Note on the behavior of trypan blue injected into the developing egg of the hen.код для вставкиСкачать
Resumen por el autor, George B. Wislocki. Nota sobre el comportamiento del azul tripan inyectado en el huevo de gallina en vfas de desarrollo. Una soluci6n coloidal, el azul tripan, fu6 inyectada bajo precauciones asepticas en huevos durante el und6cimo dia de incubaci6n. El pequefio orificio abierto en la cAscara, a trav6s del cual se inyect6, fu6 cementado, volviendo a colocar el huevoen la incubadora. Despu6s de dos dias se abri6 el huevo examinando el embri6n. El colorante fu6 inyectado en la c6mara abrea, sac0 vitelino, sac0 alantoideo, sac0 amni6tico y en el mesoderm0 del alantoides. En cada una de estas regiones se comporta de mod0 diferente. La membrana de la cascara que rodea a la cAmara a6rea le absorbe rApidamente. Escapa desde el sac0 vitelino a travds de las cklulas epiteliales endod6rmicas que forran el sac0 interiormente, pasando a1 mesodermo subepitelial, donde es absorbido y almacenado por las c6lulas mononucleares que rodean a un plexo de vasos vitelinos. Cuando se inyecta en el sac0 alantoideo el colorante no es absorbido. Cuando se inyecta en el sac0 amni6tico tifie alepitelio amni6tico y tambikn penetra en el tracto gastrointestinal del feto por la boca. Cuando se inyecta directamente en el mesodermo de la membrana fetal se difunde en la corriente sanguinea del feto tifiendo vitalmente a1 embri6n. Translation by JoSe F. Nonidez Cornell Medical College, New York A C T H O H 3 ABSTRACT OF THIG P h P E R IGSUED B Y T H E n i n L I o G R . % P i i i c S E R V I C E , OCTOBER 17 KOTE OK THE BEHAVIOR OF TRYPAN BLUE INJECTED IKTO THE DEVELOPING EGG OF THE HEN G. B. WISLOCKI Anatomical Laboratory, Johns Hopkins University THREE FIGURES Attempts have been made by several investigators to determine the fate of dyes injected into the egg and their effect upon the embryo, but the experiments, though yielding a few results, were very brief. Zaretsky ('10) injected trypan blue, trypan red, neutral red, methylene blue, fluorescin and eosin into developing eggs, and obtained results of greatest importance with the first trvo. He injected 0.5 to 1 cc. of one of these dyes into the air chamber of the egg, and after a period of days noted a slight staining of khe amniotic fluid, but never any staining of the embryo itself. I n other instances, after injecting the dye into the outer wall of the allantois, he observed a faint staining of the entire embryo, fetal membranes, amniotic fluid, and albumin sac. He stated that of the tissues of the embryo the kidneys were the most deeply stained, indicating a rapid excretion of the dye by that channel. Granules of dye could not be identified microscopically in the tissues. Griiper ('la) reported the staining of early chick embryos with neutral red, trypan blue, and trypan red, but nothing more than diffuse staining of membranes and embryo alike was observed. The most valuable results following the injection of dyes into the developing chick so far reported are those obtained by Bakounine ( '95). She injected indigo carmine intravenously into a series of chicks ranging in age from three to fifteen days; excretion of the dye by the tubules of the wolffian bodies was demonstrable in the entire series. 267 268 G . B. WISLOCKI The present paper is a preliminary account of some observations upon the behavior of trypan blue injected into developing eggs. The experiments were undertaken in spite of the failure of previous investigators to make any significant observations with vital dyes in the chick. It was hoped that by injecting the dye into different regions of the egg, some knowledge concerning t,he functions of the fetal membranes, and possibly a successful vit,al staining of the embryo, might be obtained. Chicks of eleven days’ incubation were used. Trypan blue, which forms a colloidal sol, was made up in 1 per cent strength in sterile distilled water. The injections were made with a syringe bearing a 26-gauge needle, 1 inch in length, through a nick in the shell, the opening being no larger than necessary to admit the needle. The trypan blue solution (0.2 cc.) was injected into each egg, whereupon the needle was withdrawn and the tiny opening in the egg-shell sealed with a drop of melted paraffin. The eggs were then returned to t h e incubator to be opened at the end of forty-eight hours-the thirteenth day of incubation. By this method, of course, it cannot be determined exactly at the time of injection into what region of the embryonic membranes the trypan blue has been injected. If, with a general knowledge of the orientation of the fetal membranes and embryo, a number of eggs are injected, each locality which it is desired to inject will be reached in a certain number of eggs. This is made plain by reference to the diagram shown in figure 1, which illustrates condit)ions in the egg about the eleventh day. Thus i t will be seen that by stabbing a