dal Pensa iirllc cellule cartilaginea." If Cajal is correct in his identifica- tion, it would api)ear that th(> canaliculai' api)aratus and the mitochondria are identical. 1 have already discus.sed (,1U12, p. 4UUJ the older statements of Popoff (1906, p. 258), Smirnow (1906, p. 153), Van Durmc (1907, p. 84), Meves (1908, ]). 846), and Hoven (1910, p. 479), who are incHned to believe this to be the case. It is liard to see how these two views can be reconciled. I am inclined to think that the well-known lack of s])ecificity of the methods of silver impregnation which Pensa (1913, ]). 5()0) and Rina Monti (1915, ]>. 45) have emi)loyed are the cause of the confusion. 1 do not believe that the (iolgi method can be trusted invariably to demonstrate a certain structure within the cell, like the canalicular api)aratus; and, for this reason, I can not accei)t unreservedly Cajal's identification of the canalicular apparatus with the CJolgi apparatus. 1 agree with Duesl)erg that a more precise definition of the "Apparato reticolare interno" is highly desirable, but I do not agree with him in his attempt (1914, j). 37) to define it in terms of its relation to the centrosome, because our knowledge of the centrosome itself is so dejjlorably inadecjuate. We re(|uire. above all else, more accurate metliods before th(> matter can be cleared up. (4) This discussion of the structure of chromophile cells may be profitably con- cluded by a statement of our present knowledge of the cytoplasmic structure of living nerve-cells of vertebrates not in the chromophilic condition. Mitochondria unciuestionably occur and may be .seen as such in living nerve-cells even without any vital stain. The Nissl .substance is usually present in solution, not in the form of discrete masses as seen in fixed preparations. I believe that there is also an amor- l)hous argentophilic material which (when treated by ai)propriate but very capricious methods) a.s.sumes the form of fibrils. The canalicular api)aratus, like the neuro- Hbrils, is an unknown (juantity in living nerve-cells, although it may l)e demonstrated in fixed tissues with considerable regularity. These structures, or more correctly speaking substances, are distinct and should not be confused with one another. Although the mitochondria alone have a definite morphology and can usually be seen in living ner\e-cells, imder ordinary conditions, with the present means at our disposal, it would be arbitrary in the extreme to say that the others can never be seen. Pigment, fat, lipoid, etc., may of course be seen in variable amount in living nerve-cells. It is the more fundamental constituents with which we are concerned. The recent work in bio-chemistry, summarized l)v F. (iowland IIoi)kins ( 1913, p. 663) in his jiresidential address before the Physiological Section of the British Association, has, I believe, an important bearing here. The cell is regarded as a dynamic system of co-existing phases in more or less stable ecjuilibrium.the condition of which is altered, from moment to moment, by ))roces.' grams. The brain was cut into serial sections 4 /j in thickness and stained with fuchsin iind methyl green. All the figures were drawn from cells in the .same section to insure uniformity in the action of the stain and of the difTerentiator. The mitochondria are stained red, the Nis.sl .substance green, and the canalicular apjiaratus persists, in some of the cells, in the form of clear, uncolorcd spaces. Figures 7 to 9 represent cells from the cerebral cortex of a male white mouse, 29 days old and weighing 10 grams. Portions of the brain were prepared by the uranium-nitrate method of Cajal and were cut into serial sections 4 >i thick. These figures were also drawn from a single section to insure uniformity in the action of the counterstain, methyl green. The canalicular a|))iaratus is in the form of a blackened network and the \is.sl subst.ance is colored green. Fi<:. 1. Two cells, having a distinct increase in amount and intensity of the .stuining of the mitochondria. This change may mark the first stages in the assumption of the chroniophilic condition. 2. A nuich greater increase in amount of mitochondria and a slight increase in intensity of the staining of the Nissl substance and the nucleus. 3. The Nissl sub.stance is more abundant. It is diffuse and stains more brightly. The outlines of the mito- chondria are indistinct. The nucleus stains darkly. \ few clear canals are visible near it. There is what appears to be a shrinkage space on either side of the cell. 1. .'^till greater clianges. The mitochondria appear to be going into .solution; outlines of nucleus barely dis- tinguishable. 5. The mitochondria have almost all gone into .solution. The Xi.ssl substance is almost entirely obscured by the cloud of mitochondrial material which stains with the mo.st energetic of the two dyes, acid fuchsin. The nucleus is invisible. t>. A complete "chondriolysis" or .solution of the mitochondria. The canalicular ajjparatus is present in the vicinity of the nucleus. 7. The increase in amount of the Nissl substance indicates a slight degree of ehromophilia. The canaUcular appa- ratus is blackened and shows no changes. 8. Greater increase in the Nissl substance. It is diffuse, with marked hyperchromatism. The nucleus stains diffusely with methyl green. Its outlines are obscure. The canalicular apparatus, in black, is unaltered and the cell as ;i whole is shrunken. 9. Cell so intensely stained with the methyl green that the nucle\is can not be seen. Canalicular apparatus slightly condensed, othenvise unchanged. There is a considerable shrinkage of the cell. < ./('^ ' T* — -'-^V /■ re" ^ ^- -^^ /5^. 8 > ^V; •tf^ C. A. HOex A CO., BALTIMOnC THE STRUCTURE OF CHROMOPHILE CELLS OF THE NERVOUS SYSTEM. 43 BIBLIOGRAPHY. Altmann, H. 1890. Die ElementarDrnanismfU unci ihre beziehungeu zu don Zellen. 14.5 pp. Leipzig, Veit & Co. Barker, Lewellys F. 1899. The nervous system and its constituent neurones. 1122 pp. New Yorlc, .■\pploton & Co. Bensley, R. R. 1011. 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ON THE DEVELOPMENT OE THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. By R. S. Cunningham. \\ itli tivf plates. 45 CONTENTS. PAGE. Methods 50-52 Vessels ari.-;i!)g from the left liuct 52-64 Lyiiij)hatics of the bronchi 63 Lymphatics of the veins 63 Lymphatics of the pleura 63 Summary 64-GG Bibiiogi-aphy '. 66 Explanation of plates 67-68 1).\ THE DEVELOIMIENT OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO ?I(i. Bv R. S. 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 lymj)h-vessels in the time of their appearance, and hence constitute what may be called a primary lymjihatic 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 cys- terna chyli. The further development of the lymj^hatic 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 lymi)hatics grow by a centrifugal sprouting of pre-existing endothelium, the other believing that these vessels are formed by a coalescence of numerous isolated spaces develoj^ing 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 sjjrouting of the endothelial walls of the sacs and of the right and left thoracic 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-lymi)hatics) agree in claiming that lymphatics do not grow by the centrifugal s]irouting of the i)re-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 Lymphgefiissystems.) Though primarily concerned with the problems of origin and the method of growth 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 LIXCIS L\ THE EMBRYO PIG. theory is correct, it is clear that it should be iK)ssible to follow the growth of lym- phatics from the sacs into any organ or group of organs. It shoukl also he i)ossible to demonstrate in i)r()gressively older stages constantly increasing lymphatic zones and decreasing non-lymphatic zones. The development of the lymi)hatics of the skin, of the intestine, and of the lung has now been studiinl in this manner. In 1904, Dr. F. R. Sabin demonstrated that the skin receiv(>d its lym])hatic supi)Iy 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 (inall\- unite, so that the entire skin is supplied with lymi^hatics. The differentiation wliich 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 lymphatics 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- jjreted 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 i^erii^heral vessels, only those of the skin and the intestin(> 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 develojjment 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 lymi)hatics. 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 ])rimary 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 is not 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 develo])s from three sources: the thoracic duct, the right thoracic duct, and the cejjhalad portion of the retroperitoneal sac. In 1913, i^abin remarked: "The right lymi)hatic duct curves ventralward and grows to the heart and lungs." This is the onh' statement which I have been able to find DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS L\ 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 patholog3^ On account of the comjilexity of the development of the lung lymphatics, it has seemed Ijest to i)resent 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 cm. (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 tlic 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 cm. (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 lymphatics 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 Schaeffer for the illustrations. 50 DE\ELOPME.\T OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. METHODS. Tlu> injection method has lieen principally used, hut it has been siip])lemented 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 nimiber of especially prepared scries. 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 i)repared series the blood-vessels were injected. All these embryos were fixed in Carnoj^'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 embrj^o immediateh' in the fluid and allow it to remain there G to 8 hours; thcni transfer directly to 70 per cent alcohol; dehj'drate 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 httle shrinkage. The stains used were Ehrlich's hematoxyUn and a mixture of eosin, aurantia, and orange G. Thehijection masses used were india ink, a saturated solution of prussian blue, a 5 per cent aqueous solution of silver nitrate, and an acjueous 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 nearlj' 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 i^recipitates 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 earhest injections were made bj^ filling the jugular sacs from the superficial 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 cm. long, because the injection mass usually follows the path of least resistance, which is into the jugular vein. In injecting embrj'os between 3 and 6 cm. 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 retroiieritoneal 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 Ij^mphatics up from the retroperi- toneal sac to the posterior 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 cerebral wards; the right plexus is some- times filled also, and often the vessels of the left lobe of the lung. DEVELOPiMENT OF THE LYMPHATICS OF THE LUNGS IX 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 cm. (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 s,ystems 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 500 ^t); these were mounted in balsam but not stained. Other lungs were cut at 10 to 20 ju 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 l)c 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 inunediate neighborhood of the main bronchi and their chief subdivisions. Stage 7 cm.: The most interesting change, however, lies in the further growth of (he jyiiiphatics, which in the earlier stages are found in the root of the lung in the neighborhood of the pulmonary ves.sels and the large bronchi. As they grow in, they accompany these structures for a distance; 52 DEVKT-OPMENT OF TTTK T.YMPIIATICS OF THE I>U\GS IX THE EMRRYO PIG. then apjiioacliing the end blanches the\' leave them and run in a plexiform manner midway between the broncliial tubes until they reach the pleura. This gives the lung now an indefinitely lobulated appearance in which the perijihcry of the simple lobule is indicated by the lymphatic vessels and the center by the l)ronchi. The lymjihatics are lined with flattened eudotlieliuin; 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 cm.: The lymphatics, forming a plexus around the l)ronchial veins and arteries at the root of the lung, accomijany them towards the perijiherj', giving off branches to the interlobular spaces en route. * * * On reaching the peri])hery of the lung they leave these structures and pass out, as in the preceding stages, to the pleura. Thej' 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 lung as well as in those on the surface under the pleura. Stage 19 cm.: In general the relations of the lymphatic system have not changed. Stage 23 cm.: At 23 cm. 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 iiiterloliular system which Miller has lieen unable to find in the human lung. Injections pointing to such a relationship he has iiiteri)reted as artefacts. If Miller's conclusions prove correct, then the Ij'mphatics 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 handicajiped by having onlj^ 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 {i. 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 reconcilal:)lc when we have followed the develoi^ment of the lymphatics through the various stages that lead to the adult form. The literature on the lymphatics 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 mesenchAniial 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 sac with the retro- Ijeritoneal sac — in a pig embryo 2.5 cm. long. Very soon after this the first evidence of the i^ulmonary supply may be found. I have obtained partial injections at 2.8 cm., and have found some small vessels in serial sections at 2.6 cm.; 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 ]:)osition it comes down behind the arch of the aorta. This transition is shown by l^abin (1913, figures 12 and 13). 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 thoracic 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 cm. From this plexus vessels grow across the trachea to anastomose with other vessels from the similar plexus on the opposite side; other Ij'mphatics 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, arc 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 bifurcation 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 supiily. 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 thoracic duct and its plexus surrounding the aorta, to join with the large 54 DEVELOPMENT OF THE LYMPHATICS OF THE LUX(!S IN THE EMBRYO PIG. vessel which has been described as the first to the hing and which comes over the arch to reach the hiluni. These vessels from the duct below the hilum form a l)lexus with the vessel from above, as has been described. It is well known that the thoracic 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 anlajje of the vessels that surround the aorta in the adult. This relation has been figured by Ileuer (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 consccjuently very conspicuous in injections of this region. Heuer has figured this lymiihatic as one that goes to the heart, a conclusion entirely justifiable from the general appearance of the inject(>d specimen. Figure 1, plate 2, is from a dissected embr3'o 4 cm. 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 l)e seen some of the vessels that grow up to meet the branch from al)o^•e. 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 cm. long, and can be seen in sections at 3 cm. (see figure 1, plate 1). The lung-tissue is at this time very slightly difTerentiated mesenchyme, containing the earlj' 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 cm. in sections. But I have not been able to inject them earlier than 3.3 and 3.5 cm. Figure 1, plate 1, is of a section from an embryo 3 cm. long, in which the blood-vessels were injected wliilc 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 comi^lete the description of this plexus before considering the portion of this study which relates to the lung proper. The development of the vessels within tlie 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 Ix-eii 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 tlie left, but differs in a few particulars, chiefiy relating to and in consecjuence 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 duct 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 L\ THE EMBRYO PIG. 55 cardiac division, after reaching the base of the heart, along the posterior wall of the vena cava, i)asses 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 dorsal to the vena cava and injecting towards the heart, I was able to fill this plexus in a pig 3 cm. 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 cm. long. In that paper attention was called to the fact that the duct grows towards the heart and that it probably represents the cardiac supplj'. 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 probablj' 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 {i. 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 directlj' 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 lymjihatics 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 di^•ido into several branches, some 56 DKVKI.oi'MKN r OK THK LYMI'lIATiCS OF Tllli LLXCS IN TlIK EiMBUYO PIG. of which anastomose with those mentioned above as growing down into the hilum of the hmg from the i)iilmonary hmb of the right duct. Other vessels turn outwards along the bronchi and veins and grow into \\w lung-tissue of the ujiper 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 Ij'ing along the tracheal wall and coming from the vessels described above, while others grow to the tracheal wall at varying i)ositions along the section h'ing 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 lymphatics of the right side were at 2.8 and 2.9 cm. Figure 2, plate 3, shows an embryo of 3 cm., 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 an,y 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 jilexus 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 imtil, 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. Thei'(>fore 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 bifurcation, 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 cm. 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 m(n-ged into a sheet-like i)lexus that surrounds the trachea and passes down into the lungs as tubes of vessels surrounding the bronchi. Above the liifm'cation 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 i)lexus on the left side of the trachea just at the bifur- cation a grouji of lymphatics pass up over the left stem bronchus and sweep across to the right bronchus, forming the ujiper 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 l)e seen that the left supply is a more imi)ortant part of the general origin than the right, supplying, as it does, all of the left lung and part of the right. DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS IX THE EMBRYO PIG. 57 It is of importance to note here that the heart is migrating downwards (i. e., caudalwards) during this period, and, by the time the embryo has reached 4.5 cm. 