THE VALUE OF THE INJECTION METHOD I N THE STUDY OF LYMPHATIC DEVELOPMENT' OTTO F. KAMPMEIER From the Laboratory of Comparative Anatomy, Princeton University SIX FIGURE@ Two papers have recently been published by Professor Sabin3 and Dr. Eliot Clark4 in which they advance several criticisms against the theory of the direct mesenchymal origin of lymphatics, and attempt to prove that blind and discontinuous anlagen of an incipient lymphatic vessel do not exist as such, but in reality are continuous with one another. Probably their strongest contention claims that whenever a developing lymphatic duct exhibits discontinuity such a condition rests on a faulty interpretation, or is due to limitations in the study of serial sections and to the inability of the eye to discover the continuity even with the aid of high magnifications. They see in the injection method the power to demonstrate that the anlage of a lymph channel during its genesis and growth never manifests a phase of consecutive isolated segments or spaces, and they maintain that wax reconstructions which show such segments are consequentlyinadequate, inaccurate and fragmentary. They further contend that many of the spaces described as lymphatic anlagen by Huntington6 1 Read before the American Association of Anatomists a t their twenty-eighth session, December, 27, 28 and 29, 1911. 2 Expense of illustrations borne by author. Florence R. Sabin: A critical study of the evidence presented in several recent articles on the development of the lymphatic system. Anat. Rec., vol. 5, no. 9, 1911. 4 Eliot R . Clark: An examination of the methods used i n t h e study of the development of the lymphatic system. $nat. Rec., vol. 5 , no. 8, 1911. 5 George S. Huntington: Ueber die Histogenese des lymphatischen Systems beim Sauger-embryo. Verhandl. d. Anat. Gesellsch., 1910. The anatomy and development of the systemic lymphatic vessels in t h e domestic cat. Memoirs of The Wistar Institute of l n a t o m y and Biology, May, 1911. 22:$ a i d McClure,R in their studies on the developnient, of the t.horiicic duct and mesenteric lymphatics in the cat, are quite probably artifacts or shrinkage cavities resulting from unequal fixation, silice they occur in the center of the body where the fixing fluid prnetrrates last.7 During the course of the last two years the writ,er has studied the development of the thoracic duct in pig embryos and will soon publish in more complete form a paper dealing with the results of this investigation. Having observed the origin of the duct in the appearance and confluence of discontinuous niesenchymal spaces, he can entirely confirm Huntington’s%essential conclusions in this respect. Invariably all of the pig embryos belonging to the critical period of the thoracic duct, that is, to the period of its inception and actual genesis, positively show a nu)iiher of blind lymphatic anlagen in the pathway subsequent.ly occupied by it. But the writer will not at this time enter into ;t detailed description of any of the specimens examined by him, except of series 23a of the Johns Hopkins University Embryological Collection. This pig embryo was injected with India ink m d prepared by Professor Sabin and was sent t o the Princeton Lahoratory to illustrate, with the developing thoracic duct as $1) e x m i ple, the ‘centrifugal growth’ theory of the origin of lymphatics. This theory holds that the entire lymphatic channel system arises from various radiation centers or lymph hearts by buds or sprouts, which by continual proliferation of their endothelial cells dOllg:tt(? peripherally and, by repeated branching and rebranching, in\-a.de gradually both the deeper-lying and t,he superficial regions of t h e body. “ The thoracic duct,” according to Professor Sabiii’s most recent view, “develops in part as a down growth of the jugular sac and in part, especially its dilated portion or cistern:t chj-li, its a direct transformation of the branches of the azygos w i i i , . . * ‘ g Charles F. W. McClure : The extra-intinial theory and the tle\-elol)nic.n~of t,he mesenteric lympho.tics in the dornestic ent. Vcrimndl. d . .In:~t.( ; c ~ e I l ~ i ~ h . , 1910. Huntington and AIcClure : The dcrclopincnt of the main lyniph clixrIric~ls of thc cat in their relation t o the rciioiis, tenl. .in?. . f o u r . .!n:it., vol. 1 ; . ‘ ~ ~ 0 7 . .ibstr. : h i t . Rcc., vol. 1, 1006 -0s. Sabin: 1911, p. 440. Huntington: 1911. 9 s:Ll)irl:I!lll. p. 421. It n-ill b~ w ~ w hon-c.\-(~, , that one can scarcely imagine a more radical rpfutation of the criticisms mentioned, as well as of the view of the centrifugal budding of lymphatic ducts, than that offered by series 23a. Brforc. presenting the evidence, I wish to acknowledge my jidebte(1iiess to Professor Sabin for the privilege of studying the errhryo in question. Series 23a, an embryo measuring 23 mm. in length,1° displays meellent, fixation and preservation. The injection was also succe4’ully carried out and is as perfect as a developing lymph vessel permit.;. The evidence for the discontinuous and direct mesenchj-ma1 origin of the thoracic duct is distinctly portrayed in the acroiiipaiiying figures. Fig. 1 represents a simplified or schematic drawing based on an accurate reconstruction of the thoracic duct regioii, and the microphotographs (figs. 2, 3, 4 and 5) picture actual cross-sections taken a t four different levels, as indicated