needle into the egg, with a knowledge of the orientation of the structures within, one will in many instances inject, with precision the yolk sac, allantoic sac, amniotic sac, etc. Orientation is made easier if one remembers that, after the egg has lain undisturbed in the incubator for a few hours, theembryo floats to the upper surface; hence the amniotic sac is more readily injected from above, and the yolksac from the side or below. Suffice it to say that in a series of five dozen eggs, injected as described above and killed on the thirteenth day of incubation, all of the selected points have been successfully injected. X description of the findings in each of the groups follows. TRTPAN BLUE INJECTED INTO EGG O F H E N 269 Injection into the air chamber. The air chamber is a cleft between the two layers of the shell membrane. The membrane is composed of matted fibers of organic substance which cross one another in every direction. When trypan blue is injected into this chamber it is rapidly absorbed by the shell membrane, much as it would be by blotting paper, staining it a deep blue. The adjacent structures beneath the shell membrane, namely, the chorion and allantois, as well as the embryonic fluids, remain Fig. 1 Schematic representation of a chick approximately on the tenth day of incubation, showing the technique used in injecting diffcrent localities of the egg. unstained and consequently also the embryo itself. When the shell membrane is removed, the inner surface of the shell is frequently found colored in the neighborhood of the air chamber. Injection into the yolk-sac. On injecting trypan blue into the yolk-sac the dye remains somewhat localized in the yolk in the neighborhood of the injection, lending a greenish appearance t o the yolk. The trypan blue, together with the yolk, is absorbed by the endodermal epithelial cells lining the interior of the yolk-sac. The dye imparts a greenish-blue coloration to the wall of the yolk-sac. Microscopically, a greenish, diffuse coloration 270 G . B. WISLOCKI of the cytoplasm of the yolk-sac epithelium is visible. The dye enters the epithelium most abundantly in the region of the area vasculosa, where the mall of the yolk-sac is thrown into numerous folds covered by large columnar cells which possess swollen ends and the cytoplasm of which is closely filled with droplets of yellow substance. Fig. 2 Photomicrograph of the wall of the yolk-sac of a thirteen-day chick, showing a portion of the areavasculosa. A group of cellswhich ingest vital dye (a) is seen adjacent t o a blood vessel. Other groups of cells (b) m e hematopoietic and do not phagocytize dye. The dye eventually penetrates the basement membrane upon which the endoderm rests and reaches its final destination in groups of cells surrounding the rich venous network in the yolk-sac wall. These cells are closely associated witJh groups of hemoblasts which invest the blood-vessels. The former are round or polygonal and possess a central or slightly eccentric round nucleus. Their cytoplasm absorbs the vital dye and stores it in the form of closely packed blue granules (fig. 2). These cells appear to be the ultimate destination TRTPAN BLUE INJECTED INTO EGG OF HEN 27 1 of the dye injected into the yolk-sac, as no evidence of its further passage is discoverable. The endothelial cells lining the blood-vessels remain unstained and, since the embryo itself does not become vitally stained, it is unlikely that any of the dye escapes into the vitelline vessels. It is of interest to note that in spite of the fact that throughout incubation the yolk-sac possesses a connection through its stalk with the intestine, none of the dye injected into the sac enters the intestine through this opening. Injection into the aZZantoic sac. When trypan blue is injected into the allantoic sac it mixes uniformly with the allantoic fluid, coloring the latter dark blue. The dye does not escape from the allantoic sac-a fact which can be demonstrated in several ways. If the discolored allantoic fluid is drained off and the sac washed out with physiological salt solution, the allantoic membrane is found to be unstained. The fact that the embryo and all its membranes are unstained confirms the view t,hat no dye has escaped from the allantoic sac. Furthermore, microscopic examination of the wall of the sac reveals the absence of the dye in the delicate, flat polygonal cells which cover its surface, or in the layer of star-shaped cells just beneath the surface. These observations accord well with the view that the allantois is a reservoir for the excretory products of the rnesonephros and met anephr 0s. Injection into the amniotic sac. Trypan blue mixes uniformly ith the amniotic fluid. The amniotic membrane becomes pale blue due to the presence of dye, which in some instances is visible under the microscope as blue dust in the delicate, flattened epithelial cells lining the surface of the membrane. The stomach and intestines invariably contain dark blue stained mucoid fluid which indicates that the amniotic fluid is swallowed by the embryo. Stained fluid is also observed in the lumen of the trachea and primary bronchi. A pale blue staining of the entire embryo is frequently observed, but the quantity of dye is insufficient to make it visible in the tissues when examined under the microscope. 