Talk:Paper - On the development of lymphatic nodes in the pig and their relation to the lymph hearts (1905)
The Development Of The Lymphatic Nodes In The Pig And Their Relation To The Lymph Hearts
Florence R. Sabin. From the Anatomical Laboratory, Johns Hopkins University.
With 17 Text Figures.
Notwithstanding the numerous investigations on the lymphatic nodes, there are many points in regard to their structure and development in which our knowledge is not yet clear.' For example, such a fundamental question as the relation of the nodes to the lymphatic system as a whole and to the vascular system, in other words, the prohlem of general morphology: or, more special questions in regard to the nodes themselves; primarily the existence of a structural unit; and secondarily the relations of the endothelium of the channels to the reticulum within the node, whether the channels are open or closed, and the origin of the lymphocytes.
In regard to the problem of general morphology, we have previously shown " that the lymphatics are modified veins. They develop as bloodvessels do, by the budding of endothelial cells, and the direction of their growth is determined by the arteries and veins. In the lymphatic system there develop four lymph hearts which pulsate in the amphibia; but in the mammalian forms, at least in pig and human embryos, have no muscle in their walls. These lymph hearts drain the body, that is to say all the lymph passes through them before entering the veins. It will be shown in the present paper that the first lymph nodes in the body develop from the lymph hearts. That is, the organ which was a pulsat ^ During the past eleven years, there have been four extensive researches on the development of lymph nodes by Gulland, Saxer, Retterer, and Kling. Each of these authors has reviewed the literature. Gulland, Journal of Pathology and Bacteriology, Vol. 2, 1894; Saxer, Anatomische Hefte, Bd. VI, 1896 Retterer, Journal de L' Anatomie et de La Physiologie, 1901 Kling, Upsala Lakareforenings Forhandlinger, 1903, and Archiv f. mik. Anat. u. Entwicklungsgeschichte, Bd. 63, 1904.
^Sabin: American Journal of Anatomy, Vol. I, 1902. American" .Tocrnal of Anatomy. — Vol. IV.
356 Developiiiont of tlie Lyniphatie Xoilos in the Pig
ing heart in the aniphil)ia becomes transformed into a node in the higlier forms. The lym])li heart is the point from which ducts radiate in development to drain a large area, generally speaking a quarter of the surface of the body, and here the primary lymph node develops. Subsequently there are secondary points, from which ducts growing from the Ivmph heart radiate to drain lesser areas, and here other nodes are formed. These are subcenters through which lymph passes before reaching the primary node.
As to the nodes themselves, the study of their development Ijrings out that they are made of two fundamental structural elements. First, a lymphoid or adenoid tissue, consisting of lymphocytes in a reticulum around the blood-vessels making the lymph cords and germ centers; and secondly a lymphatic tissue, or sinus, made of large numbers of lymph ducts closely packed together. In brief, an ordinary lymph node is a blood vascular organ, made in part of structures derived from the bloodvessels and in part of structures derived from the lymphatics. The vascular unit of the node is the terminal artery and its capillary plexus, the artery being bordered by the cord and the capillary tuft surrounded by the germ center. These two elements, the vascular and the lymphatic, are found in varying proportions in the ordinary node. However, both in the embryo and in the adult, either element may be found alone. In the emliryo and probably in the adult pig, there are small lymph follicles consisting of a tuft of blood papillaries surrounded by lymphocytes and entirely without a sinus. In the hfemolymph node and in the spleen the same type of lymphoid tissue is found, but here the sinuses are made, not of lymph ducts, but of veins. Thus in the blood vascular organs the lymphoid element consisting of lymphocytes in the adventitia of an artery is constant, while the sinus element varies, being absent, or made of veins, or of the modified veins wdiich are called lymphatics.
Throughout the paper certain terms have been adopted. Lymph node is used as a general name to cover all lymphatic glands; the term follicle is used to represent a simple node consisting of the structures that go with a single artery. The simplest node consists of one follicle; other nodes are groups of follicles. The follicle is the anatomical or structural, unit; it is also the vascular unit. The follicle may be without a sinus, or surrounded by a lymphatic sinus or surrounded by a venous sinus.
The term lymph heart has been retained notwithstanding that in the pig there is no muscle in its wall at any stage. It is, however, a sac from which all the ducts for the skin radiate and in that sense is homologous with the lymph heart of the amphibia.
Florence E. Sabin 357
Material and methods. — The material for the present study has been embryo pigs of all stages. In studying the development of the nodes, just as in studying the development of the ducts, it is essential to employ injections, both lymphatic and arterial, and these injections have been made in every stage. The lymphatics have been injected by means of a hypodermic syringe, with either saturated aqueous Prussian blue or with India ink. The material has been preserved for the most part by the injection of a saturated solution of bichloride of mercury, either into the aorta or into the umbilical vein. The blood-vessels are usually first washed out with warm salt solution, then the bichloride introduced and continued until the embryo is hard and white. The injection is made slowly with a pressure of about 100 mm. of mercury and the bichloride allowed to stay in the vessels from one-half to two hours. It is then washed out thoroughly by injecting 70 per cent alcohol through the same canula. The embryo is then placed in 80 per cent alcohol over night and the next day transferred to 95 per cent alcohol. This method involves the least possible shrinkage, indeed it may be made to produce a slight distension of the tissues, which is an especial aid in studying lymphatic nodes.^
In studying the developing nodes in fresh tissue, it is readily noticed that they ^are sometimes found distended with fluid and sometimes collapsed. It is just as easy to tell with the unaided eye when a node is thus distended with lymph as to distinguish between the mesenteric lymph nodes distended with chyle or collapsed and empty. This method of injection produces the same distension of the spaces that occurs normally when the node is in active function; that is to say, it makes the lymphatic ducts rounded rather than collapsed. This explains the especial value of the method as applied to lymphatic tissue.
A valuable aid in the localization of the nodes, and especially in studying the relations of the lymph hearts to the developing nodes, is found bv making injected embryos transparent.' The lymphatics are first injected with India ink and then the entire embryo is placed in 95 per cent alcohol. They are left in the alcohol until they are shrivelled. This takes at least two weeks. The embryos are then cleared in a dilute solution of potash from 1 to 2 per cent, taking from 1 to 4 hours. The specimens are preserved in glycerine, at first 20 per cent and later in pure glycerine.
^ McFarland : Jour, of App. Microscopy, Vol. II, No. 10, and Myers, Ibid., Vol. VI, No. 12, and J. H. Bull., 1905.
■* Mall: American Journal of Anatomy, Vol. IV, 1905, p. 6.
Developineut of the Lymphatic Nodes in the Pig
The hjmpli heart. — The present paper is a continuation of two papers previously presented in this journal, the first in Vol. I, 1901, and the second in Vol. Ill, 190-1. It has been shown ^ that the lymphatics bud off from the veins at the root of the neck, grow along the internal jugular vein on either side, and expand into a sac in the neck. This sac or lymph heart is shown in Fig. 1 as it appears in the neck of a pig 2.7 cm. long.
Fig. 1. Embryo pig, 2.7 cm. long, showing the anterior lymph heart injected. X about 41/1-. E, extravasation at the point of injection; Id, lymph ducts leading from the extravasation to the lymph heart.
The lymphatics were injected with India ink by a hypodermic needle introduced into the ducts over the shoulder at the point marked by the extravasation in the figure. The relation of this sac to the veins is shown in the accompanying diagram. Fig. 2. One duct of the sac liesalong the course of the internal jugular vein. Just below the ear, under cover of the sterno-cleido-mastoid muscle, this duct widens into a sac which makes an arch in the neck. The sac curves outward and down
Ibid., Vol. I.