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 cm.) 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 cm. 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 ve.ssels 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 cm. 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 cm., 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 lymi)hatics, 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 corresiwnding bronchi. This is, indeed, as far separate as is possible, since there is almost no lung-ti.ssue 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 ;\Iiller (1900). 58 DEVELOPMENT OF THE LYMI'IIATICS OF THE LUNGS IN THE EMBRYO I'lO. 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 descrilx-d. The others are, bj' 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 sui)plied 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 imtil 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 2.9 and 3 cm. These are the first branches from the vessels that are forming the ])lexus 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, l>'miihatics 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 i:)lexus that covers the bifurcation. 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 lym])hatics grow towards tlu^ i)leura; they are derived l)()th from the plexus that follows the ])rimary vein and from the vessels that sur- round the i)rimary ])ronchi. Tlu^ 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, lie 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 lymphatics 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 lymphatics 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- DEVKLOPMENT OF THE LYMPHATICS OF THE UXflS IX 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 pj'ramid-shaped lobules are surrounded by plexuses of lymphatics. Along these plexuses the differentiation of the connective-tissue layers takes place, for, when the lymjihatics 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 (juestion of tissue densit3\ 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 Ij^mph- 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 greatlj' reduced on the secondarj* 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, j^late 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 sj'stem 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 embiyos, a lymphatic sac just median to the mesonephritic vein. Baetjer (1908) showed that it arises from the ventral surface of the large vein which connects the two Wolffian bodies (embrj^os 17 to 23 mm.) ; Heuer, following Baetjer, found that num(>rous lymi)hatic sprouts arise from this sac and invade the intestine through the mesentery. This sac sui)plies lymph-vessels to the stomach, the liver capsule, the ■\\'olffian 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 DE\ELOPMENT OF THE lA-MriiATlCS OE 'JlIE LUNGS EN THE ENHJRVO I'lG. of tissue that passes down behind the diai)hragm to end in the tissue around the aorta; it corresponds to the li<>;amentuni i)ubnonale in the human. It is through this prolongation of tlie lower lobe that the lynii^hatics from the retroijcritoneal 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 jiole of the lung (figure^ 2, plate 4). There are three or four vessels that grow out from the sac and vij) 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 ligament um 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 directl}' up al^out 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 ])roduces the two plexuses on the two borders of the lung (figure 3, jjlate 5). I have injected these vessels at 3.4 cm.; 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 j)lexuses, exactly similar to those described above. These rapidly anastomose with the other lymphatics, and, by the time the embryo has reached 4 cm. in length, the entire lung is uniformly supplied. It is very pertinent to incjuire why the lymphatics that reach tlie 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 verj' 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 sac via the vessels that grow u]) from that structure, or to the thoracic ducts through the tracheal ]ilexuses and the vessels accomj)anying the veins and the bronchi. We have seen how the lymphatics grow into the lung-tissue and there form two distinct 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 j^oles 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 cm. embryos. At 6 cm. the lymphatics around the trachea form a close-meshed i^lexus 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 Kmgs is derived, but there are numerous vessels passing down into the right lung from the right i)lexus, and the two are closely bound together, especiallj" 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 supi^ly 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 closel.y l)ound, 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 multi])lication 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 accomjianied the parent bronchus and follow the new-formed structure towards the periphery. These lymphatics leave the bronchus and pass to the venous group when thej'^ reach the region where the air-sacs arc 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 princij)al 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 ]-)y numerous anastomotic vessels. 62 DEVELOPMENT OF THE LYMPHATICS OF THE LUXCJS IX THE EMBRYO PIO. In the meantime, the vessels of the pleura, which at from 5 to 6 cm. we have seen beginning to form the true pleural jjIoxus, continue to proliferate, and tlius form a fine-moshod plexus in the pleura between the blocking-off of the lobules. The completion of the i)riniary plexus is shown in figure 3, plate 1. This is the surface of the lung in a pig embryo of 6 cm. with the pleural vessels injected. Each of these uninjected areas represents a primary lobule, and the surrounding lyni])hatics mark out tiie connect ive-tis.sue ])lexuses. Figure 1, jjlate 5, illustrates one of the primary lobules, and the close-meshed plexus is the true pleural supi)ly. 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. Tiiese ]iass around the air-cells, but are never found on their walls, and, uniting with the terminal vessels of the end veins, j^ass to join those in the i)leura. These arc 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 s])ecimens, 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 lymphatics 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 lymphatics 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 embrj^os 23 cm. long. It was surprising that injections did not reveal this j^lexus very much earlier. I have not l:>een able to demonstrate any lymphatics in the submucosa before the embryo reached a length of 19 cm. 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 grouj). This process has been carefully studied by Ileuer in the formation of the niuco.=!al plexus in the intestine. The submucosal plexus is comi)letc 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 grou]) of lymi)h-ve.s.sels to be found, and these have alreadj' been described. The lymphatics of the adult lung were first described by Olaf Rudbeck in 1651-1654 (quoted from ]Miller, 1900). Since that time numerous workers have studied the.se vessels. In 1900 W. S. Miller reviewed the literature very thoroughly, and it will, therefore, be unnecessary to repeat that here. Aliller studied the lymphatics in the lungs of adult cats and dogs by injecting them from one of the pleural vessels. He divided the lymphatics into four groups, as follows: A. The lymphatics of the bronchi. ('. The lymphatics of the arteries. B. Tho lymphatics of 1ho voins. I). Tho lymphatics of the pleura. DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. 03 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 Ij'^mphatics that accompany the smaller bronchi and empty into the nodes which are situated at the hilum of the lung. While there are several Ij'mphatics 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 lymphatics. 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 Coimcilman (1900) with regard to the interlobular lymphatics, l^appy 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 l()l)ulos in much the same manner as the blood capillaries do around the air-sacs. 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 DEVF,I,OPMEXT OF THE LYMPHATICS OF THE LUXOS IX THE EMBRYO PIG. bution to the venous trunks. The fact that so careful an observer as Miller does not find these lymphatics in the sepia suggests the possibility that the assumption of mature activities in some way brings about the atrophy of all of the interlobular lymphatics except those that accompany the veins. Again, this plexus may be peculiar to the pig. It seems necessarj' that this ciuestion must remain unsettled until studied b}' some method other than simi)le injection. The ([uestion of the drainage of the lung lym])hatics is of exceptional interest and importance; and while we must dei)end, for the final settlement, upon phj'sio- 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 i)oint towards the hilum. This is assumed to be ^•ery goocl evidence that the flow is in that direction. No valves have been described in the Ij'mph-vessels which accompany the smaller bronchi, veins, and arteries. Hence it can not be stated whether the lymi)h flow, in the lymph-vessels of the veins, is towards the ])leura 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, h-'ome of these drain as do those above — towards the median and pass down in the ligamentum ))ulmonale — while others i)ass directly down from the ]K)sterior 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 ])ro])er. The lymphatics that jiass 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 u]ion the pathology of the lungs or of the abdomen remains to be settled. SUMMARY. The hniphatics of the lungs are derived from three sources — the right and the left thoracic ducts and the retroperitoneal sac. In embryos 2.G to 3 cm. 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 thoracic duct divides, in embiwos about 2.5 cm. 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 l)ulmonary 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 develojjs from the vessels surrounding the \'eins and passes to the jileura, 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 develoji mor(> 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 uj) of lymi^hatic vessels. From every point of division of the bronchi, lymphatic vessels pass to the lymjjhatics of the veins; those around the terminal bronchus leave it near its ending in the atria, and pass to join the lymphatics of the veins or septa, or, more rarely, those of the pleura. Lymphatics also arise from the retroperitoneal sac and grow u]i posterior to the diaphragm to enter the lower \)o\e 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 develoi) 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 perijihery 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 fi-om the early vessels following the veins. The lymphatics accompanying the veins remain connected with those of the bronchi and septa. The common plexus surrf)unding the artery and bronchus is separated into two individual plexuses, incitlent to the increase in size of the arteiy; however, these continue to have anastomosing branches. The vessels of the pleura mark out the early connective-tissue septa, but later there develoi:)s 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 cm. and, in general, point away from the pleura. None, however, have been found in the smaller vessels which accompanj' the terminal bronchi. In the adult there 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 lironchi 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 \hv 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. These vessels pass through the ligament of the lower lobe and behind the diaphragm. BIBLIOGRAPHY. Baetjer, Wai.tkk a. ; On tlic oriKiii of the mesenteric sac and thoracic duct in the embrj'o pig. Anier. Jour. Anat.. I'hihidelphia, 1908, vili. Clahk, a. H.: On the fate of tlie jugnlar lymph sacs, and the development of the lymph channels in the neck of the pig. Amer. Jour. Anat., Philadelphia. 1912, IX. Clark, E. R.: Observations on living growing lymphatics in the tail of the frog larva. Anat. Record, Phila- delphia, lOO'.l, III. . An examination of the methods used in the 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. .\nat., Philadelphia. 1912, xiii. Councilman-, W. T.: The loliule of the lung and its relations to the lymphatics, Journ. Boston Soc. Med. Science, Boston, 1900, IV. Cruiksh.ink, '\\'.: The anatomy of the absorbing vessels of the human body. London, 1790. Delemere, G., P. PoiRiER, and B. CrMEo: The lymphatics. 1904. FLl>fT, J. M.: The development of the lungs. Amer. .lour. Anat., Philadelphia, 1900, vi. Heuer, G. J.: The development of the lymphatics in the small intestine of the pig. Amer. Jour. Anat., Philadelphia, 1909, IX. K.\.\ip.\ieier, O. v.: The development of the thoracic duct in the pig. Amer. Jour. Anat., Philadelphia, 1912, XII. Klein, E.: Anat. of the lymphatic system. London, 1875, ii. Lewis, F. T.: The development of the lymphatic system in rabbits. Amer. Jour, of Anat., Philadelphia, 1906. V. McClure, C. F. W.: The development of the thoracic duct and right lymphatic ducts in the domestic cat. Anat. Anz., Jena, 1908, xxxii. Mascagni, Paul.: Vasorum lymphaticorum corporis humani historia et ichnographia. Senis 17S7. MiLLEH, W. 8.: The slnicturi- of the lungs. Jour. Moijjh., 1893, VIII. . The lymphatics of the lungs. Anat. .\iiz., Jena, 1896, XII. . Das Lungenliippehen, seine, Bliit- und Lymph- gefjisse. Archiv. fiir Anat. und Physiologic, 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. C'ompt. Rend., 1860, xxx. Poirier anil Charpey: Treatise of human anatomy. Sauin, F. R.: On the development of the superficial lym- phatics in the skin of the pig. .\mer. Jour. Anat., Baltimore, 1904, iii. . The lymphatic system in human embryo, with a consideration of the morphology of the .system as a whole. Amer. Jour. Anat., Philadelphia, 1909, i.x. . Der Ursprung und die Entwickelung des Lymi)hgc- fiissystems. Ergc'bnisse der Anatomie und Ent- wickelungsgeschichte, Wiesbaden, 19i;i, xxi. . The oiigin and development of the lymphatic sys- tem. The .Johns Hopkins Hospital Reports, Mono- graphs, new .series, 191.'i, v. Sappey, p. C: Anatomic, physiologic, pathologic des vaisseaux lymphatiqvies. Paris, 1874. SiKORSKi, J.: Ueber die Lymphgefiisse der Lungen. Cen- tralbl. f. Mcdicin. Wis.sensch., 1870. Teichivian, L. : L^eber Lungenlyn'phgefasse. Anz. d. Akad. d. Wissensch., in Krak.au, 1896. VON WiTTiCH, W.: Ueber die Beziehungen der Lungenal- veolen zum Lymphsystem. Mitth. Anz. d. Konigsberger Phys. Laborat., 1878. Wywodzoff, — . : Die Lymphgewege der Lunge. Wiener Medicin. Jahrbucher, 1866, XI. EXPLANATION OF PLATES. Plate 1. Fig. 1 . Diagram of transverse section of left linig of an embryo pig 3 cm. long, in which the l)l()()d-ve.ssels were injected through the umbilical artery with india ink. The lymphatics appear as dilated spaces (blue). The sec- tion is 20/i thick and is stained with hematoxylin and eosin, aurantia, and orange G. X55. Ao, aorta; T, trachea. Fig. 2. Diagram of section through loljule of lung of an embryo pig 7 cm. long, in whi<-h the lymphatics were injected with india ink through the left tracheal plexus. The veins were slightly injected by the rupture of a lymphatic ves.sel into a vein near the hilum. The section is lOO/j thick and is unstained. X47.5. A, artery; B, bronchus, V , vein; I'l, pleura. Fig. 3. Surface of lung of an embryo pig 0 cm. 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 200^ thick and is unstained. X29.4. P L, primary lobule. Fig. 4. 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 \ eins contain some blood pigment. The section is 400m thick and is vmstained. X33. 1', vein; B, bronchus. Fig. .5. Diagram of left tracheal plexus in an embryo pig 6 em. long, in which the lym])hatics were injected througli the thoracic duct. Cleared by Spalteholz method. Note that i)art of the vessel marked with an asterisk (*) has been removed in dis.secting the body-wall away. This ves.sel is the one described as the first to the lung. X15. *, first vessel to lung; T, trachea; Th D, thoracic duct; L T P., left tracheal plexus; Ao, aorta. Plate 2. Fig. 1. Dissection of an embryo pig 4 cm. 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. Fig. 2. 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 20 >i thick and is stained with hematoxylin and eosin, aurantia, and orange G. X93. B, bronchus. Fig. 3. Dissection of an embryo pig 4 cm. 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 ma.ss because the meshes are so close that they could not be analyzed in the drawing. X15. Th I), thoracic duct; R Th D, right thoracic duct; Ao, aorta; L T P, left tracheal plexus; B, bronchus. Fig. 4. Longitudinal section of upper lobe of right lung of an embryo pig 6 cm. 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 400 fi thick and is unstained. Cleared by the Spalteholz method. X3f). Plate 3. Fig. L Small block of an embryo pig 15 cm. long, in which the lymphatics were injected with prussian blue by ])unc- ture of an interlobular septum. The arteries were injected with india ink through the inilmonary 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 jarreil out of posit ion while being drawn and hence the group of lymphatics in the septum is .shown bent to one side. X40. PI, pleura; A, artery; / L .S', interlobular septum. Fig. 2. 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 SpaUeholz method. X30. Th D, thoracic duct; R Th D, right thoracic duct; V C S, vena cava superior; Ao, aorta; /' A, pulmonary artery; C L, car- diac branch of the right thoracic duct. Fig. 3. Diagram of a section of the right lung of an embryo pig 6 cm. long. This is the same specimen from whicli figure 4, plate 2, was made; the part of the section shown in that figure is indicated by an X. X20. 67 68 DEVELOPMENT OF THE LYMPHATICS OF THE LUNGS IN THE EMBRYO PIG. Plate 4. Fig. 1. Longitudinal section of the left lunj; of :in embryo pig 5 oin. long, in wliicli tlio lynipliatics were injected with pru-ssian blue throuKh the retroperitoneal sac. The veins have some blood pigment in them. The section is 4(X)m tliick ami is un.stained. X2'2. V, vein. Fig. 2. Dissection of an embryo |>ig 4 cm. loii};. in which the lynipliatics were injected with prussian blue from tlic 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; I), diaphragm; H I' S, retro- peritoneal sac; *, first vessel to the lung. Plate 5. Fig. 1. Surface of lung of an embryo pig 23 cm. long, in wliich the lymphatics were injected with prussian blue, by puncture of an interlobular septum. Cleared by Spalteholz method. The interlobular septum is indi- cated by a very large lymphatic trunk. X28 / L S, interlobular septum. Fig. 2. Longitudinal section of the upper portion of the lower lobe of the left lung of an embryo pig o cm. long, in which the lyiM])hatics were injected with prussian blue through the retroperitoneal sac, and the veins have retained a little blood iiigment. The section is 400^ tliick and is unstained. X57. 