272 G. B. WISLOCKI Injection into the mesoblast of the allantois. When trypan blue is injected into the mesoblastic tissue uniting the allantois with the chorion and amnion, vital staining of the embryo results. This is due, no doubt, to the easy access which the dye has to the network of allantoic vessels through which it is soon conveyed to all parts of the embryo. The membranes and integument of the vitally stained embryo present a light blue aspect. The yolk remains unstained, as does also the shell membrane and the shell itself. The depth of color in the various organs of the embryo proper varies: the central nervous system is unstained, the arteries are conspicuously blue, the lungs pale blue, the liver is greenish blue, the spleen reddish blue. The wolffian bodies are by far the most deeply stained organs; the metanephros is appreciably blue. Microscopic examination of the tissues reveals the presence of trypan blue in granular form in several of the organs. I n the thirteen-day-old chick it is found in most abundance in the wolfian bodies. Here it occurs in the shape of numerous tiny granules in the epithelium lining the uriniferous tubules (fig. 3). As might be expected from our knowledge of the distribution of vital dyes in the adult renal apparatus, no dye is discovered in the glomerular capsules. In the metanephric tubules, which at this period are already quite distinct, only traces of the dye are visible. It would appear, then, that a t this period of development the wolffian body still serves as the main pathway of excretion, though regressive changes in the tubules are already plainly visible. The second organ in which trypan blue appears in abundance is the embryonic liver. The dye is found microscopically in nearly all the endothelial cells lining the sinusoids and the terminal branches of the portal vein. In the liver cells themselves no particles are visible. The spleen, which in the thirteen-day-old chick is a small round organ approximately 2 mm. in diameter, contains traces of trypan blue which appear to be within cells lining the vascular channels and occasionally within mononuclear cells lying free within the sinuses. TRYPAN BLUE INJECTED INTO EGG OF HEN 273 Kowhere else in the tissues of the embryo is trypan blue abundant, although traces of dye are encountered not infrequently in the connective tissue in cells resembling clasmatocytes. That there are cells in the connective tissue of the chick capable of phagocytosis is best shown by examining the mesoderm at the site of injection of tJhetrypan blue into the wall of the allantois. Fig. 3 Photograph of a drawing of t h e wolffian body of a thirteen-day-old chick, showing a glomerulus with the bcgiiining of a tubule. The black dots represent granules of vital dye. Here numerous mononuclear cells, some of them round, others irregular in outline, are encountered with dye granules within their cytoplasm. The behavior of trypan blue in the thirteen-day-old chick seems to be of sufficient interest to warrant an extension of these observations to other stages of development. The findings described suggest paths of investigation which i t bemight of interest to pursue. For instance, an investigation of the function of the mesonephros and the permanent kidneys as excretory organs 274 G . B. WISLOCKI during embryonic life would be of considerable value. A study of the absorption of vital dyes from the yolk-sac at different stages would likewise furnish us valuable knowledge concerning the physiology of absorption in the embryo. The method used in these experiments will, however, never be applicable to chicks under four or five clays. In thesc younger stages direct observation of the living developing chick under the microscopewill always prove more satisfactory. SUMMARY A series of experiments is described upon the behavior of a vital dye, trypan blue, injected into the developing egg of the hen, and the technique of injection is given. In t.he vitalIy stained chick granules of dye are found in cells of the mesonephros, metanephros, liver and spleen. REFERENCES CITED BAKOUNINE, SOPHIE 1895 Sur l’activit6 sdcr6tice des Bpithhliums de Wolff e t des Bpithbliums rknaux dans les premiers jours de d6veloppement embryonnaire. Arch. ital. B i d , val. 23, pp. 350-354. G R ~ P E RL., 1911 Beobachtung von Wachstumsvorgtingen an Reihenairfnahmcn Iebender Huhnerembryonen nebst Bemerkungen uber vitale Piirbung. Arch. f. Entwcklsmech., B. 33, S. 303, 327. ZARETSKY, S. 1910 Versuche iiber vitale Farbung des Embryo. Virchow’s Arch. f. path. Anat. u. Physiol., Bd. 207, S. 25-45.