Florence E. Sabin 359
ward with the apex, marked a in the diagram, near the surface, adjacent to a superficial vein over the shoukler. This apex is to be found in the triangle between the sterno-cleido-mastoid muscle and the trapezius. From the apex of the sac, a duct follows along the vein of the shoulder and empties into the duct along the internal jugular vein. This duct becomes the main duct of the sac.
Figure 3 is a section of the base of the sac at about the level marked b in Fig. 2. The section is cut transversely through the neck of a pig 2 cm. long and passes through the larynx. It shows the relation of the lymph heart both to the internal jugular vein and to the sterno-cleidomastoid muscle. The lining of the sac consists of a single layer of endothelium.
Fig. 1, which shows the lymph heart and its ducts in a pig 2.7 cm. long, is to be compared with Figs. 1 and 3 in this journal, Vol. Ill, 1904, pp. 184 and 185, which show the superficial lymphatics in pigs 2.5 cm. and 3 cm. long.
From these three figures it can be seen that the lymph ducts or capillaries grow
J, ,, " J, ,, n , , ii 1 • I'lG- 2. Diagram showing- the
irom the apex Ot the sac first to the Skm relation of the lymph heart, of
of the shoulder and back of the head. Then hea'rt'is in soiid^bfacii. '!i,^apex
-, , „ -, p ., „ ,, of the lymph heart: b, base of
ducts grow forward from the apex ot the sac the lymph heart; ejv, external
, , .. I? , 1 , 1 ■ T . • 1 jugular vein : /i\ facial vein ;
over the surface of the sterno-cleido-mastoid O'l', internal jugular vein, muscle, and form a long plexus around the
external jugular vein parallel to the anterior border of the muscle. This plexus is shown in a little later stage in Fig. 6, and in section in Fig. 5. From this long plexus ducts first grow to the face as is shown in Fig. 2, Vol. Ill, p. 185. Later on it will be shown that at this stage, namely, when the pig is 3 cm. long, the apex of the sac begins to be transforined into a lymph node.
The condition of the lymphatic system in the neck of a pig 3 cm. long is as follows: There is first the lymph heart with its efterent ducts connected with the veins; then ducts have grown from the apex of the sac first to the skin of the shoulder and back of the head, and secondly to the face. The sac shows also the first rudiments of a lymph gland.
The maximum size of the lymph heart is attained when the pig is 3.6 cm. long, and in Fig. 4 is given a flat reconstruction of the sac at this stage. It was made from a set of serial sections and gives the size more accurately than the potash specimens. Fig. 5 is a section from the
Development of the Lymphatic Nodes in the Pig
same series taken at the level marked h on Fig. 4. Tt corresponds with Fig. 3, and shows a similar relation to the internal jugular vein and the sterno-cleido-nuistoid muscle. It shows that the sac comes nearest the surface between that muscle and the trapezius. It also shows the external jugular vein at the anterior border of the sterno-cleido-mastoid muscle and the neighboring plexus of lymph ducts. A section about half way between the letters a and h on Fig. 4 shows the heart near the surface and the duct adjacent to the vein as two distinct cavities.
Fig. 3. Transverse section through the neck of an embryo pig, 2 cm. long, showing the anterior lymph hearts, x 15. Ijv. internal jugular vein; I, larynx; p. pharynx; son, storno-cleido-mastoid muscle; sh, sympathetic nerve; rn. vagus nerve.
Fig. G is aiiotlier specimen made transparent in potash. It is from a pig 6 cm. ](Mig and is to 1)0 compared with Fig. 5, Vol. Ill, p. 188, which is from a pig 5.5 cm. long. The former shows the lymjDhatics in the depth and tlie latter those of the surface at al)0ut the same stage. The injection for this specimen was made in two places: one just back of the fore leg as marked by the extravasation ; from this injection the ducts over the shoulder, the lymph sac, and two lapge ducts running to the long plexus were filled. The second injection was made l)etween the eye
Florence E. Sabin
. and ear, by which the capiHaries over the face and the long plexus in the neck were filled.
In Fig'. G the lymph heart is still plain, showing as a triangle in the depth. It has modified somewhat in shape, inasmuch as lymph nodes are forming at the apex and base, making the angles of the triangle appear as knobs. The apex of the sac is now labeled primary lymph node, and the base is lettered h. The actual size of the triangle as a whole is not greater than when the embryo was 3.6 cm. long. That is to say, the distance from the apex to the base is about 4 mm. in either case.
The ducts over the shoulder from the apex of the sac are well injected, these being the first set to develop. The set of ducts which grows forward from the apex of the sac over the surface of the sterno-cleidomastoid muscle to make the long plexus in the neck shows somewhat, but is not as well injected in this specimen as in Fig. 7. They are present at this stage but the injection from the region near the eye was not pushed quite far enough to bring them out well. This set of ducts develops into the long and abundant plexus which follows the course of the external jugular vein as it lies parallel to the sterno-cleido-mastoid muscle. From this long plexus, the entire face, front of the neck, fore leg, and thorax are supplied with lymphatics, and these different sets can be seen in Fig. 6. All of these sets of ducts anastomose in the skin, as can be seen in Fig. 5, Vol. Ill, p. 188.
In brief, the ducts for the shoulder and back of the head grow directly from the lymph heart; those for the face, neck, and fore leg grow from the lymph sac, but form a long plexus along tlie external jugular vein before reaching the skin. As has just been said, both sets of ducts, distinct in the depth, anastomose in the surface. The line of growth of the lymphatics has been tested by a large number of injections in every stage from the time tlie lymphatics first appear up to the time of birth. Every injection made into the ducts of the skin of the anterior part of the body will run to the lymph sac or the gland derived from it, if pushed far enough. The different systems of ducts of the neck can be brought out by injecting in four different places. When the needle is entered over the shoulder the injection mass invariably runs to the apex of the 28
Fio. 4. Diagrara of the anterior lymph heart in an embryo \ng, 3.6 cm. long. X 10. A, apex of the heart ; h, base of the heart and level of Fig. 5; ijv, internal jugular vein.
Dt>velo])iiu'iit of tli(> TAiiii)liatic Xoiles in tlie Pig
lymph sac; occasionally it enters tlie surface duets that anastomose with the long plexus. When the needle is introduced into the layer of the lymphatics between tlie eye and ear, or over the lower jaw and front of the neck, or into the pads of tlie fore feet, the injection mass runs into
Fig. 5. Transverse section through the neck of an embryo pig, 3.6 cm. long. X about 11. The shape of the entire heart of which this figure shows a section is given in Fig. 4, in which the line b is the level of Fig. 5. Ca, carotid artery; ejv, external jugular vein; ijv. internal jugular vein; Z, larynx; Ih, lymph heart; Ip, lymph plexus along the external jugular vein; nv, vagus nerve; oe, oesophagus; scm, sterno-cleido-mastoid muscle; sn, sympathetic nerve.
the long- plexus and across the sterno-cleido-mastoid muscle to the apex of the lymph sac. This general relation is not only true in the stages already pictured, but in the later stages when the apex of the lymph heart is a lymph node and the long plexus has been replaced by a chain of lymph nodes.
Florence 1». Sal)in
As is seen in Fig. (!, the duets which connect the lynipli sac and the loiiii' plexus, join the ])lexus half way between the ear and the fore leg. Once or twice, out of many injections in which the needle w^as introduced between the eye and I'ar. tlie injection mass lias reached the veins in two ways: one the usual course through the lymph heart, and secondly, through ducts that follow the course of the external jugular vein to its junction with the internal jugular, showing that the ducts along the two veins anastomose.
Fig. 6. Lymphatics in the neck of an embryo pig, 6 cm. long, showing the modified lymph heart in the depth and the plexus of lymphatics along the external jugular vein, x about 3. B. lymph node developing in the base of the lymph heart; e, extravasation at the point of injection; Ip, long plexus of lymphatics along the course of the external jugular vein; pin, primary lymph node developing in the apex of the lymph heart.
