$3 .1 2;... {.1 l? » ~ _ 9“. ‘ *3: f ‘ ‘5" -ur— vp “V? .. ,I . in I its/“.3155 .1711...er1 1. 16.0.33}... 90...? .l. )D.V.’If.: a” 1 71¢ . , $31.31 . H5355. .. r) x. z! « , 9\ vb?! . . .\. . 1’ K J: .3‘F\w Joan/‘1'»: 3*.) 1.3.1 . ‘. .u Fin a. .i‘Px‘iflan: 3!.5fkwhawlufiurfirfi‘flflq ((1 (or III! I... .Jir v9. 35.322111 A. ‘ . 49h: Virfimr} .. ‘_ Ox‘ . f 4 3829 7"ummmmmmlulu»\Irmnwmml' ' 3 1293 1001 This is to certify that the thesis entitled AGE CHANGES IN TI—IE PIG LIMPH NODE presented by A FREDERICK LUDWIG BOUWIAN JR. has been accepted towards fulfillment of the requirements for M;— degree in MEL ,/ aim/NZ Major professor .5». Date August 2;, 1952 .‘KJ. I owe LIBR; M ichigar Unive: AGE CHANGES IN THE PIG LYMPH NODE BY FREDERICK LUDWIG BOUWMAN)JR. A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology 1959 ‘ AGE CHANGES IN THE PIG LYMPH NODE BY FREDERICK LUDWIG BOUWMAN)JR. AN ABSTRACT Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department 3f Zoology 1959 APPROVED: &/ / gn/hfié/ AH nBSTRACT AGE CHANGES IN THE PIG LYMPH-NODE BY FREDERICK LUDWIG BOUWMAN, IR. A total of thirty-eight lymph nodes, one or more from ten different body regions, were used in this study. Nodes were obtained from various ages of pigs (late fetal life to three years of age). Tissues were fixed in Lavdowsky's for- mal-acetic acid alcohol fixative and stained with either Harris' hematoxylin, Weigert Van Giesen connective tissue stain or Bielchowsky's reticular stain. Late fetal nodes and those from young animals (1 to 6 days of age) exhibited a medullary region with an abundance of sinuses and scattered reticular cells. In nodes from - older animals (3 weeks of age or older) lymph sinuses were occluded in part with reticular cells and lymphocytes. Thus the medullary sinuses, which characterized nodes of man and the dog, were not identified. The medulla usually surrounded the cortical tissue. However, in popliteal, superficial and deep inguinal nodes, the cortex was fre- quently at the periphery. In many of the older nodes ex- amined, some of the nodules were surrounded by either col- lagenous fibers, reticular fibers or both. With increased age the stroma became heavier and at the age of seven months elastic fibers were present as thin layers which in the capsule were not always near the inner edge. Elastic fibers were present in the parenchyma, par- F. L. noun-ma, JR. ABSTRACT ticularly in older nodes. Most of the changes noted above occurred most rapidly after birth, but continued until the age of sexual maturity at six or seven months. Mesenteric, pharyngeal, mandibular and lumbar nodes were characterized by extensive fusion. The two bronchial nodes that were examined were small and showed no evidence of fusion. Popliteal, parotid, superficial cervical and deep and superficial inguinal nodes were conglomerates of many fused and partially fused nodes. Grossly these nodes had a lobed appearance. In some nodes the cortex was ad- jacent to the subcapsular sinus, and was divided into am- pullae or alveoli as in the typical lymph node. Afferent lymph vessels extended through the trabeculae to Open, deep in the node, into sinuses adjacent to corti— cal tissue. Then lymph flowed to the medulla and drained into efferent vessels from several locations on the sur- face of the node. ACKNOWLEDGEMENTS I wish to gratefully acknowledge the assistance and many kindnesses of: Dr. R. A. Fennell, Professor of Zoo- logy; Dr. M. Lois Calhoun, Professor and Head, Department of Anatomy and Mrs. Bernadette Henderson, Secretary, De- partment of Zoology, all of Michigan State University and my usually patient wife, Gloria Jane. iv CURRICULUM VITAE Frederick L. Bouwman, Jr., born in Grand Rapids, Michi- gan on January 16, 1916. Attended public schools and grad- uated from Ottawa Hills High School, Grand Rapids, Michigan. Received an A. B. Degree from Calvin College in 19%0. Serv- ed in the Medical Department of the Army of the United States, in the Mediterranean Theatre of Operations, from l9h2 to l9h6. Awarded the degree of M. S. in Zoology by Michigan State University in 1947. At present, Professor and Chairman of the Department of Biological Sciences at the Detroit Institute of Techno- logy, Detroit, Michigan. /"-’ ,.,A~~. ~_. U_\J.u.a_n.a.u Abs ract .