on t hc diagram by the transverse lines and numbers. T h e jugular lymph sac and that segment of the thoracic duct, arilage connected with it were injected, and therefore they are shon-n iii black on the drawing. Near the sac the most anterior portion of the embryonic duct is in the form of a broad and extensive plexus, a typical section of which is reproduced in fig. 2, illustrating the large size of the channels (t.p.) and the extravasatioiis of the injection inass ( X ) into the surrounding mesenchyme. To consider the right limb of the thoracic duct anlage first, the injected vessel extends unbrokenly backward and dextrad ton-ards the right postcardinal (future azygos) vein as a slender chaiiiicl (fig. l), and terminates shortly below the level of the ( ’uriwian duct in a ‘mossy7area produced by slight extravasations. Kegoiicl this point we meet with the most decisive evidence in favor of the non-venous origin of the thoracic duct namely, a clear case of discontinuity, than which nothing could be more concluI d When it is considered t h a t the processes of fixing and hardening reduce thc length of pig embryos, for example, bv one t o one and a half millimeters, it is beeii thnt this 23 iniii. embryo (scrip5 2 3 : ~ uicasured ) before fixation, is approxim:ttel> equivalent 111 :\ptx to 21 5 timi. embryos, the length of mhirh was oht:tinrtl xftct flutiori. 226 o m v .'1 KAMPMEIER sive. Immediately following the injected vessel is a long blind fusiform space, but in no way coniiected with it, as exemplified by the drawing and the microphotographs. I n fig. 3 the position of the mossy extravasations from the end of the injected vessel is indicated at X , ventral to the broad lumen of this Iongindependent space (id), the section having just passed through its most anterior tip or beginning (fig. 1). That there is absolutely no open comniunication between these two segments of the thoracic duct anlage is strikingly confirmed by both observation and experiment. In the first place the most critical examination with the aid of high magnifications was not able to detect continuity, and secondly, not a particle of the injection mass was found to have entered the cavity of the blind space, although the pressure of the injection was sufficiently great to produce the extravasations referred to above. The long fusiform lymphatic space of the right side is of considerable length (fig. 1),capable of being followed through thirtyseven sections (thickness of sections: 20 micra), and is variable in diameter, a t times being broad, and at other times narrow and not so sharply demarcated from the intercellular lacunae of the tissue surrounding it. In form it is very irregular, and its lumen is often bridged by mesenchymal strands of greater or lesser thickness which give to it a multilocular appearance as shown in figs. 4 and 5 (t.d.). This condition, coupled with the fact that it is bounded by ordinary unspecialized tissue cells, supplies strong proof against the view of its venous origin. Fig. 5 also illustrates the occasional circumclusion of venules (v.) by this space and draws even more plainly the distinction between lymphatics and venous channels, where the latter are replete with blood and possess clearly defined boundaries as compared with the often indefinite outlines of a lymphatic anlage. Caudally the long space becomes more indistinct until it vanishes by the loss of its cavity in the confusion of the interstices of the mesenchymal reticulum, but after a number of sections it is followed by a second space, which though shorter and simpler (fig. 1) exhibits the same peculiarities of character. This again is followed by tissue, which, as yet undifferentiated, is coarsely reticulate and persisteiitly sug- INJECTION METHOD-LYMPHATIC DEVELOPMENT 227 gests the potentiality of further lymphatic anlagen. Both of the spaces described and figured are situated in the same axis of the injected channel and consequently in the axis of the ultimately complete thoracic duct. On the left side n series 23a we find the principle of development to be the same and to be expressed fully as well. The injected segment of the left thoracic duct is shorter (fig. 1) and more slender than its homologue on the right side, and often it can only be traced by a mossy path due to slight extravasations. At intervals beyond the farthest extent to which the injection mass has penetrated and located in a line destined to become the pathway of the future thoracic duct are a number of small blind mesenchymal vacuoles, the largest one extending through eight sections at the level of the Cuvierian duct. The conspicuous size of the lumen of the last space, the mesenchymal strand bisecting it, and the compactness of the neighboring tissue are clearly shown in the photograph, fig. 4 (t.s.). From the observed facts it is seen that the argument for continuity in the anlage of a lymph channel and its demonstration by the method of injection do not hold, at least not in the case of the developing thoracic duct in pig embryos. Instead, the use of the injection method coupled with the study of serial sections emphasizes the discontinuities in the incipient duct. Herein lies the primary value of this technic; it defines the territory in which the fundamental period of development of such a channel has already been completed. But to arrive at a knowledge of the histogenic