Fig. 7 is from a pig 11 cm. long and shows an injection of the lymphatics made from two points : one between the eye and the ear, and the other into the foot pad. The injection mass, both from the ducts of the face and from the fore leg, has entered the long plexus and then
Development of the Lympliatic Nodes in the Pig
passed through ducts that lie over the sterno-chleido-mastoid muscle into the node representing the l3anph heart (pin). At this stage there are two lymph nodes at the angle of the jaw, nf, one deeper, receiving the ducts around the eye and cheek, the other more superficial, receiving the ducts just in front of the ear. The rest of the long plexus is also being modified into lymph nodes, one of which is in the middle of the plexus where the ducts join with the lymph sac, the other is at the posterior end of the plexus and drains the fore leg (nfl).
Fig. 7. Lymphatics in the neck of an embryo pig, 11 cm. long, x 1%. Lp, lymph plexus which lies parallel to the external jugular vein; nf, nodes developing in the long plexus draining the face; nfl, node developing in the long plexus and draining the fore leg; pin, primary lymph node between the trapezius and sterno-cleido-mastoid muscles.
Since the spread of the superficial lymphatic capillaries in the skin of the pig is practically complete when the embryo is 6.5 cm, long, it may be well to sum up the superficial lymphatic system at that stage. In the neck there is, in the depth, the lymph heart now considerably modified by the formation of lymph nodes. It has one large efferent duct along the internal jugular vein and one along the main superficial vein of the shoulder. Secondly, there is a plexus of ducts along the external jugular vein; this plexus connects freely with the lymph heart. Lymphatic nodes in the neck are to be found developing first from the lymph sac, secondly in the long plexus on the course of the external jug
Florence E. Sabin 365
ular vein, and thirdly in the depth along the internal jugular vein. In the surface the capillaries have grown from the apex of the l3-mph heart to the shoulder and back of the head and from the long plexus to the face, neck, thorax, and fore leg. The capillaries of all these sets of ducts anastomose freel}^ in the skin and there are no valves to check the spreading of an injection mass. The lymphatics of the axilla belong to the deep set which grow along the arteries rather than the veins.
The spreading of the lymphatics for the lower part of the body can be constructed from Fig. 5, Vol. Ill, p. 188. The position of the posterior lymph heart is just caudal to the kidney, and at this point a h'mpli node develops. The superficial lymphatics for the lower part of the body grow in two directions, one set following the vein to a point over the crest of the ileum, where a node is formed which drains the skin of the back and hip; the other set coming to the surface in the inguinal region where a long node is formed which drains the abdominal wall and the hind legs. These three nodes with the abundant chain of nodes along the aorta represent the distribution of the glands which drain the skin of the lower part of the body. In following the histogenesis of the lymph nodes all of these different nodes have been studied, but most of the figures presented are from different stages of one node, namely, the first one to develop in the body.
Histogenesis of the primary lymph node. — We turn now to the histogenesis of the lymph node, which will involve determining the structural unit, and tracing the two elements, the vascular element with the adenoid tissue, and the lymphatic element or sinus.
The first lymph node in the body develops from the apex of the lymph heart and will be referred to as the primary lymph node. This node will be traced in its development until its condition, is practically adult.
The first evidence of the formation of lymphatic nodes occurs when the embryo is 3 cm. long. At this stage the lymph heart, which has been a smooth walled sac, as shown in Figs. 3 and 5, lined with a single layer of endothelial cells, begins to show a slight modification at the apex in that bands of connective tissue begin to push into the lumen Avithout destroying the lining. The apex of the lymph sac is pictured in Fig. 8 from a pig 3.6 cm. long. The section is taken from the same series as Fig. 5, by which the transverse plane of the section can be noted. The figure shows the character of the surrounding tissue consisting of a syncytium of protoplasm with nuclei in the nodes. The wall of the sac consists of a single layer of endothelial cells, and in the left hand side there is no perceptible modification of the connective tissue. On the right
Dcvelo])nK'nt of the Lvinphalic Nodes in the Pig
side, however, bands of connective tissue project into the sac without destroying its endothelial lining at any point. The connective tissue in these bands and on tlie right border of the sac appears different from the surrounding tissue.
Studied with the oil immersion lens, the surrounding connective tissue appears as described by J\[all " to be a network of granular protoplasm in
Fig. 8. Primary lymph node from a transverse section of the neclv of an embryo pig, 3.6 cm. long, x about 60. The left side of the figure is the mesial side of the heart, the right side is toward the skin and shows the afferent ducts. The top of the figure is the position of the hilum of the node. Ld, lymph duct (afferent) ; lU. lymph heart; v, vein.
which are distinct anastomosing fibrils. The nuclei lie in the nodes of the network and each one has around it a drop of clearer protoplasm which he calls endoplasm, distinct from the rest or exoplasm. Xear the lymph sac, on the right hand side in the figure, are numerous blood capillaries and around each one are clumps of from 8 to 20 nuclei. These
"Mall: American .Journal of Anatomy, Vol. I, 1902.
Florence R. ^^al)in 367
nuclei lie definitely within tlie tiyncytiuni and belong to the connective tissue. They are only to be distinguished l)y the fact that they are in clumps and that some show karyokinetic figures while others are smaller and take the deep stain of a newly divided nucleus. In short, cell proliferation takes place around the capillaries.
Passing now to the bands or bridges of connective tissue, the first point to be noted is that there are numerous blood capillaries filled Avith red blood cells, many of them nucleated. The same sort of protoplasmic network is present as in the surrounding tissue, but the network is denser and the meshes finer. The increase seems to be in the granular protoplasm rather than in the fibrils. In this dense network of protoplasm are crowded many connective tissue nuclei ; the mature ones are oval in shape and take the stain faintly. Many of the nuclei are dividing, and there are numerous small, round, deeply staining, young nuclei. These round nuclei belong, however, to the connective tissue and there are no true wandering cells outside of the blood-vessels. Thus the modification of the tissue around the sac consists merely of an increase in the blood capillaries, and in the connective tissue protoplasm and nuclei. The cell increase does not take place independently of the blood capillaries. There is no muscle in the wall of the sac at any time. By the time the embryo is 3.6 cm. long a second node is Just beginning at the other end of the sac. This second node from the lymph sac develops in the same manner as the first, but slightly later.
We pass now to the primary lymph node when the embryo is 4.9 cm. long, as shown in Fig. 9. The section is taken in the same plane as Fig. 8, that is, it is from a set of transverse sections. The efferent ducts are on the right and the hilum at the top of the section. There are no striking changes between this and the preceding stage. The node as a whole has increased considerably in size. The lymph heart is about the same actual size as in Fig. 8, but the lymphatic plexus is greater. From Fig. 1 it can be seen that when the ducts first start out from the sac they grow directly to the skin, but in Fig. 9 there has been an anastomosis or plexus formation of the ducts on the border of the sac. This greatly extends the area of the node. On the left side of the sac there are a few blood capillaries with clumps of dividing nuclei around them. The bands of connective tissues shoAv the same abundance of blood capillaries and increase in the protoplasm and nuclei. Young and dividing nuclei are abundant, Init no true wandering cells are present.
A more important stage is met with when the embryo is 7 cm. long. From this stage on, the development of the primary lymph node is shown
Develo])nu'nt of the Lviiiplialie Xodcs in the Pig
in a series of five diagrams. Each diagrain is made from a single section traced with the aid of the camera lucida. The blood-vessels are put in freehand from the study of the complete set of serial sections from which each diagram was made. All the figures are of the same magnification.