>.. 4- f; .. o3 ACliIiO‘.:l.:CL;' _. .. Curriculum Vitae Centenss I. Introduction II. Material: :nd Methods III. ObserVati us A. General Description of the Pig 3. The Mesenterie Lymph Node C. THC Popliteal Lymph Rode 1. The Paretid Lymph Modes 3. The Pharyngeal Lymph Hedes F. The SuperfiCial Cervical Lymph G. The Mandibular Lymph Node H. The 3ronciial Lymph Mode l. Jhe Lumbar Lympfi Lode The Deep Inguinai Lymph Node H, ,. . 3 ie Suoerficial Inguinal Lymph Lymph Flow Through the Iode VI. Literrture Cited Page iii iv 5 Lymph Node 5 N 9 30 none #3 liode FIGURE 1. Mesenteric 15 cm. fetus Trabeculum with afferent vessel 2. Same node as fig. 1 Nodule 3. Same node as fig. 1 Megakaryocyte #. Mesenteric 24 cm. fetus Afferent vessels in trabeculae 5. Mesenteric 2 days Plexus of lymphatics 6. Same node as fig. 5 Afferent vessel with valve 7. Mesenteric 3 weeks Fusion 8. Same node as fig. 7 Enlarged View of fig. 7 9. Mesenteric h months Heavy stroma 10. Same node as fig. 9 Light stroma ll. Mesenteric 5 months Origin of efferent vessel 12. Mesenteric 7 months Elastic tissue layer 13. Same node as fig. 12 arteriole 14. Popliteal 2 days Medulla and cortex 15. Same node as fig. 14 Trabeculae with afferent vessels l6. Popliteal 6 days LIST OF FIGURES Reticular cells FOLLOWS PAGE 10 IO [.4 It 20 (.1) PC (1) l\) “Y. ,- u. . .~ ,, u: we , w , , .. , 7' 17. 18. 19. 20. 21. 22. 25. vii Popliteal 3 weeks Lobulated appearance of node Popliteal 4 months Elastic fibers Same node as fig. 18 Lymph channel to medulla Superficial Cervical 7 months Capsule around nodule Mesenteric 5 months Trypan blue in node Po;>.‘.7'-t“t~1 3 weeks Phagocy’cosiS Parotid 4 months Capsule around nodule Mesenteric 5 months Afferent vessel with valve 0) O? vili LI‘T OF TABL" F (I) TABLE OPPOSITE F "1 11. #‘ CD A. Age of pigs an; region: 1T0m which nodes 2 were taken l 1. INTRODUCTION There is no adequate description of the pig lymph node in the English language and no thorough investigation of the structure of this organ has been made for about twenty years. The pig node differs so much in structure from the typical mammalian node that descriptions of the typical node are useless in attempts to interpret this organ. Further, there are no descriptions of the node in very young animals. Thus it was considered worth while to OXpmine the organology, his- tology, and cytology of the node in pigs ranging in age from late fetal to seven months postnatal. In addition one node from a three year old pig was examined. Dr. R. A. Runnells stated that the direction of the flow of lymph through the node is important :0 the patholo- gist. He suggested that a confirmation of Trautmann's (1926) and Goldkuhl's (1927) description of the lymph flow would be useful. HMGHflwdH .msm m H H H HmsHSwnH @009 H H umpssq 7 N H H HMHnodonm H H H , andnHeawz N H H HmUaneo .95@ N N Hmewdhnmnm : H H H H Boost. a N H H H N H H HmopHHaom HH H H N u H H H H H OHAopsomoE .mph .05 .os .05 .03 .mms .mzz .mxs .me .56 .me .50 HN .so mH H309 m a m is m a o m e m H HSom Hate moxmp ones mowos JOHQB Scum mQOHmen cum wme mo em< ¢ Hflm4a 2 II. MATERIALS & METHODd Lymph nodes were removed from nineteen pigs obtained from the following sources: i.e. the autopsy laboratory of the College of Veterinary Medicine (M.S.U.), the Depart— ment of Animal Husbandry (M.S.U.), a local meat packing com- pany, and the General Biological Supply Compan‘. Ten of the pigs were males, four were females and four were cas- trates. They ranged in age from late fetal to three years postnatal. Since many were crossbreeds, no attempt was made to classify them according to breed. One or more lymph nodes were obtained from mesenteric, popliteal, parotid, pharyngeal, superficial cerviCal, mandi- bular, bronchial, lumbar, superficial inguinal and deep in- guinal regions. A total of thirty-eight nodes were obtained. For further details see Table A. Examination of Table A disclosed that nodes with a wide range of ages were obtained from two regions in order that age changes could be studied. These were the pcplitetl as an example of a superficial node and the mesenteric as an example of a deep node. The other nodes were obtained to learn if differences exist between nodes from different re- gions. Animals from