processes in this history it is found to be practically useless; for it is obvious that the injecting substance obscures the exact outlines of a channel, and therefore a comparison between the cells bounding the lumen and the cells of the surrounding tissue is made impossible. On the evidence of the few microphotographs inserted here the conspicuous presence of the spaces designated by the writer as thoracic duct anlagen can not be denied. Nor can they be regarded as artifacts, for not only is their normal appearance in the figures contrary to such a view, but also the fact that they occur only in the pathway of the subsequent duct and its tribu- 222) OTTO F. KAMPYEIER taries, and are not found elsewhere in this region. Moreover, froin the evidence of a large number of pig embryos it can he firnily established t.hat these multiple spaces or anlagen appear only at a definite period in embryonic history, during the critical genetic stages of the thoracic duct, or in other words, just precediiig it.s completion as a continuous structure. An examination of embryos of the proper consecutive ages will show plainly such anlagen beginning as minute mesenchymal vacuoles, which become progressively larger (figs. 1, 4 and 5), longer, and finally confluent with one another in a general centrifugal direction. The extent to which the injection mass will penetrate a t any given niornerit of development therefore indicates or measures the distance in which such confluence has already occurred. Fig. 6 represents a t'ransverse section from a later stage (26 mm. pig embryo), the thoracic duct of which has become a continuous channel throughout from the jugular lymph sac to the cisterna chyli and the inesenteric and posterior lymphatics. Taken at approxiniately the same level as fig. 4, this figure may be compared with it, as showing the right and left limbs of the duct in the same topographical position as the spaces which have been described as its anlagen. If the discontinuous anlagen of the thoracic duct, as shown in the sections of series 23a, were to be regarded as art,ifacts, it appears to the writer that the same contention should apply with equal force to the continuous thoracic duct as shown in transverse section in fig. 6, especially to the right limb which is seen to be no more definite in outline or boundaries than t'he space illustrated in figs. 4 and 5 (id.). Vig. 1 Aisimplificd or aclicmatic drawing of an accurate reconstruction (dorsal view) of the thoracic duct region in the pig embryo, series 23a (Johns Iiopkins University Embryological Collection). The lymph sac and the injeckd portion of t h e thoracic duct anlage are shown in black; the uninjected lymphatic spaces arc tliscoritinuous but are located in the axes of the injected channels, and consequeritly in the pathway of the future complete thoracic duct.. 'The cross liiirs indicntc the lcvcls :It which figs. 2>3, 4 and 5 were takcn. J U G U L A R L Y M P H SAC (IN J ECTEO) E X T E R N A L JUGULAR L L.THORACIC DUCT UNI NJ E C T E D L Y M P H A T I C SPACE LEFT P R E C A R D I N A L I ~ ~ R-THORACIC DUCT - UNINJECTED LYMPHATIC SPACES RIGHT DUCT O F CUVIER 3 4 L. L E F T POSTCARDINAL U N l N J E C T E D LYMPHATIC SPACES L E F T SUPRACARDINAL ( AZYGOS ) R I G H T SUPRACARDINAL [A Z Y W ) 229 Fig. 2 'h:insvcrse section through the left lower cervical region in pig embryo 2% (J. 11. U.E. C., slide 21, section 16), 200; t.p., anterior lymphatic ~ 1 ~ x of 3s t h e thoracic duct, injected; X., extravasations of the injection mass int,o the SICmunding nitwnchyme; .J'.v., internal jugular; s.n., sympathetic n r r w trunk; 0.. x oesoph:~~iis. Fig. 3 'L'r;~rtsvc~rsc swtion taken iie~trtlic level of thc: riglit C'rivicri:in drict in pig cwibryo 233 (.7. H. 1.E. C., slide26, section lo), X 200; t.d., anterior tip of the long fiisiforrn lympliutic space of the right thoracic duct line; X . , positiori of extravas:tt(~lp:trt,iclcs from the injected portion of thc right thoracic duct; v., venules, deriv:itivcs ;md tributaries of the postcardinalvein ( p . d . ) ;a., aorta; o.,oesophagus. T h e IIIOI'(L delicate lining of the lymphatic space as compnrrd with that of the veins anti vc,niilcs c:in hc v l i ~ : ~ r ltlist,ingnished y in the figurr. 2:w Fig. 4 Transverse section taken shortly beyond t h a t of the preceding figure (series 23a, J. H. U. E. C,, slide 26, section 12), X 200; t.d., long fusiform space in the right thoracic duct line, and mesenchymal bridges traversing its lumen; t.s., blind lymphatic space a t t h e level of the left Cuvierian duct (d.C.), and in t h e path of the left thoracic duct; o., venules, branches of the postcardinals; p.d., P.s., right and left post-cardinals; a., aorta; o . , oesophagus. Fig. 5 Transverse section through the thoracic region in pig embryo 23a (J. B . U. E. C., slide 27, section 18), X 200; t d . , long fusiform lymphatic space in the right thoracic duct line, and surrounding a venule (u.),or tributary of the right post-cardinal vein (p.d.,); a., a0rt.a; o . , oesophagus. 2:11 232 OTTO F. KAMPYEIEII Fig. G 'I'ransvcrse scction t a l ~ e nne:tr the l c ~ do f t,hr left Cuvieriaii duct in R 26 min. pig embryo (series 69, Princeton Embryological Collection, slide 12, section 9), X 150; t.d., t s . , right and left thoracic ducts; p.d., P.s., right and left postcardinals; d.C., left Crivieri:in duct: u., aort:r; o., oesophagus.