1', vein; A, artery; /■'/, pleura; B, brondius. Fig. 3. Lower portion of left lung of an embryo pig 5 cm. long, in whidi the lymphatics were injected with india ink through the retroperitoneal sac. Cleared by the Spalteholz method and mounted in balsam. X28. CUNNINGHAM Fig. 5 (C. R. 6^) Fig. 3 (C. R. 652) Thra Shar/ir full Cjmthilt jirl dmfany CUNNINGHAM R. Th.D. -LT.P. Th.D. Fig. 2 (C. R. 7™) Fig. 4 (C. K. 6™) /. f. Diduuh /tcil Camphtil A> l Ctmpanj CUNNINGHAM F. Th. D. Fig. 1 (C. R. ISan) Fig. 2 (C. R. 3™) -s. ^^■"t'|,:^yi ^'SS^L \ Fig. 3 (C. K.6?n!) /. f. Dlduiih fiM Camp' lU An Umruny CUNNINGHAM — 'Ei^^^CS s ^' CUNNINGHAM PLATE 6 Fig. 2 (.C. K. ■/■--'i'; F.g. .^ (^ . K.o--) /. f. DiJuitli /nil Campl,fll jtit Ccmfanj CONTRIBUTIONS TO EMBRYOLOGY, No. 13. BINUCLEATE CELLS IN TISSUE CULTURES. By Charles C. Macklin. Four plates, containing seventy figures. 69 CONTENTS. PAGE. Introduction 71 Method: (a) Preparations of the cultures: (1) Tissue and media 71 (2) Cultivation 72 (6) Observations: (1) Livinp;: Continuous observation 73 Vital standing 74 (2) Fixed 75 The binucleate cell 76 (d) Incidence 76 (6) Morphology 77 The binucleate cell — Conlinunl. page. (c) Origin: (1) Theoretical 80 (2} Observations: Living 82 Fixed 83 (3) Mechanism SC (rcentage of growths was obtained. Hence this addition to the media was generally made. Ordinary bouillon had a similar activating effect. The embryonic extract was ))repare(l as follows: After the embryo had been removed from the egg, under sterile conditions, and with as little contamination with yolk as possible, it was placed in a Petri dish containing 10 c.c. of sterile Locke's media and washed. The tissue to be planted having been dissected out and removed to another dish of media, the remainder of the embryo was cut up and placed in a small, sterile test tube with a little media, and carefully ground up with a glass rod. This mixture was next centrifugalized, and the supernattuit fluid added to the culture, generally in the proportion of equal parts of this fluid and Locke. Too high a proportion of embryonic extract was undesirable on account of its richness in food material, in that it produced a cell overloaded with fat globules, which interfered with observation. The advantages of glycosahne over plasma have been noted by Lewis and Lewis (1912a, p. 10). It is more transparent and practically all of the growth is upon the lower surface of the cover-slip — not scattered throughout the hanging drop, as in the case of the plasma clot. The cells, unim]X'ded by the fibrin network, migrate freely along the cover-slip, ujjon which they spread themselves flat and thin, thus facilitating observation. The ([uantit}^ of fat being much less than in plasma- grown cells, the cj'toplasmic constituents, such as the centrosphere and mitochondria, are much more easily observed and studied. There are, too, the additional ^■erv considerable advantages that the media is more convenient to handle, fixed prepa- rations are more easily made and are not marred by stained fibrin and coagulated albumen, and experimental operations, such as staining with vital dyes, are more satisfactorily carried out. Immediately after planting, the cultures were transferred to a warm box, kej^t at a constant temperature of 39 to 40 degrees bj' means of an electric thermo- regulator. With the microscope inside this box it was not necessary to remove the cultures from their warm environment for purposes of observation, and it was owing to this manifest advantage that the earlier method of observation of the living cultures upon a warm stage outside the incubator was discontinued. In addition, the warm-stage method of heating, from one side only, was found to be inferior to that of the warm box, in which the culture was completelj^ surrounded by an environment of uniform temperature. Illumination was fiu-nished by daA'light, Tungsten globe, or Welsbach burner. A raj' filter, consisting of a glass vessel filled with a solution of copper sulphate or BINUCLEATE CELLS IX TISSUE CULTURES. 73 copper acetate, placed between the source of illumination and the condenser, when artificial light was used, was found to be an advantage (Kite 19136, p. 149). In studying the grosser changes, such as the variation in shape of the nucleus, the 4 mm. Leitz apochromatic objective was used; for the finer details the Leitz ^\y oil- immersion objective was found to be satisfactory. Oculars were Leitz Compen- sating Nos. 4 and 6. For observation of the living cells the cultures of the second day were generalh' the most favorable; the growth was then usually abundant and the cells in a healthy condition, with a fair proportion of mitoses. It is quite evident that continuous observation of the living cell, provided it can be carried out successfully, is the ideal method of studying the sequence of changes occurring therein. Indeed, for the study of amitosis it has been regarded as indispensable, as witness the statement of Richards (1911, p. 125): "For ami- tosis there is but one absolutely certain criterion, the observation of living material and subsequent study of material fixed under observation;" he adds, "this is, of course, impossible in most cases." The method has already' been used in the study of living multinucleate cells of tissue cultures, Lambert (1912a) having attempted to settle the question of the origin of giant cells growing from explanted tissue by its aid, and the character of the results attained through its use was sufficiently encouraging to warrant its application to the problem in hand, though not altogether satisfactory in view of the obvious difficulties. It was hojied, too, that these difficulties would be mini- mized by the use of glycosaline media, which produced films of tissue sufficiently thin for study in the living condition. It was first planned to ascertain the full history of the binucleate cell by selecting a cell with a single nucleus and observing it continuousl}' on the stage in the warm box till either the nucleus divided and formed a double-nucleated cell or the cytoplasm became merged with that of another mononucleate cell to form a single cell containing two separate nuclei. Observations upon this binucleate cell were then to be continued until the ultimate fate of the double nucleus was disclosed. Drawings were to be made from time to time with the camera lucida. This ideal was found to be impossible of realization, on account of the technical difficulties. Cultures under continuous observation, exposed, as they were, to strong light, often showed evidences of degeneration; even daylight seemed to cause this and the use of ray filters did not altogether eliminate it. Degeneration was noticed at times even when the plan was followed of making short observations and immediately turning the light off, the culture remaining continuously on the stage. Living cells show a marked tendency to migrate; hence the cell under observa- tion had to be closely watched to j)revent its escape from the field of vision. Other cells often wandered over the cell under inspection, and so interfered with the work. Added to these difficulties is the length of time involved in the process, which necessitates many hours — even days — of continuous observation. Then, too, the minuter cell changes are very difficult to follow, even for short ]ioriods, the only 74 niNUCLEATE CELLS IN TLSSUE CULTURES. optical i)icture presented being very delicate shades of difference in refractivity. The obscure and peculiar optical properties of living matter, as Kite (1913, p. 148) points out, give rise to an important source of error. The ideal procedure having to be abandoned, the alternative practice was adopted of following shorter periods of change and piecing tiu^ records of these together. A start was made with the formation of the double nucleus, and here another difficulty was encount(>red; it was manifestly impossible to tell which of the thousands of nuclei in the culture was about to divide, and by selecting nuclei at random, months might be spent without getting one which ultimately divided. It was thus necessary to select a nucleus which gave some indication of being on the way to di\ision, i. c, by elongation, or equatorial constriction; such a cell was observed continuously until it divided or became degenerate. The subsequent history was studied by selecting a double-nucleated cell and observing it con- tinuously. In the observations the shape of the nucleus was particularly noted, and with this was considered the behavior of the centrosphere, mitochondria, fat glolniles, nucleoli, shape of the cell generally, and whether or not the cell itself ultimately divided following nuclear division. Cells on or near the outer border of the new growth were found most favorable, since they were larger and more free from sur- rounding cells. Thej^ appeared to be usually quite healthy during the first 48 hours at least. As has here been noted, the morphology of the cell is difficult to make out in the living and unstained condition, and it was thought that insjjection would be much easier and more accurate if the details could be rendered visible by the use of stains which would not impair the vitality of the cell. Churchman and Russell (1914) and Russell (1914) have recorded satisfactory results with gentian violet in staining embryonic and adult tissues of the frog growing in vitro. They stated that endothelium from adult frog pericardium in frog's plasma to which gentian violet had been added grew definitely when the strength of gentian violet was 1 in 2,000, and actively in a dilution of 1 in 20,000. Furthermore, their records show that they were able to follow cell division in their stained preparations. Clear karyokinctic figures were not seen in growing adult frog tissue, but in embryonic frog tissue these figures were found in the dividing stained nuclei. They beheve that the nucleus is stained intravitally and that growth continues in the i^resence of the dye. Toxic action was shown when this stain was used upon Paramecium, even in dilutions of 1 in 1,000,000. They believe that "the use of stains in the plasma in which tissue is grown will ])robably facilitate the study of nuclear growth." My results with gentian violet in chick tissue growing in Locke do not bear out those of Churchman and Russell, for the staining could not be considered as in any sense intra vitam, under the conditions existing in my experiments. A solution of Griibler's gentian violet was made uji in a strength of 1 in 100,000 with slighth' diluted saUne. Without removing the culture from the warm box, the cover-slip was lifted off and a small drop of the stain (warmed to the same tem- BINUCLEATE CELLS IN TISSUE CULTURES. 75 perature as the culture and of about the size of the hanging drop) was added to the latter, the excess fluid being withdrawn. The dilution of the stain was thus approxi- mately 1 in 200,000. The culture was immediatel}' examined under the microscope. The dye rapidly diffused through the cytoplasm into the nucleus, the nucleoplasm taking on a finely granular appearance; this latter was, apparently, the result of the coagulative action of the dye. The nucleoli were distinctly marked out, and stained much more darkly than the nucleoplasm or cytoplasm. The nuclear membrane, too, was sharply outlined as a dark violet ring. This staining was very valuable in delineating indistinct nuclear boundaries, since these, in the living unstained culture, are often obscure. Irregularities in outline, such as indentations, were rendered very plain, and the method was of assistance in studying the relationship to one another of double nuclei. The cytoplasm, after this treatment, consisted of coarse violet granules in a very faintly stained matrix, showing at times a slightly fibrous structure. Cell borders were well marked, especially the pseudopodia, which, however, lost their power of movement upon being stained. Intercellular bridges could be studied. Mitochondria were not specifically stained, and degenerated in a short time. In a culture so stained evidence of life, such as pseudopodial and mitochondrial move- ment, cell migration, and mitosis, ceased almost at once, and in a few minutes vacuoles formed in the cells and the entire culture became degenerate. Though gentian violet staining is of great assistance in obtaining a conception of the morphology of the cells rapidly, under the conditions of the experiments its toxic action precludes the possibility of the stained cell undergoing vital changes. Owing to its coagulative action the appearance of the living protoplasm is not accuratelj' reproduced in the stained preparation. In spite, however, of these disadvantages, the use of gentian violet enables one to inspect portions of the cells which, in the living condition, are almost, or quite, invisible, and also to examine more accurately and easily some of the visible parts. M. R. and W. H. Lewis (1914 and 1915, p. 376) used janus green as a vital stain in tissue cultures growing in Locke solution, and found that, although the mitochon- dria were specifically stained, the dye was toxic in as low dilutions as 1 in 200,000, and caused speedy death of the cells, as well as distortion of the mitochondria. Janus green (Hoechst), di-ethyl saffranin azo di-methj'lanilin, in Locke's solu- tion, in a strength of 1 in 40,000, was applied to living cultures in the same manner as the gentian violet, and was found to stain the mitochondria specifically in about 5 minutes, but no movements of these bodies could then be noted, and the threads broke up into a row of granules. The cells soon died, as evidenced b}' their vacuo- lated and degenerated appearance. While janus green staining provided a rapid and convenient method of observ- ing mitochondria, its toxic action rendered it valueless as a means of studying vital changes; moreover, the stained mitochondria soon lost their normal optical char- acters, thus prohibiting extended observation. Some of these living cultures were fixed and stained. Osmic-acid vapor was used for fixation, and Heidenhain's iron hematoxylin was found to give the best staining. 76 BINUCLEATE CELLS IN TISSUE CULTURES. The culture, growing in the hanging th-op, was removed froin the vaseUned slide and exposed to the fumes from a 2 per cent aqueous sohition of the tetroxid of osmium. Thi.s may be done b\' placing the cover-slip, drop down, over the mouth of a bottle containing the fixative (the vaseline adhering to the cover-slip and pre- venting the escai)e of the vapor) or, as suggested by ]\I. R. Lewis, by floating the cover-slip, drop up, upon the osmic solution. Fixation is complete in 5 or 6 minutes, and the preparation is then dark brown or black. It is now rinsed off with distilled water, and pai^sed raj)idly tlirough ethyl alcohol solutions of 35, 50, and 70 per cent. To the latter a few drops of hj'drogen jjeroxid are added, which bleaches the prepa- ration. It is then passed back rapidly through the same alcohols, rinsed in distilled water, and washed in running tap-water for 5 minutes. Too long immersion in alcohol will cause the mitochondria to become dissolved out. The cover-slij), cultiu'e downward, is now floated upon a solution of 4 per cent iron alum and allowed to remain for 12 to 24 hours; next it is washed in running tap- water for 5 minutes and then immersed in 0.5 per cent aqueous hematoxylin for 24 to 48 hours, after which it is washed in running tap-water for 1 minute, differ- entiated in 2 i)er cent iron alum and again washed in tap-water for 10 minutes, dehydrated through the alcohol series, cleared in xylol, and mounted in balsam. The hematoxyhn solution is prepared as follows: Hematoxjdin (10 per cent in absolute alcohol), 0.5 c.c; distilled water, 10.0 c.c. These fixed i)rei)arations were used to make clear the morphology of the living cells, especially such details as nuclear membranes, nucleoli, mitochondria, and cen- trosomes. An attenijH was made to inck out the successive stages in the process of direct division of the nucleus for comparison with the observations upon Uving material, and thus to build up a series exhibiting the various changes. The phases of mitosis were also studied, and drawings were made of interesting cells. For statistical purposes cell counts were made of some of these preparations by jjlacing a glass disk, upon which squares had been ruled, in the ocular, and using the mechanical stage. THE BINUCLEATE CELL. INCIDENCE. The frequency of occurrence of binucleate cells varies within wide limits in cultures from different tissues. They were foimd to be most numerous in mem- branes growing from the heart, and were not uncommon in cells of the connective- tissue type from this and other tissues, but in the endodermal membranes from stomach and intestine they were exceedingh^ rare. They may be even altogether absent from the new growth. Lewis (1915, p. 156) notes that in growths from the leg of chicks no amitotic forms were noted. To get an idea of the relative number of these cells as compared with the total number of cells in the new growth, careful counts were made of 20 fixed cultures from chick heart. Imperfect cells and those situated so close to the original piece as to be indefinitelj' outlined were omitted. In these 20 preparations there was a total of 41,725 cells, of which 375 were binucleate, or an average of 1 binucleate to BINUCLEATE CELLS IN TISSUE CULTURES. 77 each 111 cells; thus the binucleate cells made up 0.9 per cent of the total cells appearing in the new growth. Even in different preparations from the same tissue binucleate forms were found with varying frequency. Among the 20 cultures of heart mentioned above, one preparation showed 1 double nucleus to each 28 cells, while in another the ratio was 1 to 1,180. Age of tissue, too, in these 20 heart preparations, had a bearing upon the inci- dence of binucleate forms, new growths from the younger hearts showing a some- what greater proportion of double nuclei than those from older cardiac tissue. In hearts from chicks of 5 days' incubation there was, on the average, one binucleate to each 105 cells; in 7-day hearts the ratio was 1 to 123, and in 8-day hearts it was 1 to 233. Finally, duration of growth seemed to be related to the relative frequency of occurrence of these cells. In the same 20 j^rej^arations it was found that cultures of the first 24 hours showed one double nucleus to each 183 cells; in cultures of the second 24 hours the ratio was 1 to 86 cells. This seems to point to a considerable amount of nuclear splitting in the second 24 hours, of which some at least probably occurred within the new growth. Cultures of older duration were, in the slides counted, not sufficiently numerous and tjqiical to base accurate conclusions upon. MORPHOLOGY. The average binucleate cell (figs, la, 7, and 9) is somewhat larger than the average mononucleate, the area occupied by the nucleus being approximately twice as great. Each nuclear part is, in size, shape, and general apiicarancc, very similar to the nucleus of the mononucleate cell. The nuclear parts are often pressed close together (figs, la, 60) and their adjacent surfaces are consequently flattened, the intranuclear pressure in each being evidently equal. When thus related, the appear- ance of the double nucleus in the living preparation (and indeed in some of the fixed preparations) simulates a single nucleus which looks as though it were separated by an equatorial membrane. Such an appearance has been interpreted as a nuclear plate, or intranuclear membrane, and so described by Child (1904, 19075, and 1911, p. 283), and others; but for reasons which will appear later on, I believe that such appearances in tissue cultures are due to the apposition of nuclear surfaces, as above described. In an elongated nucleus which has become bent upon itself the folded free edge of nuclear membrane, projecting into the karyoplasm, may simulate a partition which seems to be growing across the nucleus from one side to the other. A nuclear configuration of this character is presented in Child's (1911) figure 16, page 293, and in other of his figures. It is not to be wondered at that the approximated areas of nuclear wall at the folded edge are somewhat attenuated and appear thin, as Child (1911) has observed (p. 283). Such reduplications of nuclear membrane are not to be looked upon as intranuclear membranes which cleave the nucleus ])y growing across its equator. I have seen no evidence of a type of amitosis of this kind. Sometimes an equatorial membrane is simulated by an elongated nucleolus lying across the nucleus. Again, as Richards (1911, p. 124) suggests: "A strand of 78 BINUCLEATE CELLS IN TISSITE CULTirRES. linin stretched across a luiclcus with chromatin granules upon it often gives the ai^pearance of a membrane dividingthe nucleus aniitotically(endogenous division?)