pecking plants had very recently been slaughtered. Those from the autopsy laboratory were elec- trocuted. Others were killed by injection of an overdose of Halitall, a barbitol anesthetic, into the peritoneal 1. Jensen Salsbery Laboratories, Inc. 3 cavity. Two of the very young pigs, presumably killed by being rolled on by the sow, were found dead in the farrowing pens . In most cases the desired nodes were removed from the animal and immediately placed in Lavdowsky's Formol-acetic acid-alcohol fixative, which consisted of commercial forma— lin (40 percent formaldehyde) 100 parts, 95 percent alcohol 500 parts, glacial acetic acid 20 parts, and distilled water #00 parts. The nodes were left in Lavdowsky's fixative for 2% hours and then stored in 70 percent alcohol. The smaller pigs were fiXed and preserved entire in 10 percent formalin after the body cavities were opened. In order to study the direction of the flow of lymph through the node, certain pigs were injected subcutaneously with trypan blue solution or India ink suspension before the animal was killed. The trypan blue was employed as a .05 percent solution in distilled water and the India ink as a 50 percent suspension in distilled water. The pigs were killed ten to thirty minutes after the injection. After ten minutes trypun blue was present in the node, but no appreci- able quantities of India ink were found until 20 to 30 mi- nutes had passed. One animtl was kept alive after injection by removing the node surgically after intraperitoneal in- jection of Halitol. This procedure was followed under the direction of C. N. Titkemeyer, D.V.M. and was repeated until the proper time between injection and removal of the node was established and all of the desired nodes were obtained. s; h. All nodes were dehydrated in a mixture of one—half 95 percent ethyl alcohol and one-half butyl alcohol followed by two changes of butyl alcohol. The nodes were left from one to three hours in each liquid. Following dehydration they were infiltrated in butyl mush, a 50 percent solution of paraffin in butyl alcohol, for ten to twelve hours, pass- ed through three changes of paraffin remaining several hours in each change and then embedded in paraffin. Sections were cut at five, six or seven micra on a rotary microtome. In some cases about a dozen sections were cut and mounted on three or four slides. Other nodes were cut into serial sec- tions. Usually sections were cut from two, three or four parts of one node so that various parts of one node could be compared. Sections for routine examination were stained with Harris' hematoxylin and eosin. To study cornective tissue, sections were stained by the Wicgert Van Gieson method. To Show reticular fibers, the sections were stained by Biel- chowsky's method as described by Bensley and Bensley (l93b). 3 III. OBsdenTIOhb A. General Description of the Pig Lymph Node The gross appearance of the pig lymph node differed from the typical node of other mammals only by the lack of a clear- ly defined hilus. The hilus was not found in the pig node because the entrance of blood vessels was not confined to one area of the node. They entered both with the afferent lym- phatic vessels and where the efferent lymphatics took origin. Furthermore, arterioles and capillaries were seen which en- tered the parenchyma of the node through the capsule (fig. 13). Because efferent lymphatic vessels left the pig node at many locations the nodes did not possess a group of blood and lymph vessels which entered or left a specific area of the surface. Thus a hilus was not formed. On the other hand, microscopically visible hilus-like indentations were seen where afferent lymphatics entered the node. Geldkuhl (1947) referred to them as pseudohili. This terminology was unde- sirable since these indentations were, from a morphological point of view, essentially similar to the hili described by other‘authors. Koelliker (1902) described the microscopic appearance of the mesenteric lymph node of the pig as follows. ”Eigenertig sind die mesenterialen Lymphknoten des Schweines, wie v. Reck- linghausen und Chievitz hervorheben und neuerdings wieder Ran- vier bemerkt, dadurch, dass so zu sagen kein Earkgewebe vor- handen ist und Follikel durch die ganzen Drusen