Fig. 9. Primary lymph node from a transverse section of the neclv of an .embryo pig 4.9 cm. long, x about 44. The section is placed similar to Fig. 8. Ld, afferent lymph duct; Ih. lymph heart; v, vein.
about 33 diameters. The lymphatic vessels are shown in solid black as if injected, while the connective tissue of the hanph cords and follicles is dotted. In the later stages the increased number of the dots represents the increase in lymphocytes and the lines show the beginning capsule and trabecule.
Florence E. Sabin
In the first diagram, Fig. 10, tlie step in advance beyond the stage of Fig. 9 is in the proliferation of the lymphatic capillaries. The sac has been completely cut up into ducts. The entire node consists of a plexus of lymphatics which differs in no way from the plexus in the skin pictured in my first paper. There are the same swollen bulbs, the same blind sprouts and slender channels. The connective tissue bridges are similar to those of the preceding stages. They contain many dividing cells but no true wandering cells. In the bridges is an abundant plexus of blood capillaries which are not shown in the diagram. This diagram might also represent any lymph node which develops in a plexus.
To sum up, the figure marks the culmination of the first stage of the development of the lymphatic nodes in early embryos, namely, the stage in which the node consists of a plexus of lymphatic capillaries separated by bauds of connective tissue which is denser than the surrounding tissue. This stage is shown in Kling's ' models. Fig. 1. The connective tissue is embryonic in type, consisting of a net work of granular protoplasm with a few fibrils and with many nuclei. The bands or bridges have blood capillaries and the increase in connective tissue does not take place independently of them. There are no true wandering cells outside of the blood capillaries. It is the stage of lymphatic ducts and pure connective tissue bridges. All of the nodes of the early embryos, the primary nodes in the sense of Gulland pass through this stage. That is to say, the nodes which develop in the long plexus in the neck from which ducts radiate to the face, neck, fore legs and thorax (Fig. 6) ; or the node which comes in the inguinal region at the point where tlie ducts radiate over the abdominal wall and hind legs; or in the node over the crest of the ileum where they radiate over the back (see Fig. 5, Vol. Ill, p. 188). All of these nodes come in places, where plexuses are formed because ducts radiate over a wide area, which is shown well in the figure just quoted. They are primary nodes in the sense of Gulland because they develop early and drain large capillary areas. It will be shown subsequently that lymphatic
Fio. 10. Diagram of the primary lymph node in an embryo pig- 7 cm. lonjr. X about 53. The lymphatics are in solid black and the connective tissue bridges are dotted.
Development of the Lvniphatic Xodes in the Pig
nodes which develop later in the life of the embryo, after lymphocytes occur, hurry through the prinuiry process, and show a considerable modification of it.
Up to this time the node has had none of the structures characteristic of the adult node; there are no lymph cords, nor germ centers, no lymphocytes, and no sinuses.
The next stage, pictured in Fig. 11, shows the beginning of some of these structures. The diagram is made from a section of the primary lymph node in a pig 8 cm. long. In the center of the node the blood capillaries have proliferated, giving a tuft of capillaries surrounded by
Fig. 11. Diagram of the primary lymph node in an embryo pig, 8 cm. long. X about 33. This represents the primordial follicle. The hilum is marked by the artery. A. artery; aid. afferent lymph duct; eld, efferent lymph ducts; f. follicle.
connective tissue. The artery is shown leading up to the node but reduced to capillaries on entering it. The vein is not shown in the diagram, but the artery and vein lie parallel, up to the point where the node or follicle is entered, where they separate. This is an important ancT characteristic point in the relation of the blood-vessels. At this stage there are only capillaries within the node.
Here for the first time we can speak of the lymph follicle, wliich is the vascular unit and consists of the structures that go with a single artery. At this stage the entire node is one follicle. Here also for the first time two elements are differentiated, a lymphoid element connected with the artery and a lymphatic element made of lymph ducts.
Florence 11. Sabin 371
The point of entry of the artery determines the hihim of the node. The position of tlie hiiuni is determined from the lieginning of the formation of the node hy the lines of growth. Hh^od-vessels and lymphatics grow from the center of the body to the periphery, so that the proximal surface of the gland has from the start the entering blood-vessels and the efferent lymphatic ducts, while the peripheral surface of the node is the place from which the efferent lymphatic ducts radiate to the area they are to drain. In the central core of connective tissue the lymphatic capillaries are reduced in number and size; they are never quite absent but do not appear except in well-injected specimens. The presence of these ducts within the connective tissue core may have some liearing on the pathology of lymph nodes. The disappearance of the lymph capillaries in the center of the node involves the retrogression and absorption which is characteristic of developing tissues. Throughout the evolution of the lymph node there is continnal building up and tearing down. This will be evident in later stages where there is a continual change in the proportion of the lymphoid structures or cords and the lymphatic structures or sinuses.
Beside being the stage which marks the beginning of the adult structures of the node, that is to say, of the follicle, this stage also shows fundamental changes in cell differentiation. It marks the beginning of the wandering cell in lymph nodes. Up to this time the connective tissue part of the node has consisted of a network of granular protoplasm with many nuclei, young, dividing, and old. At this time three types of wandering cells appear, the lymphocyte, the polymorphonuclear form, and the eosinophile.
Lymphocytes are present in the thymus at a much earlier stage, they are abundant there in the sections from the enil)ryo 3.6 cm. long. In the sections of the lymph node at 8 cm., there are a few lymphocytes in the connective tissue core of the node, and in little clumps in the connective tissue just without the node. These little clumps of cells are found near the capillaries. The differences between the connective tissue cell and the lymphocyte are as follows: The nucleus of the former is large, faintly staining, and oval in shape, and the protoplasm belongs definitely to the network, while the latter has a small, round, deeply staining nucleus, with a more distinct nuclear membrane. The nuclear network and the chromatin granules are coarser, and there are one or more nucleoli. Moreover, the protoplasm makes a narrow but definite rim around the nucleus. Between the connective tissue cell, especially the _young forms, and the lymphocyte one can see every possible transition.
3T2 Development of the Lviiipliatic Xodes in the Pig
Often the connective tissue nuclei appear as if being extended from the protoplasmic network of exoplasm, the irregular endoplasm still clinging to the nucleus.
This form of observation cannot be considered as proof of the origin of the lymphocyte from connective tissue — it is obvious that the position of a wandering cell cannot give evidence of its origin. With the same t3'pe of tissue to examine, Gulland, noting the occurrence of the lymphocytes in clumps around the capillaries, concluded that they were filtered from the blood stream. The evidence does not suffice to prove either that the lymphocyte develops from the connective tissue in the lymph node, nor that it reaches the node through the blood stream. "We must await some new method of attacking this problem. One point is, however, definite in my specimens — that cell division in the connective tissue takes place in little clumps around the blood capillaries, and, as will be shown later, the division of the lymphocytes takes place also around tufts of capillaries.
Besides the lymphocytes there are a few polymorphonuclear cells at this stage, perhaps not more than twenty or thirty in each section. They are quite typical, having irregular nuclei and finely granular protoplasm. They occur within the follicle. Eosinophiles appear also for the first time. Within the follicle there are numerous red blood cells outside of the capillaries, showing signs of degeneration, that is, a vacuolization and a breaking up of the protoplasm into granules. These granules are all of the same size and cannot be distinguished from the granules of the eosinophilic cell. This is the same evidence that has led Weidenreich to the conclusion that the eosinophilic granule comes from the red blood cell. It is suggestive, but not conclusive.
To sum up the stage represented by Fig. 11, it marks the beginning of the dift'erentiation of the node into its two elements, lymphoid and lymphatic. It shows the beginning of the follicle and of the wandering cell. There is a marked proliferation of the blood capillaries and a consequent increase in the connective tissue in the center of the node. This involves a retrogression or destruction of some of the lymph ducts. At the same time wandering cells appear, lymphocytes in greatest numbers and also polymorphonuclear leucocytes and eosinophiles. There is also evidence of degeneration of the red blood cells.