." Ho also states that he has found no evidence of the "endogenous" division of Child (1907a, p. 95); nor have I seen anything of this kind in tissue cultures. Optical appearances similar to Child's (1911) figure 6 have been seen in living cells and interprct(Hl as indentations and infoldings of the nuclear membrane. All these conditions can be made clear by the use of a dye like gentian violet upon the living culture, or by proper fixation and staining. In no case has a bona Jidr intranuclear membranous partition been found in any kind of i)rei)aration. I may also state here that my ()l)servations upon fixed and stained cells in tissue cultures have not disclosed cases where one nuclear half was more darkly stain(>(l than the other, such as those mentioned by Child (1904, p. 549; 190(5, ]). 595; 1907c, p. 171 ; and other i)laces) and which he believes to indicate "a certain degree of physiological independence before separation of the parts." In the living condi- tion, too, the nuclear jiortions present no evident difference in cytoplasm. The contents of the nuclear ])arts are in every way similar to those of the single nuclei. The nucleoplasm ;i])i)ears homogeneous during life and when fixed with osmic-acid vapor is finely granular. This method of fixation {)reserves most accurately the details of the living cell (Lewis and Lewis, 1915). There is usually at least one nucleolus or karyosome in each nuclear portion, and more often two (figs. 7 and 9) or even more. The nucleoli of the connective- tissue type of cell are irregular in shape, often elongated, and vary greatly in size (fig. 8). In the living cell they are highly refractive. They continuously undergo changes in shape, size, and number during the life of the cell (figs. 24 to 35, and plate iv), as can be seen by watching the living nucleus. It is then apparent that their outline is "ragged," as Lewis and Lewis (1915) describe it. The bodies even appear to break up from time to time, and afterward to recombine (figs. 24 to 35). At times the nucleolus comes to lie ver.y close to the nuclear membrane (fig. 29) and it may even appear to be attached to it. These bodies take the gentian violet dye very well and stain darkly with hematoxylin. If overdifTerentiated with iron alum the nucleolus appears as an agglomeration of small granules of about equal size (fig. 10); it is jirobably to be regarded as a gel of varying density, the densest portions being represented by these darkly staining granules. During mitosis the nucleolus disappears with the formation of the s]nreme, and the daughter nucleoli reappear in the reorganizing daughter miclei. The nuclear l)ortions may be separated by an interval (fig. 9), or simply touching one another (fig. 7), or may be pressed so close together that their adjacent surfaces are flattened, similar to the condition in the early cleavages of Moniezia, as mentioned by Harman (1913, p. 221). They tend to remain close to one another, and do not migrate far apart, as nuclei in a .syncytium. When separated, the nuclear portions show mito- chondria betw'cen them (fig. 9) and usually the centrosphere is situated either in the interval between the nuclear portions, or opposite this interval, as in figures 7 and 59. In the living condition the centrosphere or "central body" of Lewis and Lewis (1915) appears as an area of .slightly greater refractivity situated at one side of the nucleus in mononucleate cells; this side is frequently concave, with the centro- BINUCLEATE CELLS IN TISSUE CULTURES. 79 sphere situated in the concavitj' (fig. 24c). This concave side then appears indis- tinctly marked out in the living culture, the close proximity of the centrosphere and mitochondria obscuring the nuclear outline. Its relation to the parts of the double nucleus has been noted. I have not observed the centrosome (centriole) in the living cell, but when stained with iron hematoxylin this body appears usually as two minute dark granules, lying close together (fig. 7). The centi'osphere takes a slightly darker stain than the area surrounding it, and thus appears to be a somewhat more concen- trated area of the protoplasm. From this area mitochondria radiate, as seen in figure 8. In the living condition the centrosphere shows an indefinite, irregular, apparently serrated edge, the toothlike processes of which undergo a curious con- stant, slow, almost imperceptible indrawing and outpushing. The mitochondria seem to be intimatelj^ connected with this body, as observed by Lewis and Lewis (1915, p. 349), but they differ from it in their reaction to janus green and to certain methods of staining in the fixed condition, such as iron hematoxylin. Mitochondria in tissue cultures have been described at length by Lewis and Lewis (1914, 1915). Their curious movement, mentioned by these authors, is plainly evident. The special relation of these bodies to the binucleate cell is their position between the nuclear portions, as in figures 8 and 9, unless, as in figure 7, the parts of the nucleus are too close together to permit of this. The relationship of the mitochondria and adjacent centrosphere to the portions of the double nucleus is similar to that of the Netzapparat of Deineka (1912, figs. 2 and 12) under similar conditions. Fat, though not so abundant as in plasma-grown cultures, nevertheless occurs as fine globules which tend to crowd together at the nuclear poles (fig. 32) and often become arranged in rows between the mitochondria. The other details of the binucleate cell are very similar to those found in the mononucleate. Occasionally cells are found which contain three or more distinct nuclei (fig. 16) and the evidence seems to indicate that the binucleate cell is the first stage in the formation of the giant cell; this stage, however, is seldom passed, for giant cells are comparatively rare. Such multinucleate cells are quite different from the foreign- body giant cells of Lambert (1912 o and b), which have been shown b}^ him to arise by fusion of i^reviously separate wandering cells. Binucleate cells, and the intermediate stages leading up to them, have long been known in embryonic tissue. Child (1907c) shows several such from chick embryos in his figure 12. IVIaximow (1908) describes and figures double nucleated cells, similar to those found in tissue cultures, in mesenchj-me of embryo rabbits of 11^ to 13§ days, and he has found araitosis also in the guinea pig in the same region and stage. Patterson (1908) shows illustrations of cells of the same type in develop- ing pigeon's eggs, and such cells have been described by many others. Thus it is certain that, since the paired nucleus occurring in the tissue-culture cell is similar to that found in the cells of embryonic tissue, it can not be considered as an abnor- mality due to its artificial mode of life. 80 BINUCLEATE CELLS IN TISSUE CULTURES. Harrison (1913, p. 67) has shown that the behavior of cells growing in culture media is comparable to that of cells growing in the embryonic body, and it is reasonalile to assume that the beliavior of these binueleate cells in tissue cultures a])pr()ximates the beliavior of similar cells in the developing embryo. Hence the vital i^henomena manifested liy such binueleate cells in tissue cultures afford n^liable evidence as to the changes which take place in similar cells livhig under normal conditions in the corresponding embryo. ORIGIN. If we inquire as to the origin of these binueleate cells of (lie new growth we are confronted with four possibiliti(>s, viz: (a) Migi'idioii as a binueleate coll from the oxplanted tissue. (b) Fusion of the cytoi)lasni of two previously separate cells without fusion of the nuclei. (c) Division of the nucleus by mitosis without division of the cytoplasm. (d) Division of the nucleus hy amitosis witliout division of the cytoplasm. This list does not include the theoretical origin of nuclei de novo from the cyto- plasm, or their development from extruded chromidial substance (Young, 1913). These hypotheses do not app(>ar to have been substantiated, and no evidence in favor of either appears in tissue-culture jM-eparations. First, considering (a), we fintl that twin nuclei occur in the area of new growth immediately surrounding the original tissue, and such forms are well known in embryonic tissue. Thus it is i)rol)able that many of the binueleate cells in the new growth have migrated as such from the explanted tissue. The great increase in l)r()])ortion of double-nucleated cells in the second 24 hoiu's, however, as has been noted, suggests that not all of these cells are of migratory origin, but that some have probably arisen in the new growth itself. This view is borne out by observa- tions upon the living cell, as will be shown, where a single nucleus has been seen to become divided directly- into two parts, and also by the finding of nuclei in the act of direct division in the fixed preparations. The binueleate cells which have migrated as such from the original piece have probably originated therein in the same manner as those arising in the new growth. Giant cells can hardly be considered to have migrated as such from the original piece, for in the zone immediately surrounding the latter they are not found. Regarding {!>) , it may be said that no ajipearances which could be interpreted as transitional forms have been found in fixed and stained preparations or in cultures vitally stained, neither has the process been observed in the living culture. I therefore regard it as an im]irobable hypothesis. This could hardly be considered as an explanation of the formation of giant cells, for that would postulate the fusion of a multitude of previously separate cells, of which there is no evidence in the material examined. It may be noted that Lambert (1912?>), who brought about the formation of giant cells by fusion of monomiclear cells in cultures from chick spleen, failed to get such cells in cultures from chick heart. Furthermore, Lambert (1912a) recognized three other types of giant cells in ti.ssue cultures, besides this. 7^ BINUCLEATE CELLS IX TISSUE CULTURES. 81 Considering next (c), we find that this also is improbable. It is easy to observe tiie process of mitosis in vitro, and to follow the various changes. IMany such cases have been observed, and in none has there been seen a failure of the cytoplasm to divide following separation of the chromosomes. This process of cytoplasmic con- striction is well shown in figures 68, 69, and 70, and in the living culture it is very evident and easy to watch. In no case has it been observed, in following these cells dividing by karyokinesis, that a binucleate cell was formed; always the end result was two distinct daughter cells, often widely separated, connected by a thin strand of protoplasm (fig. 1, /). If crowding of the cells occurs, separation of the daughter cells may be interfered with to some extent, but it is doubtful if this interference ever is so serious as to prevent cytoplasmic fission altogether and thus result in the forma- tion of a single cell containing two nuclei. At least no evidence has been found from observation of the cells of tissue cultures that this is ever the case. Upon this point my observations agree with those of Child (1911, p. 283). He says: "In Moniezia nuclei which arise by mitosis are separated by an appreciable dis- tance when they form." Again (p. 292), in describing a "double" nucleus, repre- sented in his figure 1 1 , he says : "The two parts of the nucleus . . . are in immediate contact and flattened against each other. It is difficult to understand how they could attain such a position as the result of mitotic cleavage, like that of the earlier stages." It must be said that my observations upon living cells have principally been made with cells of the connective-tissue type. In the case of membranes, however, there is always a well-marked dividing line between the cells, which is made evident by staining with iron hematoxylin or the use of silver; also this potential isolation of the cells is made apparent by the fact, when cells do separate, that the cleavage is along this line of partition, as is shown from the study of fixed preparations (Lewis and Lewis, 1912c, figs. 14, 13, and 12). No such partition is ever found between the nuclear parts of binucleate cells. In fixed preparations of connective-tissue cells there is no indication of an\' failure of the cytoplasm to divide in the later stages of mitosis; that is to saj'-, we find no telophases where separation of the cytoplasm is not evident (fig. 17). Again, these double nuclei almost always have only a single centrosj^here (fig. 7), whereas nuclei arising by karyokinesis have each a centrosi^here. This finding as to the centrosphere agrees entirely with that of Deineka (1912) for the Netzapparat in the dividing epithelial cells of Descemet's membrane and connective-tissue cells of the cornea. This author is of the opinion that the Netzapparat surrounds the centrosome, and its changes ai)i)ear to follow the variations of the latter body. In binucleate cells of these tissues, in which the nucleus divides by amitosis, the Netz- apparat remains single, whereas if the nuclear division takes place by mitosis each of the daughter nuclei obtains a separate Netzapparat. By reference to this dis- position of the Netzapparat, Deineka is even able to tell the mann(>r of origin of such double nucleus, whether by amitosis or mitosis in which cleavage of the cytoplasm has been delayed. I liave never observed this cell organ in living tissue-cultures. The fact that the centrosphere in the binucleate cell is single seems to indicate that the twin nucleus is single so far as its reproductive capacity is concerned. This infcronre is borno out bv olisorvations. later to be referred to. 82 JUNUCLICATE CKLL.S IN TIS.SUK CULTURES. Considering finally (d), it seems probable that these twin nuclei arise through direct equal l)inary fission of the nucleus without division of the cyt()i)lasm. The evidence upon which this assumption rests is, first, the inadequacy of other explana- tory hypotheses; and, second, the observation in living cells of a process which is apparently direct nuclear division, and the occurrence in fixed preparations of tissue cultures of what must be regarded as transitional forms between single and d()ubl(> nuclei. It is true, as Harman (1913) remarks (p. 219), that "the fact that two nuclei lie in contact is no evidence that they have arisen by amitotic division," and in the material which she studied, viz, early cleavage stages of Tcenia teniceformis and Moniezia, she imd()ul)tedly presents convincing evidence that nucl(M which have arisen by mitosis may lie quite close to one another within the same cell. This, however, is a case of delayed cleavage, for she states (p. 215) : "In cleavage, nuclear division takes place very much in advance of cytoplasmic division. In the early divisions it is the exception and not the rule to find even a constric- tion in the cytoplasm. This gives rise to a syncytial condition. This syncytium persists until very late cleavage." This is quite a different condition from that obtaining in the cells of tissue cultures. Then, too, many of her nuclei contain spiremes. Her contention in no way counts against the view that the double nucl(>i of tissue cultures are of amitotic origin. Observations on direct nuclear fission will now be recorded, first to be described being the process as it was seen to occm- in the living cell. As has been pointed out, it is impossible to tell from inspection of the living culture which of the thousands of mononucleate cells will divide directly, and so to follow the process of nuclear amitosis in the living cell it is necessary to select a cell which shows some indication of beginning direct division, i. e., by elongation and constriction. Figures 24 and 25 appear to be typical of the early stages of direct division of the nucleus. Many attempts to trace the changes in such a cell were made, with, however, onlj' partial success, for in almost every case the nucleus lost its constriction and became rounded again, or the cell degenerated. However, one case was found where what appeared to be direct division of the nucleus occurred during observation. The various phases are shown in the series of figures 24 to 35, which were drawn at 15-minute intervals from a single cell growing in a culture from a 5-day chick heart in Locke solution with extract from chick embryo. The culture was of 57 hours' duration. A cell was first selected which contained an elongated nucleus with a marked notch in one side. In this notch the centrosphere was situated, and conse- quently this side was .somewhat indistinctly outlined (24). Instead of dividing, the cell straightened out, almost losing the indentation (25). It contained two nucleoli, one situated in the uj^permost pole, and the other, which was paired, about the equator. The nucleus next became rounded (26 and 27) and, after one hour's obser- vation, its outUne was almost circular (28) . In the latter figure there appeared tf) be only a single paired nucleolus. lUXUCLEATE CELLS IN TISSUE CULTURES. 