verbreitet und dadurch die Lymphbthnen sehr eng sind.” Prepared sections of tissue from pig lymph nodes when eXamined under the mi- croscope demonstrated that the region which corresponded to the cortex of other species may be IOCated in the center of the node rather than adjacent to the Capsule (figs. 4 and 7). The cortical tissue Wes well filled with lymphocytes and con- tained the lymph nodules. The other tissue of the node, which may be found at the periphery, did not resemble the medulla of the usual node in structure. In the literature it was usually termed the medulla, although Trautmann (1926) con- sidered it to be a special tissue. Injection experiments to be described below showed that this tissue corresponded to the medulla of the typical node because lymph flowed into it from the cortical region and then into the efferent vessels. (fig. ll). This was in agreement with the opinions of many authors who stated that this tissue should be termed the me— dulla. Goldkuhl (1327) stated that this region must be con- sidered as medulla in order that the structure of the pig node could fit into the general structural scheme of the mammalian lymph node. In the medulla lymphocytes existed as small clumps of cells distributed uniformly throughout this region (fig. 7). Small sinuses were numerous (figs. 10 and 14). The predomi- nating cell was the reticular cell those cytoplasm took a characteristic bright pink color with H. and E. stain (fig. 16). The color was evident because few lymphocytes were pre- sent to mask it. This region has been aptly described by the ., German histologists as the ”helle" or "zellarme" region. R 7 Silver precipitation stains showed that the network of reti- cular fibers found in nodes of other species was present in the pig lymph node. Collagenous fibers were common in the medulla and elastic fibers were present, particularly in nodes from older pigs. Large trabeculae occurred in the medulla only where the efferent vessels formed and during fusion of two nodes (fig. 11). This along with the presence of reti- cular cells suggested that the histological structure of the pig node was fundarentally like that of nodes from some other species, c.g., dog and man. However, is an organ the struc— ture of the pig node was different i.e., the Carter was frequently surrounded by the medulla. The capsule consisted chiefly of collagenous fibers with varying quantities of elastic fibers and smooth muscle fibers. It was thin when compared to the capsule of the nodes of oth- er species (figs. 7, 10 and 12). The subcapsular sinus was not regularly present. When it was present and adjacent to cortical tissue injection ex- periments demonstrated an abundance of dye particles in both lymph vessels and the subcapsular sinus. 0n the basis of the arrangement of valves within the lymph vessels, we interpret these observations as an indication of lymph flow from affer- ent vessels into the subcapsular sinus. Thus, from a func- tional point of view, the subcapsular sinus functions in essentially the same manner as the sinus of typical lymph nodes. Richter (1902) suggested that lymph could flow from the afferent vessels, through the subcapsular sinus into efferent vessels thus bypassing the lymphoid tissue of the node.’ The lymphatic bypass around the lymphoid tissue was not seen in sections prepared from injected nodes. However, as these exist in some other Species, i.e. the rat and dog, further investigation may disclose their presence in the pig. Trautmann (1926) maintained that efferent vessels leave the subcapsular sinus, but observations made during the course of this investigation did not confirm this point of view. In the pig as in most other mammals the nodes possessed two systems of trabeculae. One of these arose at the hilus, or in the pig where the afferent vessels entered, and the other from many locations on the capsule. The latter system was usually poorly developed in the pig as few trabeculae of this type were seen. Branches of these two systems rarely joined. The system of trabeculae that arose where the affer— ent vessels entered the node carried these vessels, as well as blood vessels, into the interior of the node. In the old- er and larger nodes this system, at least near the