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===Observations on the Development Of The Earliest Lymphatics In The Region Of The Poster Lok Lymph Heart In Living Chick Embryos===

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PRELIMINARY NOTE

VAAOT n. CLARK AND ELEANOR LINTON CLARK

Fi())/i Un- Anatomical Department of The Johns Hopkins University

The discovery made by one of the authors that, in the chick' the earliest lymphatics are non-functioning, that they are usually packed full of stagnant blood and that they can therefore be seen in the living chick and distinguished from the blood-vessels with their circulating blood, suggested the possibility of actually watching the de\'elopment of the very earliest lymphatics. The region of the posterior lymph heart, where, as Sala and Mierzejewski have shown, lymphatics develop in connection with blood vessels which are comparativel}'- superficial, offered itself as the most favorable place for such a study.

The problem before- us, then, was to keep the chick alive and under continuous observation for a sufficient length of time; to make careful records of the blood vessels during the period preceding the development of the lymphatics; to watch for the earliest' appearance of the lymphatics, noting their relation to and behavior toward the blood vessels; to find out, if possible, the mode of extension, character and behavior of the earliest lymphatics; and to make tests by various injections and by the study of cross-sections in order to supplement and control the stud3' of the living structures. •This program w^as carried out with the following results:

By using a warm chamber it was possible to keep chicks alive and under observation for from five to seven hours. This proved to be long enough for our purposes.


The lymphatics in the posterior Ijnnph heart region first appear in chicks of approximatelj^ five days of incubation, measuring immediately after being placed in the fixing fluid, between 12 and 13 mm. greatest length; though we have found considerable variation in both age and length. The embryo is at approximately the stage represented in fig. 29 of Keibel and Abraham's 'Normentaf eln . '

The first evidence of lymphatics in the tail region of living chicks is the appearance of a number of separate knobs, filled with stagnant blood, a little darker in color than the circulating blood, just lateral to several of the most anterior of the dorsal intersegmental coccygeal veins. The connections with the veins cannot be seen, since the knobs lie between them and the observer, but ink injected into the knobs can be seen to pass directly into the main intersegmental veins. ^ Between the separate knobs no anastomoses can be seen, nor can any be discovered by injection.

Soon after these knobs appear (in about fifty-five minutes), similar ones develop about them which have fine connections with them, thus forming a small cluster. The new ones are located partly on either side of the first and partly superficial to them. Their injection now shows discreet tiny clusters, somewhat like bunches of grapes, connected, as were the earliest knobs, with the same intersegmental veins. These clusters are still separate from one another.

There is a rapid extension of these blood-filled structures, and soon, in about an hour-and-a-half after their first appear ^ The injection tests were made with extremely fine capiUary glass canuhie (12 to 20 micra in diameter at the tip), which were inserted directly into the bloodfilled sprout. With care the blood capillaries in their vicinity may be avoided. Bj' using a very slight pressure extremely delicate structures may be filled wthout extravasation. The injections were made under the high power binocular microscope, .so that the progress of the injection might be watched and controlled. This refinement in the injection technic makes possible the direct, clean-cut injection of a .selected capillary, without extravasation. Our method of injection differs only in minor details from that described by B. Mozejko, "Ueber mikroskopische Injektionen nach der Methode dcs Prof. Heinrich Hoyer in Krakau," Zoitschrift fiir wissenschaftliclio ]\Iikroskopie. Bd. xxviii, H. 4, 30 Marz, 1912.



EARLIEST LYMPHATICS IN LIVING CHICKS



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connortions bctwooii lUM^hboriug clusters iiui}' be seen. Injection at this stage with India ink shows an anastomozing plexus, connected, as before, with the intersegmental veins. Injection with silver nitrate shows distinct endothelial niarknigs in the walls of the plexus.