83 The nucleus now became elongated and a refractive mass appeared in the lowermost pole — apparently another nucleolus; at the same time the central nucle- olus became a single mass, and was somewhat longer than before (29). Next, a shallow notch formed in one side, and the nucleus became shorter and thicker, its nucleoli undergoing minor changes (30 and 31). At the end of two hours the nucleus again elongated and a deep notch appeared, indistinctly marked out on one side (32). This seemed to become shallower in 33, but the presence of the centrosi)here pre- vented this portion of the nuclear membrane from being well defined. The next change was the formation of another notch on the opposite side, both notches forming what seemed like a zone of constriction about the nucleus. A refractive mass stretched across the equator of the nucleus between these notches (34). This is apparently a strand of mitochondria rather than a nucleolus, for, in the next drawing (35), 2| hours after the observation began, this strand is situated between two apparently separate nuclear portions, the nucleus having divided directly. In no fixed and stained cell has a nucleolus been seen to occupy this posi- tion; on the other hand, mitochondria have frequently been seen between these nuclear parts, as in figure S. There was here no evidence of the formation of either a spireme or an amphiaster, and thus Wilson's (1900) criterion for amitosis was fulfilled. It may also be noted that the centrosphere did not divide and the nuclear membrane remained intact. The final division apparently took place very rapidly, since the actual separation was completed in the 15-minute interval between 34 and 35. This rapidity of the end process of nuclear cleavage accounts for the infrecjuency of such terminal con- stricting forms as figures 6 and 8, and makes the relatively small number of these later transitional forms adequate to account for the number of binucleate cells which originate therefrom. The cell was allowed to remain on the microscope stage all night, but unfortunately wandered away and was lost, so the subsequent changes could not he followed. The drawings were made from direct observation, but not with the aid of the camera lucida. Mitochondria and centrospheres are partially diagrammatic. This process, though traced with difficulty, and though somewhat obscure, seems to follow the classic descrij^tions of amitotic division of the nucleus, viz, elongation with equatorial constriction, forming a somewhat dumb-bell shaped figure, and final se]iaration of the two nuclear portions. A similar elongated nucleus in a connective-tissue cell was followed for 6| hours, and did not divide, but finally degenerated; in the meantime it underwent various changes in shape and was rounded when last observed. Th(^ changes in nucleoli were similar to those in figun^s 24 to 35. Thus it appears that a nucleus in a condition of elongation and constriction may remain undivided for a long time and may even return to the rounded form without dividing at all. In cases, however, where the constriction has passed a critical point, as apparently was the case in the nucleus represented in figure 34, the process of division i)r()ceeds rapidly. The study of fixed preparations, too, throws some light on nuclear amitosis, for ill these one frefiu(>ntly finds nuclei evidently undergoing direct division. Such 84 lilxrCLKATIC CELLS IX TISSUE CULTURES. forms are to be regarded as transitional stages between the mononucleate and binucleate cell. Figure 2 shows a nucleus which has undergone elongation and equatorial constriction, so that there is an indentation on either side. The nucleolus appears to be dividing also; this condition of the nucleolus is, however, not constant. Figure 3 shows a cell in which constriction is somewhat farther advanced; here the nucleoli have apparently divided, two being seen in each nuclear portion, in these cells the method of jireparation does not show cytoplasmic details. Figures 6 and S show nuclei in which direct division is almost com])lete, the nuclear parts being held together only by the fniest filament. Similar nuclear iigures were found by IMaximow (1908) in embryonic rabbit tissue, as shown in his figure 1, and the upper two nuclei in his figure 10. In figure 8 the nucleus has divided unequally, and the larger ]iortion contains two nucleoli, while the smaller h;is but one. In figure 6 only one nuclear portion contains a nucleolus, and this is single. In both cells the unchanged centrosphere is situated in its characteristic position between the two nuclear portions, while the mitochondria radiate out from this bodj^ and a strand of mitochondria passes over the bridge connecting the nuclear parts. There is nothing in the api)earance of these nuclei to suggest the late telojihase of an intranuclear mitosis, such as those shown by Cary (1909) and referred to by Richards (1911, p. 158). The clearness characteristic of the cells of tissue cultures prevents confusion of nuclear amitosis with the late telophase of mitosis, such as has been shown by Richards (1909) to be jjossible in the cells of Tcenia. In figure 9 nuclear sejiaration has been completed, the two portions being ([uite free from one another. These are of about ecjual size and appearance, and each contains two nucleoli. Mitochondria and centrosomes occupy their typical posi- tions; the former are short rods, this being a cell from heart membrane. In figure 7 the separate nuclear parts have come together and their surfaces are just touching. Mitochondria ha\'e been forced out, but the centrosphere is characteristically opposite the area of contact of the nuclear portions. Figure 4 shows a somewhat similar binucleate cell, from a Zenker and Mallor>- preparation. It would appear that the nucleus may sometimes divide by a gradually deep- ening cleavage from one side, which finally cuts it into two pieces. This may be regarded as an asymmetrical type of constriction. Figure 5 may be taken as repre- sentative of the beginning of this process and figure 6 the end. The centrosphere is found typically in the notch, as has many times been recorded in amitotically dividing nuclei, as by Maximow (1908). In the rare exceptions to this rule the centrosphere may have been originally situated in the notch and subseciuently have left it. No evidence of separation of the centriole-pair during nuclear ami- tosis has been found. Richards (1911. p. 156) finds constricted and indented nuclei in his material only in cases of imperfect fixation. \Miatever may l)e said as to the nuclear distor- tion brought about by many fixatives, this is not an explanation of such figures as 6 and 8 seen in tissue cultures, for here osmic-acid vapor was used as a fixative and this does not change the nuclear outline, as may be i)roved by ob.serving a living nucleus and the same nucleus after fixation (Lewis and Lewis, 1915). Then, too, only a small proportion of nuclei appear thus, whereas if the ap})earances were to BINUCLEATE CELLS IX TISSUE CULTURES. 85 be interpreted as due to the fixative they should be abundant. Again, the actual observation of such nuclei in living cells is proof absolute that they are not artifacts. The only type of nuclear fission which I have observed in tissue cultures is that which occurs, apparently, by constriction. An estimate of the frequency of occurrence of such transitional amitotic nuclear forms as those shown in figures 2, 3, 6, etc., was made by making careful counts, the aforementioned series of 20 heart cultures being used. Out of a total of 41,725 cells in this series, 50 cells were found to contain constricted nuclei of such a character as would warrant their being considered as amitotic. This is a proportion of one amitotic nucleus to 835 ordinary nuclei, or 0.1198 per cent. In the same series there were 375 binucleate cells, which are regarded as end products of nuclear amitosis. The proportion of transitional forms to end products is thus 50 : 365, 1 : 7.5 or 13.33 per cent. So high a percentage of transitional forms seems to indi- cate that the nuclei remain a long time in this condition, and the observations upon living cells bear this out. The final stages of direct nuclear fission, as shown in figures 6 and 8, are, as has been noted, rarely found. In this connection it is of interest to compare the incidence of amitotic with that of mitotic nuclei. In the same series there were found to be 170 cells undoubt- edly in mitosis. The ratio of mitotic cells to total cells is thus 170:41,725, or 1 : 245, or 0.4 per cent. There were probably many more mitoses than this, for some are undoubtedly rubbed off in preparation, since their rounded and thickened form exposes them to friction in washing, etc.; also 62 doubtful mitotic forms were not included. It is an easy matter to calculate the relative proi)ortion of amitotic to mitotic forms. As has been stated, the ratio of amitotic nuclei to total cells is 1 : 835, while that of mitotic nuclei to total cells is 1 : 245. It is evident that the mitotic forms are 3.4 times as numerous as the amitotic, even when we leave out the doubtful forms and the cells in mitosis which have been rubbed off. Again, when we con- sider that the amitotic process is a slow one, as has been shown, and that mitosis is relatively rapid (1 j to 2| hours according to Lewis and Lewis, 1915, p. 371), it will be realized that the amitotic method of nuclear division is unimportant, so far as nuclear multiplication is concerned, as compared with mitosis. Thus, examination of living and fixed preparations makes reasonable the view that direct division of the nucleus occurs where this structure is elongated, and sometimes bent upon itself, by a karyoplasmic streaming, away from the nuclear equator, and a gradually deepening constriction which encircles the nucleus more or less symmetrically and cuts it into two parts, the constricted area becoming a narrow tube and finally a thread, which ultimately disappears. The behavior of the nuclear membrane during amitosis in the cells of tissue cultures seems to be essentially the same as that of the same structure in the cells of the trematode described by Cary (1909) during intranuclear mitosis. There is, however, no intranuclear sjiindle in the cells which I have examined. The final separation of the consti'icted nucleus takes only a short time, as has been noted, but a nucleus may remain for a long time apparently about to divide without actuallj' doing so. 86 HIM (I, KA IK CKI.l.S IN TISSUE CULTURKS. In the process of din-ct division of the nuclens varions factors may i)lay a i)art. First, we may refer the different changes in foini of the nucleus to changes in form of the cell as a whole. It is of frequent occurrence that a cell, by reason of the tension exerted by attached cells, or of its own amneiioid movement. In'comes elongated. In conseciuence of this stretching of the cell the nucleus also becomes drawn out, it being simi)ly a sac of fluid, and it is possible that it may become broken into two parts much in the same manner that an oil globule, floating upon water, becomes broken uj) if stn^tched. It may be asi^vnned that there is a streaming of i)rotoplasm away from the ecpiator, with a constriction in this region, which beconw's deejx'r and deejier until the nucleus is divided into two more or less e(iual portions, these now tending to assume a more globular shajje. This view of the cau.se of nuclear amitosis is somewhat similar to that of Maximow (1908), who believes that amitosis in the mesenchyme cells of developing rabbits may be brought about by the stretching of such cells consequent ujion rapid growth of the adjacent liver. The jirocess of direct division of the nucleus as described is strikingly like the division of the cytojilasm of ova which had been replaccnl in normal .sea-water after having been treated with hypertonic sea-water (J. Loeb, 1906, ]). 06, figs. 10, 11, 12, and 13). It appears that here the c(>ll ftrst becomes incut from one side; the ])rotoplasm thereujion streams off in opposite directions, foiniing two globules con- nected by a narrow isthmus. This soon becomes reduced to a mere thread com- jKised of the attentuated cell membrane, which finally disapjiears, so that there remain two sacs of protoplasm, (juite without connection one with another. The physical changes involved in this process seem to be very much like those seen in direct division of the nucleus. Loeb's figun^s ar(> very similar to those illu.strating nuclear amitosis. That the size of the nucleus is not a material factor in this i)roce.ss is seen by the variation in size of the twin nuclei, some of which are quite small. Although a twin nucleus is frequently found in a cell which is not elongated, it may be assumed that such a cell has subsequently changed its form, but that it was extended when the sejiaration of the nucleus occiu-red. This hy]iothesis would not, however, explain the formation of giant cells, multinucleated nniscle-cells, etc., and it does not provide an explanation for the evident activity of the centrosphere and mito- chondria in direct division. A second hy])othesis to account for the sejiaration of the nucleus directly postu- lates the active particii)ation of the centrosphere, or mitochondria, or both, and here we may as,sume a i)urely mechanical and a purely physico-chemical activity. It has been noted that the centrosphere is found commonly in the invagination of the nucleus; moreover, its edge shows evidence of a curious type of movement — a slow, indefinite retraction and elongation of the marginal i)rocess(\s— which seems to be associated with movements of the mitochondria. It is possible that, through this mechanical influence of the centrosphere upon the adjacent nuclear membrane, the constriction of the latter is favored and the nucleus ultimately divided, and it is easy to conceive how the mitochondria may assist in this nuclear sejiaration BINUCLEATE CELLS IX TISSIE CLLTUHES. 87 through their own movements (as they have been described by Lewis and Lewis, 1915), since they are typically found between the nuclear parts when these are separated to any extent (fig. 9), and a strand of mitochondria may even be seen Ijang across the constricted isthmus of the nucleus, when this has not become com- pletely divided (figs. 6 and 8) . This position of the centrosphere and mitochondria undoubtedly seems to have some significance in sejiaration of the nucleus, and is seen even where the nucleus is dividing irregularly, as in figures 48 to 58. The relation of the Netzapparat of Deineka(1912,fig.3jto the nucleus is similar to that of the centrosphere as just described, viz, it is found in the cleft separating the nuclear portions. This author, however, does not ascribe to it any function in nuclear separation. He believes that it surrounds the centrosome. The position of the centrosphere and mitochondria may, of course, be without significance, so far as the actual division of the nucleus is concerned, and it is possible that the relationship of these cytoplasmic bodies to the amitotic nucleus is purely fortuitous, or, at most, occasioned through their adjustment to conditions of intra- cellular pressure. The occasional absence of the centrosphere from the cleft (once in each 50 cases as determined by counts) and the presence of a cleft opposite the one in which the centrosphere is found are points which count against this second hypothesis. Again, not all nuclei, in which the centrosphere appears in a concavity on one side, divide directly; indeed, this relationship of centrosphere and nucleus has frequently been noted and illustrated in cells developing, without nuclear amitosis, in their normal environment. It would seem, therefore, that this rela- tionship, of itself, can not bring about nuclear amitosis. In no case has there been noted a ring-shaped centrosjihere, like that described by Meves (1891), which encircles the constricted zone in the dumb-bell-shaped, amitotically dividing nucleus. The centrosphere and mitochondria may be assumed to act in another way in accomplishing direct division of the nucleus, viz, by bringing about a change in the surface tension of the area of the nucleus to which they are ojjposed, through the elaboration of a chemical substance, and it may be possible to e.xplain direct division of the nucleus upon some such hypothesis as that used by Robertson (1909, 1911, and 1913) to account for division of the cell in mitosis, viz, that there is produced in the region of cleavage some chemical substance which lowers the surface tension, such as soap, and that there results, in consequence, a streaming of protoplasm away from the equator, leading to separation of the cell. Robertson postulates a cholin-fatty acid soap, the cholin being derived from the splitting-up of lecithin. Since it has been shown that mitochondria are lecithinoid bodies (Cowdry, 1914, J). 18) it is not beyond the range of possibihty to assume that they may act in the formation of a cholin soap. Indeed, the relation of mitochondria to the production of chohn in nerve-cells has recently been discussed by Cowdry (1915). The position of the mitochondria, lying across the zone of nuclear constriction (fig. 8) is eminently favorable for the action of such a soap, should it be formed there. A third theory to account for direct division of the nucleus is based upon the assumption that some intranuclear change inaugurates the process. As long ago 88 lUMCLEATK CELLS IX TISSUE CULTURES. as 1855 and 1858, Remak set forth a theory to accouul for the division of the cell, which may be stated in the words of Wilson ( 1900. p. 63) as follows: "Cell-division proceeds from the center toward the peripher> . It begins with the division of the nucleolus, is continued by simple constriction and di\isioii of the nucleus, and is completed by division of the cell-body and membrane." A type of division which bears a close resemblance to this has recently been described by Howard and Schidtz (1911) in the cells of a giant-celled sarcoma from the human a'sophagus. To this type of division Schultz (1915) has proposed the name "promitosis," and these investigators believe it to be intermediate between amitosis and mitosis. This form of cell division seems to have an interesting parallel in certain protozoa, and they regard it as a reversion to a i)rimitive biological condition in which the division si)hcre is permanently intranuclear — an idea analo- gous to that of Wieman (1910, p. 175) for a similar form of nuclear division. The first ste]> in the division of the nucleus here is taken to be a separation of the karyosome into tw'o or more parts, of ecjual or uiuHiual size, followed by a breaking- up of the nucleus into portions corresponding in number and size with the fragments of the kaiyosome, each nuclear part coming to contain a portion of the latter. This function of the karyosome in initiating division of the nucleus is analogous to that of the centrosome in mitosis. This form of nuclear division is es.sentially the same as that described by C'onklin (1903) in the follicular epithelium of the common cricket, and that it is by no means infreciuent is gathered from the numerous references to it which this author has found in the literature. Conklin, however, has never seen actual cell division following nuclear amitosis, and from the fact that the cells in which direct division of the nucleus is found sjieedily degenerate after the egg is laid he belie^'(^s that it is, in the material examined, "one of the last functions of these cells and that it is therefore an accompaniment of cellular senescence and decay." Conklin, how- ever, believes that in most cases of amitosis the nucleolus does not divide. The evidence from tissue-culture cells does not lend much sui>port to a h\'i)othe- sis ascribing to the hssion of the nucleolus the initiation of nuclear division; true, we have in ttgure 2 a nucleus which shows lateral constrictions at the eciuator, and within it, lying with its long axis parallel to that of the nucleus, is an elongated karyosome, which also appears to be undergoing division in the same plane as the nucleus. This .somewhat resembles the nuclei described by Howard and Schultz; the karyosomes of t issue-cult lu-e cells, however, are decidedly simpl(>r in structure than those of the cells of the giant-cell sarcoma. Again, the fact that in the binu- cleate cell each nuclear portion is usually sui)i)lied with one or more karyosomes seems to point to this body having been divided before or during the division of the nucleus; but against this circumstance, weighing in favor of the view that the division of the karyosome acts to excite direct nuclear division, is the occurrence of sueli di\ision where the karyosome has evidently not divided (fig. 6), since it is present in only one of the nuclear parts. ■ Such nuclei are not uncommon in tissue cultures. The peculiar condition of the nucleolus in figure 2 may thus be purely accidental, since it is not at all constant. BINUCLEATE CELLS l.