The next stage is shown in Fig. 12. It was made from the primary lymph node of a pig 13 cm. long. The first point to be noted is the development of the artery. Without the limits of the node, the artery has divided into two branches. These two branches enter the node and
Florence E. Sabin 373
divide into five main branches and two mnch smaller ones. Consequently there are five definite primordial follicles, and two small ones. Both of the small ones and two of the large ones show in the section. At this stage there is no definite capsule, the limits of the node being determined by the lympliatic vessels. The nodes increase in size by invading the surrounding tissue, for example, the artery which here branches without the node is subsequently included in the gland. This stage marks several important changes. Tlie first has already been noted as being the division of the artery and the corresponding multiplication of the fol
FiG. 12. Diagram of the primary lymph node in an embryo pig, 13 cm. long. X about 33. The section shows two large follicles and two small ones. A, artery; aid, afferent lymph ducts; bf, beginning follicle; ehl, efferent lymph duct; f, follicle; gc, germ center.
licles. The vein, which is not shown in the diagram, runs parallel to each branch of the artery up to the point where the artery enters the follicle. On entering the follicle artery and vein separate and both break up into plexuses. At this stage the artery without the follicle can be distinguished by a thickening of the connective tissue around, there being no media, and by its smaller caliber. The vein has only a lining of endothelium and there is as yet no thickening of the adventitia. Within the follicle, the vessels are all capillaries, but the plexus which connects directly with the artery is made of smaller vessels than the plexus which connects with the vein.
374 l)(>V(.'Ki)iiiK'nt of the Lvnipliatie Xodos in the Pig
Tho soeoiul point in advance is the formation of the germinal center. AVithin the follicle, as will be seen in the diagram, there are small clnmps of colls, definitely lymphocytes, heaped aronnd a capillary tuft. In the entire node at this stage there are eight of these germinal centers. The lymphocytes are closely packed in them, and there are more lymphocytes near these centers than elsewhere in the node. In regard to the wandering cells, the follicles contain in general four types : First, the lymphocytes which are found in the germinal centers almost to the exclusion of any other free cell. A few of them are to he seen near the germ centers. Second, polymorphonuclear forms, which are scattered throughout the follicle except in the germ centers. Third, eosinophilic cells. Fourth, mononuclear forms which have larger nuclei and more protoplasm than the lymphocytes. All of these cells are found in the follicle. Eed blood cells are also present, many of them being free in the connective tissue meshes and showing a breaking up of the protoplasm into granules. This appearance of the red blood cell is seldom seen in the corpuscles within the capillaries. The bridges of connective tissue between the lymph ducts have fewer wandering cells than the follicle.
Beside the two changes already noted, namely, the division of the artery and consequent multiplication of follicles, and secondly, the differentiation of the follicle into germ center and lymph cord, there is a third important change, namely, the beginning of the formation of lymphatic sinuses out of the lymphatic ducts. It will be noted in the diagram that around the border of the follicle the lymph ducts are arranged in rows closely packed together and that the connective tissue bridges between them are slender. This is still more definite in the next diagrams given in Figs. 13 and l-l. In section, this point is to be seen in Fig. 15, where the surface of the node adjacent to the capsule is made of a plexus of lymph ducts, while in the depth of the node the ducts are closely packed together in certain areas making sinuses. The section shows all gradations in the width of the connective tissue bridges, some being just wide enough to contain a single nucleus while others are broad bands.
Kling was, I think, the first to note this method of the formation of the sinus, though he clouded the clearness of his picture by saying that subsequently the bridging of the sinus is made by the stretching of endothelial cells across the lumen of the ducts, so that there are bridges made of endothelium alone. This appearance is undoubtedly found in sections just as in sections of the lung the epithelial lining of an air sac sometimes shows as a membrane.
By following the evolution of the sinus it is possible to understand clearly the relation of the reticulum of the adult sinus to the endotlielial
Florence R. Sabin 375
cells. The reticulum fibers develop subsequently in the connective tissue bridges (not. as Kling says, from the endothelium). Tlie lymph ducts from which the sinus is made have a complete endotliolial lining. Moreover, the increase in the ducts which form the sinus takes place by the same process of the budding of endothelial cells which characterizes the development of lymphatics or blood-vessels elsewhere. Thus the spaces of the sinus are lined throughout by endothelium. The sinus can be pictured in three dimensions by imagining the follicle surrounded by a plexus of ducts so dense that the bridges between them are reduced to the thickness of a single network of fibers. Such a structure in cross section would give the appearance of fibers with endothelial cells around them cutting the lumen of the sinus. As a matter of fact the fibers are between the endothelial cells and without the lymph channels. They connect with the rest of the connective tissue framework of the node as is readily seen in Fig. 12.
The next stage is from a section of the primary lymph node of an embryo 15 cm. long (Fig. 13). There is now a great development of the artery. The wall of the artery has developed considerably and shows one row of smooth muscle cells beside the adventitia. The vein which lies beside it has only an endothelium and a thickened connective tissue sheath. The artery divides into three main branches on the edge of the node, and within the node each branch subdivides several times. As in the early stage, the artery and vein run parallel until the follicle is entered and there they separate. The amount of lymphoid tissue has increased parallel with the development of the artery. The sinuses are growing down into the node between the arteries, thereby surrounding and limiting the lymphoid masses around the blood-vessels. By this process the lymph cord along the blood-vessel becomes defined, as will be clear in the next diagram.
The especial advance in this stage lies in the connective tissue, for here the reticulum fibers within the node first begin. Up to this stage the connective tissue of the node has been a protoplasmic network with delicate anastomosing fibrils which, however, do not stain sharply by Mallory's method. Xow for the first time there are a few fibrils which stand out clearly in the Mallory stain. They occur in the germ centers where they are laid down in concentric circles. With the oil immersion lens it can be seen that the fibers of the germ centers make a definite mosaic of polygonal spaces in concentric rows. All of these polygonal spaces thus outlined are filled with cells. This appearance of the mosaic can be seen in thin sections of the adult node and can be brought out by silver nitrate.
376 Development of the Lymphatic Nodes in the Pig
The diagram shows the first beginning of the trabeculas in the connective tissue that pushes down between the peripheral sinuses as these surround the follicles (see dt on the figure). Neither the capsule nor the trabecuhiB have fibers different from the surrounding connective tissue at this stage.
There are certain interesting points in regard to the cells of the node at this stage. In the germ centers there is a marked division of the
Fig. 13. Diagram ot the primary lymph node in an embryo pig, 15 cm.
long. X about 33. The node shows several follicles. A, artery; aid, afferent
lymph ducts; dt, developing trabecula; gc, germ center; ps, peripheral sinus.
lymphocytes. In any one section each center contains from two to fifteen or more dividing lymphocytes. They are easily distinguished from the dividing connective tissue nuclei, which are always larger and have much more protoplasm. There are many eosinophiles in the cords, but at this stage none are to be found in the germ centers. There are numerous degenerating red blood cells.
The next diagram (Fig. 14) is from the primary lymph node in a pig 23 cm. long. Only a portion of the section was drawn, in order to
Florence 11. Sabin
fceep the diagram at the same magnification as the others. An outline of the entire section is shown in the margin. It gives the artery and a few eiferent ducts at the hihim and also a large trabecula (t), carrying alferent ducts. This trabecula connects with the cortex in another section of the sx^ries. The left hand part of the section shows a consid
Fig. 14. Diagram of the primary lymph node in an embryo pig, 23 cm. long. X about 33. The diagram was made from a segment of a section, the outline of the entire section being given in the margin. A. artery; c. capsule; cs, central sinus; dts. developing trabeculag of the sinuses; ed. efferent duct; gc, germ center; /c, lymph cord; 7p, lymph plexus; ps. peripheral sinus; t, trabecula with afferent ducts.
erable advance over the preceding stage; it represents practically adult conditions where the entire node has been transformed into lymph cords and sinuses. This change has come aljout in the following manner: The lymph duct plexus or sinus, which was only on the edge of the preceding section, making the peripheral sinus, has now grown into the 29
378 Devolopiiiciit of tlu' Ijyiii|iltatie Xodos in tlic Pig
depth botwoen the arteries, thert'hy exteiuliiii;- the sinuses and definitely limiting the lymph cords. Indeed, the true lymph cord of the adult condition now appears for the first time. The diagram shows well the nature of the lymph sinuses consisting of rows of closely packed lymph ducts. The bridges between them are still protoplasmic and slightly wider tlian they appear in the adult nodes.