During this plexus formation there is a steady extension toward the surface, and by the time anastomoses have formed between neighboring clusters, sprouts have grown to the surface and started to extend in the region superficial to the plexus and also ventralwards. It now becomes possible to study with more minuteness the changes which are going on, since these sprouts are quite superficial and are developing in a plane parallel with the surface. Observation and successive records of these sprouts in the living chick reveal a rapid extension ventrally and also anteriorly accompanied by a plexus formation. Two or three sprouts are seen to lead and soon numerous connections develop between them. Various portions of the irregular plexus thus formed enlarge and become more densely packed with blood which continues to back up from the vein. Then new sprouts grow^ out in advance and the same process of extension accompanied by plexus formation is repeated. If a single sprout is selected and frequent careful drawings are made, the changes are seen to be rapid and striking. The sprout becomes wider and longer. Branches appear, and they in turn increase in width and length. From a branch a connection forms with the original sprout, thus forming a loop. New branches and connections are formed making a plexus. Branches from neighboring loops or plexuses meet one another and anastomose. The several parts of the plexus are quite irregular in size. Most of the lymphatic vessels are several times as wide as a blood capillary, while some of the connections are as small as or even smaller than a blood capillary. Throughout, the blood in these new forming lymphatics is markedly darker in color than the circulating blood.

During their development the lymphatics show a tendency to avoid the blood vessels, for they spread out in the meshes of the blood vessels and at a slightly deeper level. This tendency



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256 ELIOT R. CLARK AND ELEANOR LINTON CLARK

is shown most markedly in a space over the postero-dorsal angle of the pelvic region. Here there is a small area which is practically without a blood capillary. Into this area one of the earliest of the superficial lymphatic sprouts grows and spreads. This area has been carefully watched for several hours preceding its invasion by lymphatics. Complete blood- vascular injections have been made at this as well as at earlier stages. These studies show that, except for an occasional very fine blood capillary, this is a pure non-vascular area. Moreover, an occasional fine capillary, when present, remains during and after the invasion by the lymphatic plexus.

The injection tests at the various stages have for the sake of clearness been inserted along with the description of the appearance in the living. It should be said that the entire process, starting about an hour before the appearance of the first knobs, and including the formation of these knobs, the clusters, the plexus, and a considerable development of the superficial extensions from this plexus, may be, and several times has been watched in the same chick. Many tests have been made on other chicks which were watched until the desired stage was reached. The time which elapses between the development of the first knobs and the formation of a considerable superficial plexus is somewhat variable, but averages about three hours.

Since the stagnant blood in the interior of the lymphatics is the index on which these studies are based, it was important to determine whether the blood always fills the lymphatics to their tips. This was tested in two ways, by pressure over the part filled with blood, to see whether the blood could be forced further; and by injection. As a result of numerous tests by both of these methods it was found that, in these early stages, practically all the lymphatics, save very fine connections, are usually filled with blood. The injections failed to reveal lymphatics beyond the blood-filled structures previously seen. Hence, since the blood fills the successive extensions of the lymphatic as soon as formed, the use of the stagnant blood as an index for the study of the development of the early lymphatics is justifiable.



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The (lovolopinoiit of the posterior lymph heart from the deep ph^xus has been traced in later stages. The plexus becomes more extensive and more dense and the vessels wider. In chicks between seven and eight days old the contractions of the heart commence while it is still in the form of a plexus. A fuller description of this later development, as well as a description of th(^ further extension of the superficial lymphatics will be given in a later publication.