\ TLSSUE CULTURES. 89 The division of the nucleolus thus seems to have nothing to do with the separa- tion of the nucleus; indeed, after the nucleolus has divided, the nucleus may not divide at all. It may, however, have to do with the size of the nuclear portions; where these latter are equal they each contain one or two nucleoli, of about equal size, whereas where they are unequal one portion — usually the smaller — may not contain a nucleolus. That direct division of the nucleus may take jilace without preliminary fission of the karyosome in tissues developing normalh' is CA'ident from the statement of Wilson (1900, p. 115): "In many cases, however, no i)rehminary fission of the nucleolus occurs; and Remak's scheme must, therefore, be regarded as one of the rarest forms of cell division." It is interesting to note that Schultz finds evidences of such a simj^le form of direct di\ision in the nuclei of cells of the same tumor in which he finds "promitosis." Summing up, then, the process of direct nuclear fission, it is probable that vari- ous factors are involved. Elongation of the nucleus is undoubtedly sometimes followed by its cleavage, and, since it is always present in nuclear amitosis, it maybe regarded probably as an essential in this. The activity of centrosphere and mito- chondria must also be considered as a factor in equal, as well as unequal, nuclear fission, and this activity is apparently made effective by nuclear elongation. Fission of the nucleolus, while possibly concerned with the relative size of the nuclear parts, is not necessarilj' associated with the initiation or carrying out of nuclear cleavage. Inasmuch as binucleate cells, and constricted nuclei which must be regarded as their precursors, are found in apjiarently normal embryonic tissue, they can hardly be considered as abnormal or as evidence of a reversion to a more primitive type of cell division; furthermore, their healthy condition is manifest from their capacity to divide by mitosis, as will be shown hereafter. Thus it is reasonable to suppose that the factors operative in nuclear division in tissue cultures are those which function in embryonic cells in vivo. Since these binucleate cells seem to represent the first step on the road to certain giant cells it may be concluded that the latter are the result of a repetition of the same processes which bring about the formation of the former. This view is in accord with that of Lewis and Lewis (1915), p. 391, who state: "These giant cells appear to be formed by an amitotic division of the nucleus without a coincident division of the cytoplasm." FATE. The nucleus having divided directly, what becomes of it? Obviously the most certain method of settling this (juestion is to select a living binucleate cell and watch it constantly as it passes through its various changes. This course has been followed with several cells, and the evidence at hand does not show that the cell as a whole divides otherwise than by the regular process of mitosis; in the early stages of this process there is a combination of the two nuclear portions to form a single mitotic figure. Plate IV is a series of camera-lucida drawings representing successive stages in the history of one of these twin nuclei, in a living connective-tissue cell, grown from 90 mxrcLEATK cells i\ Tissrr: cilti'res. a 7-day chick licart in glycosaline with autogenous embryonic extract, the culture being 19 hours old when the observation commenced. At 11'' 55'" a. m., when the observation began, the nucleus (fig. 00) was seen to be com])()sed of two portions, ai)pi-oximately ecjual, separated by what api)eared to be a single membrane, but what really rejjre.sents, as has been shown, the api)osed areas of nuclear membrane of the two portions. This double partition was seen, by focusing at different levels, to be a plane surface. The first three drawings show roughh' the ajipearance of such a double nucleus during life. The i)arts are of al)out the same size and each at first contains a single nucleolus. These latter undergo obvious changes in size, shajje, and number. There is a single centro- sphere (r). Vai globules are numerous, and the mitochondria are thread-like and ])lainly visil)le, and show their characteristic movement. The nucleus remained in much the same condition, undergoing minor changes in outline, for about 2 hours, when, at 1'' 50'" p. m. (63) the division between the nuclear i^arts was seen to become less clearly defined at one side and, gradually, refractive material from the nucleus accumulated in this equatorial plane until, at 5'' 05"' )). m. (65), there was a distinct refractive mass in this region, which was evidently chromatin. Hoon the entire cell began to contract, to become rounded, and to draw in its processes; the nuclear outline became indistinct, the i)osition of the nucleus being rejjresented by a clear space surrounded by a ring of fat globules and mitochondria (66). By focusing up and down it is seen that the cell is much thicker than before — in fact, it is almost spherical, the mitochondria and particles of fat forming a hollow globe which incloses the imclear space. The portions of the twin nucleus have (juite evidently fused and (from our knowledge of mitosis) it is plain that the cell is now in the prophase. A sijireme, however, could not be made out. The refractive material which had been seen between the nuclear portions has become indistinct. This stage was seen at 6 p. m. If we could see the cell represented in 65 in the fixed and stained condition we would tloubtless find something like figure 22; here the spireme is forming in a binucleate cell and the nucleoli are becoming smaller and are breaking up. It is evidently composed of two such nuclei as are seen in hgure 14, an early prophase in a mononucleate cell. The accumulated chromatin in the i)lane of contact of the two nuclear portions is clearly evident; this is ol)viousIy not the equatorial plate of mitosis. The nuclear membrane has almost disappeared, but the clu-omatic material is somewhat mor(> concentrated about the ])eriiihery. Figure 23 evidently represents a somewliat later stage of spireme formation in a double nucleus. Here the skein is well marked and the nuclear membrane has completely disajjpeared. These figures bear a striking resemblance to figure 6 of Rubaschkin (1905), in which he shows a spireme in a double nucleus. The stage represented in 66, if fixed and stained, would probably resemble figure 19, drawn from a mononucleate cell in the late prophase. From this point on the behavior of the combined double nucleus is identical with that of an ordinary single nucleus. As the cell was watched it was seen that a line, refractive in character, formed across its equator; this Une, represented in 67, was somewhat irregular in outline, its borders being serrated. It did not remain unchanged, l)ut on the contrary BIXIC'LKATK CKl.LS L\ TlSSUli CI I/riKKS. 91 showed almost constant minor variations in contour; it seemed to be composed of a row of small refractive bodies (chromosomes) undergoino; constant, slow, and very slight movements. From this characteristic formation, situated as it was in a diamond-shaped field, surrounded, as before, by a granular ring of refractive globules and mitochondria, the metaphase of mitosis was easily recognized. This stage was drawn at 6'> 50'" j). m. (67) and would appear like figure 15 if fixed and stained. The cell is somewhat smaller and more condensed than that seen in 66, and the appearance plainly indicates that the centrosome has divided and that each part is performing its usual function at a pole of the spindle. The actual division of the centrosome was not ob.served. After a short time the ])late was seen to split, and the two halves, retaining their parallel relationship to one another, moved to opposite poles of the cell, and there remained, thus marking the anaphase. Figure 16, from a fixed preparation of a mononucleate cell, represents this stage. Almost immediately thereafter the granules and fat globules midway from the poles of the cell were .seen to move inward as though a constriction were occurrmg about the nuclear area at this zone; the result was a dumb-bell-shaped mass within the elongated cell, formed of the nuclear area and surrounding protoplasm. Almost at once the cell membrane itself was seen to be undergoing constriction at this point, as shown in 68, at 7'" 05"' p. m. At the same time the nuclear areas at either end of the cell commenced to become free from granules of fat and other refractive material and the cell outline became larger, showing that the cell was flattening out and that the daughter nuclei were becoming reconstituted in the telophase. That the intracellular i)ressure is considerably increased during this process is shown by the bulging outward of certain portions of the cell membrane, as illustrated in 68, to form bubble-like protuberances. Frequently the granules and fat globules may be seen to rush out into these evaginations, indicating the formation of cell currents, where j^ressure has been suddenly released, through giving way and stretching of localized areas of the cell wall. These i)rotuberances soon flatten out, lie close to the cover-slip and expand, becoming armed with hyaline borders pos- sessed of amoeboid movement (Harrison, 1913, p. 67). The end of the cell opposite the connection with the daughter cell thus appears fimbriated, as shown by Lewis and Lewis (12c, fig^. 8 and 10). These refractive borders act as pseudopodia to anchor the cell to the cover-slip and to drag the daughter cells apart. The reforming nuclei, now more widely separated, and showing wider and clearer areas in the cell protoplasm, are seen in 69 at 7'' 25'" p. m., and at this time the cell was very much constricted, with the nuclei more widi^ly s(>i)arated. The constricted zone is somewhat more highly refractive than the surrounding tissue and resembles a short thread. Here also the cell proce.s.ses are seen to be feeling their way outward and to be pulling the two daughter cells apart. The stage corresponding to this in the fixed preparations is shown in figure 17; here the chromatin is a closely clumped, darkly staining mass, and the individual chromosomes are becoming resolved into smaller granules. These subsequently become scattered, and apj^ear in the later definitive, more lightly staining, nucleus as in figure 18. A marked expansion of cytojilasm is here to be noted. 92 BINUCLEATE CELLS 1\ IISSIK CTLTrUES. There ha\'e thus been formed two separate and distinct daughter ciUs, in each of which th(> nucleus is becoming gradually reconstituted. As the cell was watched the nuclear areas became clear and the membranes distinct ; nucleoli also appeared, two in each nucleus. Separation of the cells continued, their hyaline borders becoming very active, stretching away into the outlying media and writhing in a sluggish, eel-like manner. Soon the fat globules took up their characteristic arrangement in the cytoplasm, mitochondria appeared, and, in 70, at 8 p. m., 8 hours after the observation commenced, we ha^■e to recognize tw(j cells, apparently normal, each with its own centrosome. The process of mitosis was identical with that followed manj' times in mono- nucleate cells, except for the variation in the introductory stage, occasioned by the formation of the spireme from two nuclear parts instead of one. The various stages of mitosis, as it is found in the mononucleate cell, are well sliown in tlie series, figures 14, 15, 16, 17, and 18, selected from a fixed j)reparation. I have been unable to ascertain whether such spindle formations arising from the fusion of two nuclear portions are possessed of a double number of chromosomes, but the apparent identity of the mitotic i)rocess, after nuclear fusion has taken place, with that occurring in mononucleate cells, does not suggest any material variation in the chromatin arrangement. I am in agreement with Maximow (1908), when he says regarding similar spiremes (p. 95) : " Aus diesen Sjiirenien (>ntstehen immer regelmassige normale Mitosen." These cells were not followed farther. The history for the i)eriod of 8 hours, however, shows conclusi\ely that spiremes from these double nuclei may combine to form a single equatorial plate and division may occur l)y the ordinary mitotic process. That such mitosis occurs in all cases it is impossible to state from this isolated observation, but the jiresence of double nuclei (with spin^mes like those shown in figures 22 and 23) here and there in the fixed preparations no doul)t jjoints to the occurrence of such nuclear fusion as a part of the process of mitotic division in the binucleate cell. Cases have not been found where one portion only of a bij^artite nucleus was in a condition of mitosis; hence it seems reasonable to conclude that both parts are always in\olved in the process. Tliis much is demanded by our conception of the l)otential unity of the double nucleus, so far as its reproductive capacitj^ is concerned. In the case of the cells from which figures 22 and 23 were drawn, it may be argued that these represent telophases in which th(> daughter nucl(>i failed to sei)a- rate. Many mononucleate cells have been followed entirely through the mitotic process, and failure of the daughter nuclei to separate has never been noted. Again, in figure 23, drawn from an iron-hematoxylin i)reparation, there is only a single centriole-pair, not two, as would be the case in a telojihase. It might even be suggested that .such daughter nuclei have recombined, as ob.served by Kite and Chambers (_1912); here, however, artificial conditions were existent, since the cells were being forcibh* separated in the Barber moist chamber by mechanical means. Moreover, entire absence of constriction of the cytoplasm, as would occur in the telophase, jwints to the condition we are con.'^idering as repre- senting the prophase. More than this, the fact that the process has been followed UIXrCLEATE CELLS IX TISSUE CULTURES. 9'.i in the living cell, from resting twin nucleus through mitosis to two separate and distinct daughter cells, would seem to be proof absolute that these figures 22 and 23 (which represent a phase of this process) are prophases of combining double nuclei. The mere contact of two spireme-bearing nuclei (such as appear in figure 23), is of itself no evidence that they will combine, but when we bring to bear upon the interpretation of such a figure the evidence derived from a series such as that shown in i)late iv, in which a nuclear formation, like that of figure 23, represents a stage, it seems obvious that these nuclear parts are undergoing fusion to form a single plate of chromosomes. Harman (1913) shows several figures of such nuclei in early cleavages of Taenia teniceformis and Moniezia (her fig. c, plate 8), but here the sepa- rate nuclei have arisen by mitosis, according to her observations, and cleavage, which is delayed, will eventually separate the blastomeres. The nuclear memln-anes are here quite intact, and show no evidence of beginning dissolution. It may be objected that the condition of spireme is no indication that mitosis is beginning. To this the reply may be made that in the cells of living tissue-cultures a nucleus showing a spireme of this kind, no companion cell in the same condition being present, always represented the prophase of mitosis. Since mitosis occurs in binucleate cells ni vitro, it might be assumed that it would also occur in such cells in vivo, and indeed this is the case, for ]\Iaximow (1908) has found figures in fixed preparations from the mesenchyme of embryo rabbits which strongly resemble those just described. In his figure 7 (p. 93) the spireme is forming in a dumb-bell-shaped nucleus, and in his figure 8 the nuclear fragments in which the spireme is found are quite separate. Maximow believes that his pictures represent the jirophase of normal mitosis occurring in amitotic nuclei; this belief is supported by my observations upon the living cell shown in plate IV. In his figure 8 he finds the centriole-pair situated between the two coils of the spireme — a position corresponding to that characteristic for it in the amitotic nucleus, viz, in the cleft. In my figure 23, which is slightly later, the centrosome has shifted its position to the pole. He states that his results resemble the findings of Karpow (1904) for urodele amphibia; this latter author described a process of nuclear amitotic division, with subsequent formation of a spireme from the frag- ments (which may be two or more in number), with fusion to form one "mutter- stern." It may theTefore be concluded, from the finding of such double spiremes in eml)ryonic tissue, that this process of mitosis in binucleate cells occurs in lun-mal devel- opment. It is thus to be found in differentiating as well as non-differentiating cells. I regret that I have seen no other living examples of combination of the parts of a double nucleus during mitosis, but the process is so rare that its observation thus is largely a matter of chance hitting upon a favorable cell. Mitosis occurs rather infrequently in the mononucleate cell, and when it is considered that the proportion of binucleate cells to total cells is very low (1 to 11 1) the remoteness of the possibility of finding a binucleate cell which will divide by mitosis may he realized. It is only in those cultures showing abundance of both binucleate cells and mitotic figures that there is any hope of finding sucli compound mitoses. To ascertain the relative frequency with which mitosis occurred among the l)iiiuclcaf(' cells, as compared with the mononucleate, a study, by careful counting 94 BIXl'CLEATE CEI.I.S IN TISSUE Cl'LTURES. and clatisificatioii of cells, was made of tlic 20 preparations from oliick heart men- tioned before. In tliese estimates only the pro])hases were counted, since it is imjiossihle to say of the cells in the later staf^es of mitosis wheth(M- th(\v arose from a monopartite or bijjartite nucleus. Degenerate ci'lls were omitted, anil al.so the area close to the original piece was not counted, since the cells here were usually too small and clo.sely ])acked to l)e seen clearly. Nuclei with more than two i)arts of equal size were rare: such w(>re groujud with the ))iiuicleate cells in this estimation. It was found that there was a total of 41,100 mononucleate' cells (excluding the later mitotic and amitotic forms); of these 47 were in the proi)hase of mitosis, or 0.1 14 per cent of the total. In the same series there was a total of 375 binucleate cells, 2 of which were in the prophase, or 0.53 per cent. In si^ite of the rarity of occurrence of binucleate cells in prophase (there being only 2 in a total of 41 ,725 cells) it will be .seen from this result that mitosis occurred even more freciuently among the binucleate cells than among the mononucleate — in fact, 4.65 times as frequently. Thus, while it can not be stated definitely that mitosis with recombination of the nucleus always follows amitotic nuclear division, or, indeed, that it freciuently does, it may nevertheless be affirmed with confidence, even allowing for the limited extent of the ob.servation, that the incidence of mito.sis ill the l)inucleate cells is at least as high as tliat among the mononucleate. If, in addition to this division by mitosis which these binucleate cells show, they be considered as also ])roliferating by direct division of the cytoplasm, it will be readily seen that their rate of proliferation wouKl then be very much greater than that of the mononucleate cells. Tlic improbability of this excessive multi])lication strengthens the negative evidence to be put forward later that there is, in these Itiiiucleate cells, no division of the cyto]ilasm following direct division of the nucleus. We have seen that a single mitotic hgure can be formed from two nuclear l)ortions, jireviously separate, but contained within the same cell. It has also been found that the spireme may form in a bent nucleus of a shape similar to tho.se under- going direct division. Figure 20 represents an early sjjireme in such a nucleus. There is apparently but a single centrosphere, situated in the cleft. Figure 21 shows a somewhat more advanced spireme. The nuclear membrane has dis- appeared and thi> chromosomes are more definite. One centrosphere is situated above, in the cleft, and there is an indistinct trace of a second in the clear area below. We can thus construct a series, from cells taken from fixed sjiecimens, illus- trating ])roi)hases in single nuclei, in double nuclei, and in the intermediate forms connecting these. Figures 14 and 19 show .spiremes in single nuclei. In the last are two well-marked centrospheres, indicating that a spindle is about to be formed. Figures 20 and 21 show the process in intermediate forms, and figures 22 and 23 show it in the double nucleus. In figures 20 and 21 it is reasonable to sujipose that the amitotic process has ceased, since the nuclear membrane has almost or ciuite disappeared, and for the same reason the process of karyokinesis, which is .so obviously taking jilace in these cells and in those represented in figures 22 and 23, must in all of th(»se cases be considered as starting up under circumstances where amitosis of the nucleus was under way, or was completed, rather than as having the process of amitosis superpo.sed upon it. BINUCLEATE CELLS IN TISSUE CULTIKES. 