The diagram shows the rel-ition of the trabecula? to the capsule and the sinuses. Here for the firsi time there is a definite capsule and it is interesting to note that it is not complete. It extends along the margin of the peripheral sinus but ends abruptly on the right hand side of the section, where the node consists of a plexus of lymph ducts. It will be noticed that there is a definite indentation in the margin of the node where the capsule ceases. At this place the node can increase in size by invading the surrounding tissue.
The structure of the capsule itself is best studied in good Mallory specimens with the oil immersion lens. For this study it is necessary to note the condition of the surrounding connective tissue. This has been described by Professor "Mall." There is in the first place in tlie surrounding tissue a delicate network of fine but definite anastomosing fibrils. The nuclei of the network are in the nodes, and most of them form part of the characteristic spindle cells. This is the prefibrous tissue of Mall; the fine fiber network is still slightly granular, representing the protoplasmic syncytium of the earlier stages. Most of the definite protoplasm is around the nuclei. Beside this fine fiber network there are large bundles of definite fibers. The fibers of these bundles are several times the width of the fibers of the network, and the bundles themselves are often eight or ten times the diameter of a red blood cell in width. Many of these bundles have several spindle cells clinging to them. The large bundles are found near the border of some gland or muscle, while in the less differentiated areas every transition between the fine network and the coarse bundles can be made out. As Mall has shown, these are the white fiber bundles developing from the prefibrous tissue. The fibers of the bundles are close together and are straight. The capsule of the lympli node is different both from tlie fine network and from the white fibrous bundles. It consists of a dense network of anastomosing fibers. These fibers are larger and sharper than the fine fiber network, but they are much finer than the fiber bundles. They are wavy and closely packed. In other words, they differ from the fine fiber network by being larger, sharper, and more closely packed, and
Florence E. Sabin 379
from the fiber bundles by running as separate, wavy fibers, having in general the same direction but forming abundant anastomoses. These are obviously the beginning reticulum fibers.
There is one small, fat organ within the capsule. The trabeculse are formed, as can be seen in the figure, by the folding in of the capsule, thereby bringing the trabecule in the center of the sinuses (dts). This shows especially well in the border of the large central trabecula (t) of the figure and explains why the trabecule of the adult node are bordered by sinuses. The traljecula.^ often carry blood-vessels from the capsule. The tissue around the Idood-vessel at the hilum thickens to form trabecular; these trabecular follow the veins farther than the arteries for the arteries soon enter the lymph cords. The trabecular around the bloodvessels are less developed at this stage than the capsule.
The connective tissue framework within the node itself is a definite protoplasmic network with a few fine fibrils which do not stain sharply in tlie Mallory stain except possibly in the germ centers. The bridges in the sinuses are all protoplasmic at this stage. In other words, the connective tissue framework of the node is less advanced than the surrounding tissue and the reticulum fiber is a later development.
The diagram shows the relation of the lymph cords and the germ centers. As has been said, the cords are first definitely outlined and restricted to the borders of the artery by the development of the lymph sinuses. Some of the cords arc narrow and have the single central artery, but the majority have an abundant plexus of blood-vessels. The arter}' and vein never run side by side in the lymph cord, and the veins leave the cords to enter the trabeculge that grow in from the hilus. At this stage of development only the large veins are in the trabecule. The germ centers are around the capillary tufts where the lymphocytes actively divide. The lymphocytes then wander down the borders of the artery, filling up the lymph cords. In the adult node, as is well known, the germ centers may present two diiferent appearances. In the one case, the center is uniformly filled with lymphocytes which are actively dividing: in the other case the lymphocytes are crowded to the edge of the germ center, giving the appearance of a dark rim in sections stained in hfematoxylin. In the einl<ryo wliere cell division is active, the germ centers are always uniformly crowded witli Ivmphocytes.
The right-hand part of the section is far less developed. It consists of a plexus of lymph ducts with connective tissue bridges which contain a network of blood capillaries ; it is essentially in the stage of Fig. 7, llioiigli the lymphatic plexus is more abundant. IMost of the nodes in
380 Dovelopnieiit of the Lymphatic Nodes in the Pig
the pig at this stage have the center made of definite cords and sinuses and the entire periphery made of a plexus of lymph ducts; and this is the condition of the primary lymph node in a pig 2 weeks old. That is to say, there are three kinds of tissue in the nodes : first, lymph cords and germ centers ; second, lymph sinuses ; and thirdly, plexuses of lymph ducts not yet transformed into sinuses. It is clear that the lymph plexus is a stage in the development of tlie lymph sinus, and hence is a less highly developed structure.
These points are clearly shown in Fig. 15, which is taken from the inguinal node in a pig 2-1.5 cm. long. A portion of the capsule is shown which is not as yet a limiting membrane. The outer part of the node consists of a plexus of h'mph ducts with connective tissue bridges. The nuclei in these bridges are large and oval. The inner portion of the node, away from the capsule, consists of lymph cords and sinuses. Within the cords the predominating cell is the lymphocyte, with which the germ centers are closely packed. The sinuses are groups of lyiuph ducts and transitions between the lymph plexus of the edge of the node and of the developed sinus are to be seen.
In the pig the lymph plexus persists up to adult life. In specimens where the lymph ducts are all collapsed, these areas look like masses of connective tissue, hence Delamere^ pictures them and calls them homogeneous areas. Eanvier " found the same tissue in the ]uesenteric nodes of the pig, but since his specimens had the lympli ducts distended he called the areas cavernous or erectile tissue. As has been said, in studying large numbers of lymph nodes in fresh specimens one often finds the lymph ducts or sinuses so distended Avith fluid that all the spaces are rounded. The presence of this lymph plexus not developed into a sinus in the nodes in the pig is, I believe, an important point not only in the study of development but in the understanding of the structure of allied organs. In all of the hsemolymph nodes I have seen, there have been three types of tissue, the lymphoid areas, true sinuses, and zones filled with blood but not definitely sinuses. These zones appear like veins not crowded enough to be sinuses.
We have thus followed the development of the primary lymph node which comes from the lymph sac. The stages are, in brief : first, a preliminary stage in which the entire node consists of a plexus of lymphatic capillaries with connective tissue bridges containing blood
" Poirer, Cuneo, and Delamere: The Lymphatics, p. 100, translated from Poirier's Anatomie.
^"Ranvier: C. R. Acad. Sciences, 1S95, p. 800, or C. R. Soc. Biol., 1895, p. 774.
Floronce II. Sabin
« o- -i. J ! A ^t:'Mm3C
Gerra centre — >rr=:
■<iv^ ""~ ai'^-T'
Fig. 15. Portion of the inguinal lymph node in an embryo pig, 24.5 cm.
long. ' X about 200. Drawn with a camera lucida. The drawing shows the
transition between the lymphatic plexus hear the capsule and the sinuses farther within the node.