That the earliest lymphatics, those which form the primary lymph sacs, arise by the transformation of plexuses of blood capillaries, has been maintained by F. T. Lewis, Miss Sabin and by Huntington and McClure. That the extensions of these primary lymphatics into the various parts of the body may occur by the successive addition of parts of the blood capillary system which become cut off and are subsequently added to the lymphatic system, has been suggested by F. T. Lewis and maintained at one time by McClure. Our studies show that both these views are certainly incorrect. Careful observation of the blood vessels at the time just preceding and during the formation of the very first lymphatics shows that the blood vessels through which blood has circulated before the lymphatics appear are still present after the lymphatics have developed. Instead of such a transformation we have seen new blood capillaries developing almost side by side with the developing lymphatics. The same may be said of the lymphatics which extend superficially. Blood capillaries, which have been seen and recorded before the lymphatics have extended into their region, are still present after the lymphatics have reached there. This independence of the two systems is shown most strikingly in the case of the non-blood vascular area already described. If the earliest lymphatics were formed by the transformation of blood vessels, one would expect that, for a time during this

It is interesting to note that the origin of the lymphatics directly from the
veins, as deduced by Miss Sabin in her jBrst paper (Amer. Jour. Anat., vol. 1), proves to be correct. The light thrown upon this subject by means of reconstruction, while of value regarding the form and position of developing lymph channels, has left us in darkness regarding their mode of growth.



258 ELIOT fl. CLARK AND ELEANOR LINTON CLARK

transformation, complete injections of the blood vascular system would show connections between the blood capillaries and the lymphatics. We have made many such injections at each of the early stages of developing lymphatics described. The results have been invariable. The lymphatics, even from the time of their very first appearance, show no connections whatever with the surrounding blood capillary plexus. Their sole connections with the blood vascular system are the ones with the deep intersegmental coccygeal veins, and it is only through these that an injection can be made to pass from the blood vascular system over into these lymphatics. That they are not transformed blood capillaries is also shown by their irregular character, which is totally different from the pattern of the blood capillaryplexus.

Another point which comes out most strikingly in these studies on living embryos is that there is no separation whatever, either in time or manner of growth, between the development of the plexus which is to form the lymph heart and that of the peripheral lymphatics. Simultaneously with the formation of the deep plexus which is to form the lymph heart, sprouts from it may be seen extending superficially and spreading out to form the peripheral lymphatics. This spreading occurs by minute stages, in which there is to be seen the gradual centrifugal extension of the lymphatics already formed. The view held by Huntington and his pupils, and by McClure, that peripheral lymphatics are formed independently of the primary lymph sacs or lymph hearts and that they have no connections with the latter until they have formed an extensive plexus, is shown by these observations to be entirely inadmissable.

The conclusions to which these observations lead us are as follows : The first lymphatics in the tail region of the chick arise as direct lateral buds from several of the main dorsal intersegmental coccygeal veins, and not by the transformation of a previously functioning blood vessel plexus. From now on the lymphatic endothelium is specific, and spreads by a steady centrifugal extension, just as has been observed by one of the authors for the lymphatics in the tad-pole's tail at a slightly



EARLIEST LYMPHATICS IN LIVING CHICKS 259

later stage in development. The buds send out processes forming clusters. From the clusters, in turn, processes are sent out which anastomose with one another, forming a plexus. Simultaneously processes grow toward the surface from the clusters, and give rise to the superficial plexus of peripheral lymphatics of the posterior part of the body. There is no essential difference between the manner of growth of the peripheral lymphatics and that of the plexus which is to form the lymph heart. In fact, the two may be considered as but portions of a single plexus of which the part nearer the veins, from which they have budded off, and with which they maintain connections, develops into the lymph heart.




P'ig. 1 Diagram drawn I'roni a reconstruction of the veins and nerves in the cervical and upper thoracic regions of a chick embryo of five days and ten hours (13.5 mm.) Right side. 1, Precardinal vein; 2, postcardinal vein; S, duct of Cuvier; 4, intersegmental (dorsal somatic) veins; 4a, vertebral (dorsal somatic) vein; 5 lateral group of vascular islands and channels — veno-lymphatics; 9, spinal (cervical) nerves; 9a, brachial plexus.

Figure reprinted from "The American Journal of Apatomy," Volume 12, Number 4, Fig. 3, p. 479, January, 1912, A. M. Miller.

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