95 Altogether the various forms of the nucleus in which spiremes are found in tissue-culture prei^arations resemble strikingly the findings of Karpow in the leuco- cytes of urodele amphibia. This similarity is brought out in the following paragraph from Maximow (1908, p. 95): "Nun ist es aber nach Karpows Untersuchungen ziemlich sicher, dass hier die Kerna- mitose zwar zur Kernpolymorphie und sogar zu sicherer Kernteilung fuhrt, dass sie aber doch keine richtige Zellvcnnehrung nach sich zieht. Wenn die Leukocyten mit amitotisch zerschniirtem Kern sich teilen , so geschieht dies eben auf dem Wege der Karj-okinese, und aus einem zerschniirten Kern oder sogar aus mehreren einzelnen, ganz getrennten, durch Amitose erzeugten Kernen entsteht dann eine einzige, gewohnlich regehnassige, mitotische Figur. Man findet Spii'cino in ring-, hantel-, rosenkranzfoi'inigen Kernon, oft auch zwei oder mehrere einzelne Kerne in einer Zelle, alle gleichzeitig ini Zustande des Spirems, woraus dann immer ein gewohnhcher ]\Iutterstern resultiert." Maximow also shows a spireme in a dumb-bell-shaped nucleus found in his own material, and observes: "Die tief eingeschniirten, oder auch seiion ganz zerteiUen Kerne konnen in Mitose treten und man bekommt dann hantelforniige Spireme (fig. 7) oder zwei kugeUge Spireme nebeneinander in ein und derselben Zelle (fig. 8)." Thus it would seem that the nucleus enters upon the process of mitosis when- ever the stimulus initiating this process occurs, whether rounded, bent, undergoing constriction, or divided into two parts, and in all of these, after the single spireme has been formed, the process is apparently identical. The question as to whether or not the cytoplasm of the cell divides following direct fission of the nucleus, to form two separate and distinct cells, has been much discussed by vai'ious authors, among them Maximow (^1908), who found — besides the cases in which the amitotically divided nuclear portions formed a single combined mitotic figure and divided by karyokinesis— also instances where such portions simply became separated from one another and surrounded by protojilasm, to form ordinary mononucleate cells. In short, ^laximow believes that, though amitosis of the nucleus may be followed by cell division arising through a process of mitosis involving the directly divided nuclear fragments, yet it can lead directly to cell proliferation without intervening mitosis. As such a method of actual cell multiplication, Maximow believes that amitosis functions in certain areas of the normal develo))ing tissue of the rabbit,, and he has found it also in the guinea pig. Furthermore, he expresses the view that cells arising by direct division can later divide by mitosis, but his reasons for the latter assumption are not given. On the other hand ther(> are those who oppose this view and believe that nuclear amitosis is never followed by cell amitosis. P\)r instance, Karpow ( 1904), according to Maximow (1908, p. 89) came to the conclusion, based upon his observations upon the leucocytes of urodele amphibia: "dass in den P'allen, wo richtige Amitose wirklich vorliegt, man eigentlich doch nur Kernvermehrung annehm(>n kann, keine Zellvermehrung." This view is in agreement with the findings of ("onklin (1903. p. 671) for follicular ei)ithelial cells of the common cricket. No reliable evidence that fission of the cytoplasm follows that of the nucleus has been found in the tissue cultures examined by me. It is cjuite true that so-called "paired" cells ((' c, cells closely resembling one another in form, staining, etc., 96 HIM ('i.KAi'K CKLi.s IX TLssri: cri.Trur.s. lying side by side, but separated by cleavage of the cytoplasm) ma\' be picked out in the fixed i)ivparations, and it might be urged that such were of amitotic origin. This contention can not be proved, however, and it is more ])robable, in view of the lack of i^ositive evidence of amitotic division of tlie cytoplasm, that these cells are either of mitotic origin or have migrated together. The problem was attacked by the method of continuous observation of binu- cleate cells (in which the d()ul)le nucleus has ])een sliown to arise l)v direct fission), the object being to .see if the cytoplasm wouUl divide, and in this way give ri.se to two separate mononucleate cells. Several such cells containing twin nuclei were followed, but in eveiy ca.se the cell finally degenerated without dividing, after an observation of shorter or longer duration. As an example, the following may be recorded: In a connective-tissue l)inucleate cell from an 8-day chick heart of 24 hours' growth, the jiortions of the nucleus were at first ])ressed closely together, liut after 30 minutes they separated slightly, as in figure 9, and remained apart for 2 hours, when they again became pressed together. The cell was ol^served for 11^ hours, and the jirocess of se]iaration and reaiiiiroximation of the nuclei occurr(>d four times during this period. There was no trace of cytoi)lasmic divi.sion and the only changes noted were those mentioned — some shifting of position of the nuclei and a slight decrease in size of the nuclear parts; the latter is believed to be due to pro- longed expo.sure to light. Contiiuious change in shai)e of the cell was followed by change in shajie of the nucleus. This ob.servation shows conclusively that the binucleate cell may remain a very long time without direct division of the cytoplasm, and has been confirmed in the case of other binucleate cells. In living cultures the absence of evidence of direct division of the cytoplasm, coml)ined with similar absence in the case of fixed prepa- rations, leaves us with no ground for the assumption that such direct division ever occurs. Even granting that cytoplasmic division occurs at all, the process appears to be so long delayed that it can not he of much imjjortance as a method of cell proliferation. This view is in accord with that of ( 'onklin ( 1903, p. 670j, for follicular epithelial cells of the common cricket, but does not coincide with that of Child (1907, c, d. and e), who concluded from this examination of the cells of Moniezia and other animals that amitosis was a rapid method of division which occurred where the stimidus to divide was very great and the supply of nutrition was inadeciuate. Patterson (190S) and others hold similar views. From the cn'idence which tissue* cultures afford, however, I am inclined to agree with Harman (1913, )). 219) that the assumption that amitosis is a more rapid method of cell prohferation than mitosis is hardly justified. The ob.servation ju.st recorded also shows tiiat the interi)retatioii of "double" nuclei (.such as those seen in my figures 4, 59, and (50 as separate nuclear sacs touching one another) is correct, for the sacs have been seen to move apart and after- ward to return to their original contact with one another, and to repeat this process. As has been already mentioned, the ai)i)o.sed surfaces of such jiaired nuclei give ri.se to an appearance resembling an intramiclear i)late: such a |)liite has. liowever, not been found bv me in the cells of tissue cultures. BINUCLEATE CELLS IN TLSSUE CULTURES. 97 The twin nucleus is, then, to be regarded as potentially a single nucleus, in which the nuclear material is separated into two or more sacs. This nuclear material is not to be considered as in any way equally divided between the nuclear portions, which are by no means daughter nuclei. This view is strengthened by the fact that the centrosome, as has been observed, is single in binucleate cells. Before the cell containing such a single twin nucleus can divide, it seems to ho (>ssential, judging from the observations, that the nuclear material should recombine and a spireme be formed from the chromatin material in its entirety. It may be asked whether nuclear fusion, in these binucleate cells, ever occurs without an accompanying mitosis. I have seen no evidence of such recombination, either in living or fixed preparations, and regard it as improbable, because (among other reasons) the i)arts increase in size following their division and the single nucleus, which would result from their reunion, would be unusually large. Nothing was brought to light, in the material examined, which would in any way sui^port the assumjition that there are two distinct types of cell division, amitosis and mitosis, for the type of amitosis which 1 have descriljed involves only the nucleus, and mitosis was the only process which resulted in the formation of two separate cells. These observations upon nuclear amitosis do not point to its being an evidence of cell degeneration, for the cells in which it is found are not highly specialized and do not show any more tendency to degenerate than the other cells of the culture. It is generally assumed that mitosis takes place only in normal cells, so that the occurrence of mitosis in amitotically divided nuclei hardly allows them to be con- sidered as degenerate. 80, too, the occurrence of amitosis and mitosis in the same preparation (as in the culture from which figure 2 was drawn), where tlie conditions under which the cells are growing are apparently identical, militates against the view that the environment is not favorable, for the two processes are going on side by side, and mitosis demands suitable conditions. Th(> statements of Wieman (1910, p. 174), "amitosis occurs usually under abnormal metabolic conditions which are unfavorable to normal metaboUc processes" and "it can occur under circum- stances that make mitosis impossible, " are out of harmony with his finding of both direct and indirect division side by side in the same field, as shown in his figure 13. This coincident occurrence of mitosis and amitosis has been noted by other investigators. The conception of amitosis which I have advanced thus differs radically from that of Flemming (1892 and 1893), vom Rath (1891 and 1895), Ziegler (1891), and Ziegler u. vom Rath (1891). They believed that amitosis occm-red in cells which were of a transient character and in those which were very highly specialized or on the way to degeneration; and that in cells of amitotic origin the process of mitosis was not believed to take place. In their scheme the condition w hich I shall speak of as nuclear fragnnnitation seems to have a place. According to this conception, then, amitosis constitutes simply a cliange in form of the nucleus without increase in its reproductive capacity, and not an actual cell division; and division of such an amitotic cell occurs only by karyokinesis in which there is a recombination of the nuclear material. If this view be correct, 98 BINUCLEATE CELLS IN TISSUE CULTURES. and of universal application, it may be possible to reconcile amitosis with the chro- mosome hypothesis, for, since mitosis would be the only method of actual cell pro- liferation, an unequal distribution of chromatin material to the daughter cells would not be possible, according to our conception of the mitotic process. NUCLEAR FRAGMENTATION. A note may here be made regarding a curious form of nuclear division which bears some resemblance to the one just described, but which differs from it in many important particulars. It is known as nuclear fragmentation or uneciual multiple nuclear fission, and was found to occur where the conditions for growth were not favorable — for instance, in old cultures, in which the food and oxygen supply had become depleted and katabolic products had accumulated (figs. 36 to 47) and in those to which a toxic constituent had been added (e. g., ethyl alcohol, figs. 48 to 58). It thus seems to be a pathological condition and is characterized by marked malfor- mation of the nucleus, manifesting itself in lobulation and by a breaking away of these lobules, so that what was formerly a single nucleus comes to consist of two, three, or as many as seven or eight apparently separate pieces. The forms in which fragmentation presents itself are various, as may be seen by reference to figures 36 to 47, drawn from a 6-da3^ growth from the stomach of a 5-day chick. The nucleus may be but moderately deformed, as in figure 49, where a small bud has become constricted off, or there maj- be two, three, or more lobes or appendages, as seen in figures 40 and 41. These small fragments are in all stages of constriction, ranging from a blunt, sessile protuberance to a small pedunculated mass, held sometimes by a mere thread, as in figure 46. Extremely irregular forms, as 37, are not infrequent, and completely separated portions, as in 36, 42, and 45, are quite often met with. Each fragment may or may not contain a nucleolus. In the smallest pieces it is absent. In some cases, as in figure 45, if the nucleolus happens to be caught in the constricting zone it may become separated, but this is a rare occurrence. Where the nucleus is lobulated the number of lobules usually exceeds the mmiber of nucleolar portions. The culture shows other evidences of degeneration. The size of the nuclear portion seems here to bear no relationship to the size of the karyosome fragment, as it does in the multiple direct division of the nucleus described bj^ Schultz (1915). The extent to which this process of fragmentation may proceed is seen by reference to the fact that 60 per cent of the nuclei were malformed in some way, and 34 per cent were actually fragmented, in ten fields from the preparation from which figures 36 to 47 were drawn. There were no mitotic figures found m this preparation. In no case was there found any evidence of division of the cell protoplasm following nuclear fragmentation; on the contrary, a sort of syncytium was formed, in which the cytoplasm was filled with nuclear fragments of varying size. The picture presented by such a nuclear complex is markedly different from that of the giant cell, among the points of differentiation being the widely varying size of the nuclei, their lobulation, and the presence of buds in process of separation from the main nuclear mass. Again, in fragmentation the cytoplasm does not increase, as in the case of the giant cell. BINUCLEATE CELLS IN TISSUE CULTURES. 99 The entire absence of division of the cell protoplasm prevents this nuclear change from being regarded as a method of cell proliferation. Again, there is no evidence that such nuclear fragments ever reunite to form a spireme after the manner already described for the ordinary type of amitotic nucleus; indeed, mitotic figures are absent from such preparations — a fact which seems to indicate that the condi- tions which bring about fragmentation also prevent karyokinesis. The differences which fragmentation presents as compared with the usual form of direct nuclear division may be briefly summarized as follows : The nucleus is of irregular contour, multilobulated, and breaks up into a number of small, unequal- sized parts, which frequently do not contain nucleoli; the nuclear parts remain small, indicating that they have little or no power of growth, for the total volume of the nuclear substance does not seem to be increased following division. There is no evidence of fusion of the fragments contained in a single mass of cytoplasm to form a single mitotic figure. Finally, the process is found in growths which are existing under abnormal conditions, such as the presence of toxins or a deficiencj' of oxygen, and such conditions act to prevent mitosis. As contrasted with this we find, in the case of the ordinary binucleate or multi- nucleate cell, nuclear portions of regular contour, few in number (usually not more than two), of almost equal size, each containing as a rule one or more nucleoli. These parts apparently possess the power of growth, for in size they are comparable with the nuclei of the mononucleate cell. The fragments of the "double" nucleus are also able to combine and form a single mitotic figure. These cells are found in normal cultures, in which mitotic figures are frequently to be seen. Fragmentation is similar to the division which produces the ordinary binucleate cell in that the position of the centrosphere and mitochondria with relation to the nucleus is the same. In figures 48 to 58 these structures will be seen occupying the cleft, as in 55 and 58, or situated between the fragments, as in 50 and 54. Nuclear forms of this character are not infrequently found in the literature. Glaser (1907) describes an analogous form of nuclear fragmentation which occurs in the degenerating food ova of Fasciolaria tulipa. This he regards as "pathological amitosis" as distinguished from physiological amitosis. Child (1907c, p. 288) speaks of "degenerative amitosis" in starving planarians, stating that these forms " differ in appearance from the amitoses in regenerating tissues;" again (1907e, p. 173) he finds that "nuclear fragmentation is a frequent accompaniment of degeneration." On the whole, therefore, judging from the prevailing views of authors, and from the conditions obtaining in the cultures in which it occurs, it seems reasonable to regard nuclear fragmentation as an evidence of degeneration. These final changes are, perhaps, to be looked upon as an active reaction of the nucleus to unfavorable conditions of its environment, as, for instance, the presence of toxins due to katab- olism, or chemical change in the media, or injurious material added to the media, as alcohol, or to deficiency in food or oxygen. In this connection it is interesting to note that Lewis (1911) and Miller and Reed (1912) demonstrated that the presence of toxins caused an