383 Devi'Idpiiu'iit of the Lyiiiphalic- Xoilo:^ in the Pig
capillaries; then a stii<ie in which the development of the l)h)oil-vessels determines the pi-imordial follicle. Followinii; the evolution of the artery the adenoid tissue is determined while the lymphatic part of the node, or the sinuses, develops from the proliferation of the lymph ducts, in the pig the adult node still contains some of the lymph plexus which in higher animals is completely transformed into the sinuses.
Derclopnieni of nodes in the [iriiunrij plexuses. — The other early lymph nodes which drain the skin develop in certain definite areas. If we limit the group to those which receive ducts directly from the skin without passing through other nodes, these glands develop in the long plexus around the external jugular vein in the neck and in the plexus of the inguinal region, and over the crest of the ileum. The development of the nodes in all of these areas has been studied. These nodes all begin in a plexus of lymph ducts rather than in a lymph sac ; the connective tissue bridges at first show no thickening but soon the nuclei become more crowded and the nodes pass through the various stages shown by the primary lymph node. The node over the crest of the ileum is the simplest, for like the primary lymph node it develops as a single node. The long plexus has the most complicated development and it drains a varied area. In the series at 7 cm. long the entire plexus shows a thickening of the connective tissue bridges. Suhsequently, as shown in Fig. 7, the plexus represents a chain of nodes. In this figure four macroscopic nodes are shown, two at the angle of the jaw, one about the center of the plexus, and the fourth at the lower end of the plexus. Of these nodes the one nearest the angle of the jaw receives ducts directly from the skin of the face around the eye ; the other node receives ducts both from the skin over the head and from the first node. The node in the middle of the plexus receives ducts from man}^ sources, in fact from all of the other glands in the chain and from the front of the neck as well, while the fourth node receives ducts from the fore leg. Thus it will be seen that some of the nodes are secondary or intermediate in the sense of receiving ducts from other nodes. Some nodes, for example those along the internal carotid artery, receive only ducts that have passed through other nodes, that is, they develop along the etferent ducts of a node, while some receive ducts both directly from the skin and from other nodes. In short, a gland may be secondary for some ducts and primary for others.
The histogenesis of these various nodes follows the same general lines as the primary lym])h nodes, beginning witli the stage represented in Fig. 10, but there is the widest possible variation in the relative pro
Floronee i\. .Sabin
portions of the lyniphoid tissue and the lymphatic tissue. Sometimes the lymphoid tissue, that is, tlie connective tissue ])ortion with lymphocytes arouiul the artery, nearly fills the node while in other cases the lymphatic ducts predominate.
Development of the folJlde liKtepenileiitli/ of the h/iitplt duels. — One im})ortant fuiuhimental jioint comes out in the study of these
Fig. 16. Group of microscopic lymph follicles in the neighborhood of the inguinal lymph node in an embryo pig, 24.5 cm. long, x about 72. The condition of the main inguinal node at this stage, is shown in Fig. 14. A. follicles consisting of lymphocytes around a tuft of blood capillaries; &. follicles with a single peripheral sinus; c, follicles which are made mainly of a plexus of lymph ducts; Id, large lymph duct; Ip, lymph plexus in the border of a large lymph node.
various mxlc^. After a certain sta.iic in development, the larg-e nodes, especially those that develop in the Ion-;- ple.xus and in the inguinal region, show many microscopic nodes in their neighborhood. These microscopic nodes are abundant in emliryos from 2"? cm. long on. Fig. 16 is a section in the ])order of the inguinal node in a pig 24..") cm. long,
384 Development of tlie Lympliatic ^NTodes in the Pig
showing seven microscopic nodes. These different small glands represent all stages in development.
In the figure there is an ahundant plexus of lymphatic ducts and around this plexus is a group of small follicles. Two of these nodes, marked a, consist of a collection of lymphocytes around a tuft of blood capillaries. Studied through serial sections, these small follicles (a) have no lymphatics whatever. Two of the small nodes, marked h. have a single peripheral sinus around the follicle while others, marked r, have an abundant plexus of lymph ducts not transformed into sinuses. The node in the lower edge of the section is shown only in part. It is the margin of a larger node and shows the peripheral zone of lymph ducts. This entire mass of follicles will be subsequently fused with the large inguinal node, which is one method of increasing the size of the nodes. The inguinal node at birth contains fewer of the smaller follicles in the border than at earlier stages. The study of these microscopic nodes proves an important point, namely, that the lymphoid part of the node or the lymphocytes in the reticulum occurs primarily with the arteries and blood capillaries rather than with the lymphatics. This was suggested by the angiogenesis of the primary node, but is proved by the fact that small nodes are found around the capillaries before they are reached by the lymphatics. That is to say, the follicle is primarily and essentially associated with the artery. That the follicle occurs in the adult without lymphatics is shown in the Malpighian corpuscles of the spleen. In Fig. 17 is a small lymph node found in the lung of an adult pig. It lies in the edge of a lymph capillary but is without a true sinus forming an integral part of the node itself.
To return to Fig. 16, the section shows that nodes which develop at later stages after the lymphocytes occur in the glands, hurry through the long preliminary process of the first nodes; that is to say, they begin at once with a heaping up of lymphocytes around a blood-vessel.
Themolympli nodes. — The study of the development of these nodes is incomplete. It has not yet been extended to all the areas in which the h^molymph nodes occur, but confined to the neck region and along the course of the thoracic aorta. The liEemolymph node does not occur in the neck of the pig until the embryo is about 23 cm long, showing that it is a considerably later development than the lymphatic node. From the time the embryo is 23 cm. long there are one or two small nodes to be found near the node which forms in the center of the long plexus (see Fig. 7). In the specimen at 23 cm. long the hffimolymph node is in the simplest possible stage consisting of a single follicle with a
Florence R. Sabiii
peripheral sinus filled with blood. It looks exactl}^ like the follicles marked h in Fig. 16, except that the sinus is filled with blood. The next stage looks like the nodes marked c in the same figure, while a tliird stage shows an increase in the lymphoid tissue.
The hfemohmiph node thus parallels the stages in development of the other nodes, except that its sinuses from the beginning belong to the blood-vessels rather than to the lymph vessels. In the lymphatic nodes the sinuses are made of the modified veins called lymphatics, while in the hffiuiolymph node they are made of the veins themselves. The espe
Fig. 17. Lymph follicle without a peripheral sinus, found in the edge of a lymph duct in the lung of an adult pig. X 66.
cial point in the development of the nodes which is not yet clear is the relation of the veins which make the sinuses to the rest of the bloodvessels of the node.
In all of the specimens of the hasmolymph nodes as far as they have been studied, that is in pigs up to 3 weeks old, the venous or sinus portion of the node predominates over the lymphoid element. The nodes show both the true sinus and the plexus formation observed in lymphatic nodes, but the developed sinus is mucli more limited in amount. This is also true in all of the htemolymph nodes I have seen from the adult pig. 'They consist of three elements, the lymphoid masses surrounded by true sinuses, and a considerable amount of a venous
386 Development of the Lvmpliatic Nodes in the Pig
plexus not transformed into the sinus. Thus the hsvmolymph node is a later and less developed organ occurring along the l)loo(l-vessels.
The luvmolymph node found in the neck of the pig is from the l)eginning a distinct organ, different in type from the lymphatic node. In the adult pig these IiaMnolymj)li nodes in the neck are sometimes fused into the same capsule witli an ordinary lymph node, hut the two remain as distinct structures with trabecuhv between.
Summary and General Discussion.
Follicle.— From the study of the development of lymph nodes we find that there are in general three types of follicles. The simplest follicle consists of a collection of lymphocytes in a reticulum around an artery and its capillaries. This type occurs in the embryo and probably in the adult. The second type consists of the follicle of the first type surrounded by the lymphatic sinus. This type occurs either singly or in groups in the ordinary lymphatic gland. In the third type the lymphoid follicle is surrounded by a blood sinus or a less developed plexus of bloodvessels. This occurs in the hai-molymph node and spleen.
Lymphocytes. — In connection with the nature of the lymphoid tissue it has already been brought out that the lymphocyte occurs in a fine reticulum around the artery and its capillaries. For the ultimate origin of the lymphocyte we have as yet no proof, but the lymphocytes divide, as Flemming showed, in the germ centers. The germ centers are definite organs around capillary tufts or glomeruli. In the embryo the division of lymphocytes is so constant that the germ centers are always filled with them. The lymphocyte develops independently from the lymph ducts.
General structure and rjroirtli. — In regard to the general structure of the node, it has been shown to consist of two elements — a lymphoid or vascular and a sinus element, which is either venous or lymphatic. Through the study of the development of the nodes, it becomes clear that the sinus comes from a plexus of ducts or vessels by increasing the number of ducts and reducing the size of the connective tissue bridges. In the pig the plexus element of the node is not completely transformed into the sinus in the adult, so that the node has three structures, the lymphoid element, the plexus, and the sinus. There are wide variations in the proportions of these elements.
The question of growth and increase in size of the lymph node is an interesting one, closely liound up with the process of absorption in development. All of the diagrams show that the nodes in the early stages increase in size by the invasion of surrounding tissue by lymph ducts.
Florence K. Sahiii 387
This point is hrou.iilit out hv coni})aring Figs. 5 and G, from the primary node at 3.(j and 4.!) em. long. The sections are cut in the same plane and can be related by the sac. It a^II be seen that the increase in size of the second is due to the development of the lymph plexus. This invasion of ducts can take place as long as there is no definite capsule. The capsule is a late development, as shown in the last diagram, where it is still incomplete. After the capsule is formed the nodes increase in size by the fusion of many small nodes in the neighborhood. The capsules of the smaller nodes become the trabecule of the larger ones. The marked tendency of the fusion of nodes in the pig has been noted by other observers. In the pig not only do the ordinary lymph nodes fuse, but a haemolymph node may fuse with a ])ure lymph node and the two remain distinct but enclosed in a common capsule. The primary lymph node from the lymjih sac, and the node over the crest of the ileum show but few of the small nodes in the neighborhood and hence few evidences of fusion. On the other hand, the inguinal node which represents a group of nodes in higher forms, appears like a conglomerate of small nodes in the new-born pig.
The lymph nodes give inuch evidence of a repeated tearing down and relniilding in the process of growth. For example, in passing from the stage of the first to the second diagram, there must be a destruction of many lymph duets in the formation of the primordial follicle. It will be noted that this destruction is not complete in Fig. 8, for there are a few lymph duets scattered through the follicle. In following through the diagrams it will be noted that there is a constant change in the proportions of the lymphoid or vascular portion and the lymphatic portion of the node. The small nodes of Fig. 16 make this point clearer. The youngest ones have a predominance of the lymphoid portion, the very youngest ones having no lymph ducts at all. Others have merely a peripheral sinus, while others slightly larger are made almost entirely of a plexus of lymph ducts. As these nodes develop farther the lymphoid tissue will increase until the balance of lymphoid tissue, sinus, and lymph plexus characteristic of the adult node is reached. This point illustrates the extreme variation met with in the development of the lymphatic apparatus.
Sinus. — The lymph sinus develops out of the lymph ducts by a multiplication of the ducts along certain lines. The areas in which the lymph ducts multiply until the connective tissue bridges are reduced to the thickness of fibers, are determined by the blood-vessels. That is, the einuses grow in between the arteries thereby bounding the lymph cords.
388 Development of the Lymphatic Nodes in the Pig
The sinus develops by a proliferation of the cndotlicliura of the ducts, and so, as Kling pointed out, each space in the sinus has its complete ring of endothelium. The fact that the sinus is made of a great number of small lymph ducts packed closely together explains why one cannot get the silver picture of a membrane of endothelium far beyond the periphery of the node. None of the membranes are large enough. In the embryonic stages the connective tissue bridges are largely protoplasmic and show connective tissue nuclei. In the adult the bridges are a network of reticulum fibrils so that the endothelial cells appear on the fibers.
The study of the contents of the sinuses will prove, I think, an important point in the physiology of the lymph nodes. There is a marked difference between the embryonic nodes and the adult in this respect. In the embryonic nodes there are few free, wandering cells in the sinuses as compared with the adult. In the early stages there are almost no free cells in the ducts. After the lymphocytes appear, they occur occasionally in the ducts and sinuses, as well as a few large mononuclear forms. As a rule the sinuses are nearly empty of the free cells, so that the bridges stand out with great beauty and clearness. In the adult node, on the other hand, the content of the sinus in free cells is most varied, both in the number and in the kind of cells. Sometimes the sinuses are so packed with cells that the reticulum and endothelium are almost covered up. It often happens that the sinuses of one portion of a section are densely packed with cells, while in another portion they are almost empty. These free cells may be any type of white blood cell or may be large phagocytic cells. The sinuses may be filled with phagocytic cells that are crowded either with red blood corpuscles or, in the mesenteric nodes, with fat globules.
Reticulum. — The complete relation of the connective tissue framework or reticulum can only be clear after noting the nature of the sinus. The reticulum fibers first appear in the germ centers where they make a mosaic pattern around the capillary tuft. The trabeculse develop from the capsule in connection first with the sinuses and secondly with the blood-vessels, especially the veins. The reticulum fibers are laid do^vm in a very close protoplasmic syncytium, and this syncytium remains protoplasmic long after the surrounding connective tissue has become predominately fibrillar. The reticulum fibers do not show until the embryo is 15 or 16 cm. long. They appear first in the germ centers and in the capsule. In an embryo 23 cm. long they are limited to the capsule and a few trabeculse, while up to the time of birth the connective tissue framework is still largely protoplasmic.
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The reticulum framework in the adult makes a complete anastomosis throughout the node. The framework can be readily traced in the last diagram. Starting from the capsule, fibers enter the trabeculae, pass between the ducts of the sinus and enter the lymph cord. The large trabeculae carry veins. In a specimen of reticulum from which the cells have been digested out, the entire node can be reconstructed for the sinuses occur in the looser reticulum that borders the trabeculae while the cords and follicles are between trabeculae and show a much finer and denser network than the sinuses. The germ centers often appear as holes, since the fibers are delicate there.
Lymph capillaries. — The subject of open or closed lymphatics, within the lymph node as elsewhere, has given rise to most definite but opposite opinions. The study of development touches but one aspect of the question, unless it is combined with many injection experiments. The lymphatics develop as blind sacs from the veins and have a complete endothelial lining. The sinuses result from a multiplication of the lymph ducts and the only difference between the sinus and the preliminary lymph plexus is in the width of the connective tissue bridges between the ducts. In the sinus these bridges are reduced to the thickness of the reticulum fibers of the adult. Thus from the purely embryological argument the spaces of the sinuses have a complete endothelial lining. Numerous injections of lymph nodes in every stage have been made with Prussian blue and with India ink. The ink always runs farther than the Prussian blue. In injecting the nodes through the afferent ducts it is always easy to avoid undue pressure within the node, for the lymphatic area of the node is so much greater than the caliber of the ducts leading to it. It is possible in all stages up to two weeks after birth to obtain injections without extravasations. But many injections do show extravasations and these occur in the walls of the sinus rather than the less developed plexus of ducts which always forms a part of the node. This shows that the wall of the sinus is weaker than the walls of the plexus.
To conclude, the present study shows the close relation between the lymphatic system and the vascular system. In the formation of lymph nodes, haemolymph nodes, and spleen, there is one fixed element in common, namely, the lymphoid tissue associated with the artery; the fluctuating element or the sinus belongs either to the venous system or to modified veins called lymphatics. The sinus may be absent, or venous or lymphatic.