increase in the number of lobes of the neutrophilic leucocyte in blood of the human subject and also in that of the guinea pig and rabbit. They look(>d upon this increase as a physio- logical reaction on the part of the leucocj'te. AVherry (1913) found that amoebae 100 lUXrCLEATE CELLS IX TISSUE CrLTrUES. grown in oxygen-poor media showed division of the nucleus without cleavage of the cell protoplasm, and \Meman (1910) expresses the view that lack of oxygen may be a cause of a similar nuclear fragmentation in the material which he examined. Again, Holmes (1914) noted such a fragmentation in tissue cultures kept a week or more without changing the medium; when, however, the medium was changed frecjuently there was no indication of such nuclear change. Fragmentation was accompanied by other evidences of degeneration. Here, too, lack of oxygen may be the underlying cause, and the increased nuclear surface due to the change in form and multiple division of the nucleus may represent the effort, on the ])art of the cell, to secure an increased respiratory area. The mechanics of nuclear fragmentation is no less comjilicatod than that of true nuclear amitosis; indeed, it is probable that new and obscure factors l)ring about a change in nuclear outline and division of its substance. The activity of the centro- sphere and mitochondria may be regarded as similar to that found in the true form of nuclear amitosis, since their relation to the nucleus is the same. Nuclear forms somewhat resembling those just described, but simpler in character, are occasionally seen in ai)i)arently normal tissue cultures; c. g., those shown in figures 11, 12, and 13. Similar forms have been described in embryonic tissue developing normally, as figures 76, 8 a and c of Child (1904) and some of tli(^ figures of INIaximow (1908). They appear to be exami)les of spoi'adic and simple fragmentation. The fate of these buds is obsciu'e, but is prol)ably degeneration. SUMMARY. The following general conclusions, based ui)oi^ the results of the fort^goiiig investigations, have been reached: BINUCLEATE CELL. Incidence: In 20 preparations the binucleate cells made up 0.9 j^er cent of the total cells appearing in the new growth. They were more abundant in membranes growing from the heart than in growths from any other tissue, and in cultures of hearts of 5 days of age than in those from older cardiac tissue. They were also more abundant in new growths from cultures of the second day than in those of the first; this suggests that some, at least, of these cells have arisen in the new growth rather than in the original piece, with subsequent migration into the new growth. The proi)ortion of cells containing amitotic (constricted) nuclei to the total number of cells was 1 to 835; that of amitotic nuclei to bipartite nuclei was 1 to 7.5, and that of amitotic to mitotic nuclei was 1 to 3.4. Origin: The paired nuclei of binucleate cells in tissue cultures arise by direct division of the nucleus, or nuclear amitosis, without division of the cytoplasm. This occurred in perfectly normal cells. Constriction of the nuclear membrane, from one or both sides, which seems to be associated with a karyoplasmic streaming away from the nuclear ('((uator, was the onh' mechanism observed in direct nuclear fission, and in this process an activitj' of the centrosjihere and mitochondria, combined with elongation of the nucleus, appeared to be the principal factors. The centrosphere does not di\-ide, nor do the centrosomes separate. BINUCLEATE CELLS IN TISSUE CULTURES. 101 The process of nuclear amitosis is slow, excepting the final stage, which is rapid. There seems to be a critical point in nuclear constriction ; before this point is reached the nucleus may return to its original form, but after it has been passed the cleavage of the nucleus jjroceeds rapidly, and results in two separate nuclear parts. Division of the nucleolus is not an essential of amitotic nuclear division; it may, however, be concerned with the size of the nuclear fragments. There is no evidence of a form of nuclear amitosis that depends upon the for- mation of an intranuclear membrane which subsequently Rjjlits. Such a structure is simulated by the a})posed surfaces of the nuclear membranes of the parts of the nucleus of a binucleate cell, when they are in close contact. Sometimes, also, nucleoli, mitochondria, and inbending of the nuclear wall may resemble such a membrane. Fate: There is no e\idence that direct division of the cytoplasm follows direct division of the nucleus; thus amitosis is not a method of complete cell division, but is to be looked upon as a change in form of a healthy nucleus. The regular process of mitosis may occur in binucleate cells. During this jjrocess the chromatin material from both nuclear portions is merged into one equatorial plate of chromosomes, the spiremes, which begin to arise separately in the two nuclear parts, joining together to form the chromosomes. Furthermore, this is the only kind of cell division which was found to occur in binucleate cells; they either divide by mitosis or remain as they are, without fission of the protoplasm. The separate parts of the double nucleus have no reproductive independence (though they may have metabolic independence), and act as a unit in mitosis. Hence the reproductive capacity of the bipartite and monopartite nucleus is the same. Mitosis occurred as frequently in the binucleate as in the mononucleate cells. Nuclear fusion, without mitosis, has not been foimd to occur. GENERAL. Mitosis occurs in a nucleus irrespective of its shape; thus the spireme was found in nuclei of rounded form, in those presenting equatorial constriction, and in those divided into two portions. Chromosome hypothesis: Nuclear amitosis is not incompatible with theories of inheritance which assume that the chromosome is the bearer of hereditary characters. (riant cells: The binucleate cell seems to be the first stag(^ in the formation of the giant cell, which probably arises by a repetition of luiclear amitosis. This conception does not include the formation of the foreign-body giant cell. Nuclear fragmentation was found to occur where conditions for life were not favorable, and was thus a form of degenerative change. Fission of a healthy nucleus (amitosis) must thus be distinguished from fission of an unhealthy nucleus (frag- mentation) . Karyosomes of the cells examined were irregulai- in shape, underwent continu- ous change in morphology, size, numbei-, and position, and were made up of numer- ous closely j)acked masses of gel, encli with a core of greater density. The centrosphere in the cells examined was a slight!}' concentrated gel containing a centrosome (usually paired). Its border is irregular, and this undergoes continu- 102 BINUCLEATE CELLS IN TISSUE CULTURES. ous change in outline and appears to be intimately associated with adjacent mito- chondria. Vital dyes: Gentian violet did not prove to be a true vital dye. While it stained the intranuclear bodies and nuclear membrane, its action was toxic and coagulative, and the cells speedily degeneratetl. Janus green, in low dilutions, was found to stain mitochondria specifically, but its action was destructive, causing speedy death of the cell, with dissolution of the mitochondria. Enibryonic alls: Many, at least, of the facts obtaincnl from observation of cells in tissue cultures may be applied to the interpretation of similar cells de\eloping normally in the embryo. In conclusion I wish to record my indebtedness to M. K. and \\'. H. Lewis for the loan of their splendid collection of fixed preparations, and for their valued guidance; also to Dr. F. R. Lillie for the courtesy of a room at the Marine Biological Laboratory at Woods Hole, where some of the work was carried on. LITERATURE CITED. f 'arrel, Alexis, 191:5. Coiitriljutions to the study of the mechanism of the erowtli of connective tissue. Jour. Exper. Med., Lancaster, Pa., xviii, 2>i7-299. C.^RY. L. R. 1909. The life history of Diplodiscus temporatus Stafford. Zool. .lahrl)., .lena, .\lit f. Anat., xxviii, .595-659. Chii.k. C. M. 1904. Amitosis in Moniezia. .\nat. .\nz., Jena, xxv, 545-55X. . 1906. The development of germ cells from dilTeren- tiated somatic cells in Moniezia. .\nat. .\nz., Jena, XXIX, 592-597. . 1907a. Studies on the relation i)ctween amitosis and mitosis. I. Develnpnieiit of the ox'aries and oogene- sis in Moniezia. Biol. Hull., Woods Holl, xii. 89- 114. . 19076. Studies on the relation between amitosis and mitosis. II. Development of the testes and sperma- togenesis in Moniezia. Biol. Bull., Woods Holl, XII, 175-224. . 1907c. Amitosis as a factor in normal and regulatory growth. Anat. .\nz., .lena, xxx, 271-297. . 1907d. Studies on the relation between amitosis and mitosis. III. Maturation, fertilization, and cleav- age in Moniezia. Biol. Bull., Woods Holl, xiii, 1.38-160. . 1907c. Studies on the relation between amitosis and mitosis. IV. Nuclear division in the somatic struc- tures of the proglottid of Moniezia. V. General discussion and Cf)nclusions concerning amitosis and mitosis in Moniezia. Biol. Bull., Woods Holl, xiii, 165-184. . 1911. The method of cell-division in Moniezia. Biol. Bull., Woods Hole, xxi, 280-296. Churchm.^n, J. W., and D. G. HrssELi., 1914. The effect of gentian violet on protozoa and on growing adult tissue. Proc. Soc. Exper. Biol, and Med., N. Y., XI, 120-124. CoNKLiN, E. G. 1903. .\initosis in the egg follicle cells of the cricket. Amer. Natural, Boston, xxxvii, 667-675. CowDRy, E. V. 1914. The comparative distribution of mito- chondria in spinal ganglion cells of vertebrates. Amer. .lour. Anat., Phila., xvii, 1-29. . 1915. The structure of chromophilc cells of the ner\-ous system. Contributions to Embryologj-, No. 11, Carnegie Institution of Washington Pub. No. 224. Deineka, D. 1912. Der Netzapparat von Gnlgi in einigen Epifhcl- und Bindegeweliszellcn wiihrend der Ruhe und wiihrcnd der Teilung derselben. Anat. .Vnz., Jena, xli, 289-.309. Flemming, W. 1892. Entwicklung und Stand der Kennt- nisse iiber Amitose. .\nat. Hefte, 2. .Vl)t., Wiesb., II, 37-82. . 1893. Morphologic der Zelle und ihrcr Teilungsere- scheinungen. Anat. Hefte, 2. Abt., Wiesb., in, 24- 131. Glaser, O. C. 1907. Pathological amitosis in the food-ova of Fasciolaria. Biol. Bull., Woods Holl, xiii, 1-4. Harman, Mary T. 1913. Method of cell-division in the sex cells of Tsnia teniieformis. Jour. Morph., Phila., xxiv, 205-243. Harrison, RossG. 1913. Tholifeof tis,suesoutfldctheorgan- ism from the cmbryologii-al standpoint. Trans. Con- gress .\mer. Physic, and Surg., N. Haven, ix, 63-76. Holmes, S. J. 1914. The behavior of the epidermis of am- phibians when cultivated outside the body. Jour. Exper. Zool., Phila., xvii, 281-295. Howard, W. T., and O. T. Schultz. 1911. Studies in the biology of tumor cells. Monographs Rockefeller Instit. Med. Research, No. 2. Karpow, W. 1904. [Untersuchungen iiber direkte Zell- teilung.) Inaug.-Di.ss. Moskau, 1904. Rrf. in Jahresber. u. d. Fortschr. d. .\nat. (.Schwalbe), Jena, n. F. x, 42. Kite, ('•. L. 1913. Studies on the physical properties or protoplasm. I. The physical properties of the protoplasm of certain animal and plant cells. .\mer. Jour. Physiol.. Bost(.n. xxxii, 146-104. . and R. Chambers, Jr. 1912. Vital staining of chro- mosomes and the function and structure of the nu- cleus. Science, N. V., n. s., xx.xvi, 6.39-641. Lambert, R. .\. 1912a. V'ariations in the character of growth in tissue cultures. .\nat. Rec. Phila., vi, 91-108. . 19126. The production of foreign-body giant cells in vitro. .lour. Exper. Med.. Lancaster, Pa., xv. 510-515. Lewis, M. R. 1911. The blood picture in tuberculosis. Johns Hopkins Hosp. Bull., Bait., xxii, 428-434. — . 1915. Rhythmical contraction of the skeletal muscle tissue ob.served in tissue cultures. Amer. Jo\ir. Physiol., Boston, xx.xviii, 1.5.3-161. Lewis, M. R., and W. H. Lewis. 1911. The growth of embryonic chick tis.sues in artificial media, agar and iwuillon. Johns Hopkins Hosp. Bull., Bait., xxii, 126-127. , . 1912a. The cultivation of sympathetic ner\'es from the intestine (li chick embryos in saline solutions. Anat. Rec, Phila., vi, 7-17. , . 19126. The cultivation of chick tissues in media of known chemical constitution. .\nat. Rec., Phila., VI, 207-211. , . 1912c. Membrane formations from tissues transplanted into artificial media. Anat. Rec, Phila., VI, 195-205. , . 1914. Mitochondria in tissue culture. .Science, N. Y., n. s., x.xxix, .330-3.33. , . 1915. Mitochondria (and other cytoplasmic structures) in tissue cultures. Amer. Jour. Anat., Phila., XVII. 3.39-401. Loeb, J.iCQiES. 1906. The dj-namics of living matter. New- York, The MacMillan Co. M.\xiMOW, .\. 1908. Uebcr Amitose in den embr>-onalen Geweben bei Siiugctiercn. Anat. Anz., Jena, XXXIII, 89-98. Meves, Fr. 1891. LTp|)C'r amitoti.sche Kernteilung in den .Spennatogonien des Salamanders und \"erhalten der .\ttraktionssphare bei derselben. Anat. .-Vnz., Jena, VI, 626-639. Miller, J. .\., and Mar(iaret.\. Reed. 1912. Studies of the leucocytes in pulmonary tuberculosis and pneu- monia. .\rch. Intern. Med., Chicago, ix, 609-636. Aho in Trans. .\mer. Climat. .\sso., Phila., 1911, XXVII, 192-223. Patterson, J. T. 1908. .\mitosis in the ijigcon'scgg. .\nat. Anz , Jena, xxxii, 117-125. R.\TH, O. VOM. 1891. Uebcr die Bcdeutung der amitoiischen Kerntheilung im Hoden. Zool. .\nz., Leipz., xiv, 331, 342. 355. . 1895. Uebcr den feinercn Bau der Driisenzellen des Kopfcs von .\nilocra mediterranea Leach im Spcci- elleii und di(^ ;\mitosenfrag(; im Allgemcinen. Zeitschr. f. wi.ssciisch. Zool., Lcipz., LX, 1-89. RiCH.\RDS, .\. 1909. On the method of cell division in Taniia. Biol. Bull., Woods Hole, xvii, 309-326. . 1911. The method of cell division in the develop- ment of the female sex organs of Moniezia. Biol. Bull., Woods Hole, xx, 123-178. Robertson. T. B. 1909. Note on the chemical mechanics of cell-division. Arch. f. Entwicklungsmech. d. Organ., Leipz., xxvii, 29-.34. . 1911. Further remarks on the chemical mechanics of cell-division. Arch. f. Entwicklungsmech. d. Or- gan., Leipz., XXXII, .308-313. . 1913. Further explanatory remarks concerning the chemical mechanics of cell division. Arch. f. Ent- wicklngsmcch. d. Organ.. Leipz., xxxv, 692-707. 103 104 BINUCLKATK CELLS I.\ TISSUE CULTURES. Rl'baschkix, U. 1!«I."). Itliir doppclti' iiiul polymorpho K<'iiiL' ill Triti>til)lastoniori'n. Arcli. f. ii\ikr. Anat., Honn. Lxvi. 4^5 oOO. RrssELL, D. Cf. 1914. Tho offoct of ^t'tltiall \'ioli't on i^roto- zoa and on tissues growiiiK in vitro, witli cspreial reference to the nueleii<. Jour. Kxper. Med., Lan- caster, Pa., XX, 545-55:!. ScHULT/, O. T. 1",I15. Promitosis in tumor cells. Jour. Med. Keseanli, Boston, .xxxii (n. s.. xxvii), 257-270. Wherry. W.M. H. I'Jlli. Studies on the bioloKj- of an amoeba of the Liniax group. Vahlkampfia sp. No. L .Vrcli. f. Protistenk., Jena, xxxi, 77 94. AViEMAN. H. L. 1910. A study in the Remi cells of Lcpti- notarsa -.iignaticoUis. Jour. Morph., Phila , xxi, l:i5-210. Wilson, K. H. 1900. The cell in clexelopnienf and inherit- ance. 2 ed. New York, Tito Macmillan Co., 1900. VoiNU, K. T. 1913. The histogenesis of the reproductive organs of Tienia pisiformi.s. Zool. Jahrh., Jena, Abt. f. Anat., xxxv, 355-414. ZlEULER, H. E. 1891. Die bicjlogisclKr Hediutung der ami- totiselien (dircktcn) Kernteilung im Tierreicli. Biol. Ccntralbl., Lcipz., xi, 372-389. . u. O. VOX! Rath. 1891. Die aniitotischc Kernteilung bei den .\rthropoden. Bi(jl. Ceiilrallil., Leipz., xi. 744 757. EXPLANATION OF PLATES. Plate I. Fig. 1. Area of new growth from Xo. 42, 8: 12: 14 (Lewis collection). In the field are six binueleate (a) and one quadrinucleate (b) cells. The material was heart from a chick which had been incubated for 6 days; growth was of 48 hours' duration, in Locke (0.5 per cent tlextrose), fixation by osmic-acid vapor, and staining with iron hematoxj-lin. (0 shows two young daughter cells, the product of a recent mitosis. The guide-line from (n) terminates in the centrosphere of a l)inucleate cell. Retouched photograph. X46.5. Fig. 2. Elongated nucleus with bilateral constriction — the beginning of direct bilateral nuclear fi.ssion. The nucleolus is also ap])arcntly dividing. This figure, and also Nos. 3, 4, 5, 11, 12, 13, 14, 15, 16, 17, 18, and 22 are from No. 14, 9 : 1 ; 15 (Lewis). Heart from 6-day chick; grown in Locke (0.5 per cent dextrose) with a little yolk; fixed on third daj- of growth in Zenker; stained with Mallory's connective tissue stain. On account of the technique the cytoplasmic details are not represented. This and following drawings, except figures 24 to 35, were outlined by camera lucida. X 1,012. Fig. 3. Elongated nucleus almost completely divided; final stage of direct bilateral nuclear fission. Xl,012. Fig. 4. Nuclear fission completed; nuclear parts divided and lying in contact. X 1,012. Fig. 5. Direct unilateral nuclear fi.ssion; initial stage. Xl,012. Fig. 6. Direct unilateral nuclear fission ; final stage; cell of connective-tissue type; nuclear parts connected only by the merest filament; centrosphere between nuclear sacs; mitochondria streaming across the narrow- connecting strand. Drawn from preparation No. 2 (Lewis); 7-day chick heart grown for 5 days in Locke (1 per cent dextrose); osmic-acid vapor and iron hematoxylin. X 1,0.32. Fig. 7. Nuclear fission completed; growth from heart membrane; cell similar to that shown in figure 4, but prepared to show the centrosphere and mitochondria; the single centrosi)here contains two centrosomes; its position, opposite the line of contact of the two nuclear portions (below and to the right), is characteristic. No. 42 (Lewis) (see fig. 1). Xl,032. Pl.\te n. Fig. 8. Final stage of direct bilateral nuclear fission in a cell of connective-tissue type; shows the somewhat unequal nuclear parts joined by a very slender thread, apparently the attenuated nuclear membrane; overlying this are several strands of mitochondria, a similar relationship to that of figure 6; the larger nuclear sac contains two nucleoli; the smaller but one. .\ large centrosiihere, from which many mitochondria radiate, is conspicuous. The entire cell is very thin and shows mitochondria streaming out into the ])roces.ses. The morjjhology and arrangement of the mitochondria is characteristic for the connective- tissue cell growing in vitro, at periods other than mitosis. No. 17, 24 : 1 1 : 14 (Lewis). 6-day chick stomach; Locke (I per cent dextrose); 3-daygrowth; osmic-acid vapor and iron hematoxyhn. Xl,012. Fig. 9. A binueleate cell from heart membiane; the two parts are somewhat separated, and lying between them a single centrosphi're and-mitochondria are to be seen; the latter resemble cocci or short bacilli and show the characteristic radia arrangement about the centrosphere. This tj'pe of mitochomlria is found in cells of membranes growing from chick hearts. No. 42 (Lewis) (.see fig. 1). X 1,012. Fig. 10. Nucleus of distorted form in cell of connective-tissue type found in a culture growing in a weak alcoholic medium. The nucleoli in this preparation show as aggregations of granules; this appearance of the nucleoli in connecti\e-ti.ssuc cells stained in this way is found when difTerentiation with iron alum is carried too far. Mitochondria are apparently uninjured. No. 23, 24: 11 : 14 (Lewis). 6-day chick stomach grown in Locke (1 per cent dextrose) to which ethyl alcohol had been added to make approxi- mately 1 per cent; 3-day culture; osmic-acid vapor and iron hematoxyhn. X 1,012. 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