surface, was very large and may have had a central cavity filled with loose fibrous connective tissue and fat (fig. 17). These trabeculae branched extensively throughout the cortex, but rarely entered the medulla. The trabeculae, especially the larger ones, were usually surrounded by a sinus which re- ceived lymph from the afferent vessels (figs. 9, 10, 15 and 21). Injection experiments as well as observations made on serial sections have demonstrated this connection. Thus, these sinuses should also be considered as corresponding to 9 the subcapsular sinus of the typical node as they received lymph directly from the afferent vessels. The other system of trabeculae when present arose from the capsule and usually penetrated cortical tissue (figs. 1% and 17). The tissues of the trabeculae were the same as those of the capsule, i.e., collagenous fibers, elastic fibers and smooth muscle cells. Both collagenous and elastic fibers left the trabeculae singly and in groups which penetrated the parenchyma. The medullary cords and sinuses of the typical lymph node were not found in the pig node. Deposits of adi- pose tissue were characteristic of nodes from older pigs in a few cases. Extensive fusion was characteristic of many pig lymph nodes. By this process many small adjacent nodes fused to form one large node. Observations made in this study indi- cate that fusion of the nodes began before birth. No evidence of continuing fusion was seen in the mesenteric node of a three year old pig. Trautmann (1926) described the process of fusion in detail. B. THE MESENTERIC LYMPH NODES The mesenteric lymph nodes of the pig were located in the mesentery of the small intestine. They were situated near the root of the mesentery and according to Sission and Gross- man (1953) were adjacent to the anastomotic arches of the in- testinal blood vessels. The nodes were arranged in a long chain that roughly paralleled the intestine. The afferent and efferent vessels of this group of nodes were the lacteals 10 of the small intestine. The efferent lymphatics carried lymph to the cisterns chyli. l. The mesenteric lymph node in a 15 centimeter pig fetus. In the lymph node of the 15 cm fetus which would be approximately 30 days old, the capsule had not yet appeared, so the boundaries of the node were formed by sinuses at its periphery and by the surrounding connective tissue (fig. 1). This observation agreed with those of Sabin (1905) who de- scribed the capsule as first appearing in the 230 mm pig fe- tus. Trabeculae associated with the afferent lymph vessels and with blood vessels exhibited an abundance of fibroblasts and fine fibers were present (fig. 1). Trabeculae were ex- tensions of the connective tissue adjacent to the part of the node where the afferent lymphatics and blood vessels entered the node. They penetrated deeply into the node, carrying the vessels with them and they frequently exhibited sinuses adjacent to their lateral surfaces. No trabeculae were seen entering the node from the surrounding connective tissue at any place in the periphery of the node except where afferent vessels also entered. Elastic fibers were not identified in trabeculae. Both cortex and medulla could be identified in the node at this stage, but the latter comprised a very small part of the node. Within the cortex, nodules in early stages of de- velopment were seen (fig. 2). These were evident as areas where the dark stained nuclei of mature lymphocytes were closer to each other than those of the adjacent cortex. Figure 1 Mesenteric node from a 15 cm fetus. H. and E. stain 135 X A. Trabeculum B. Afferent vessel Figure 2 Same node as Fig. l. H. and E. stain 570 X Am Nodule B. Cortex C. Connective tissue around node. Figure 1 Mesenteric node from a 15 cm fetus. H. and E. stain 135 X A. Trabeculum B. Afferent vessel Figure 2 Same node as fig. 1. H. and E. stain 370 X A. Nodule B. Cortex C. Connective tissue a‘odnd node. 8 13 Occasionally, the blood capillary loop, described by Sabin (1905) around which the nodules develop, was seen. The heavy reticular and collagenous fibers which surrounded the nodule in the pig were not yet developed. The diameter of the no- dules ranged in size from 25 to 125 micra. Sabin (1905) de— scribed the nodules of cervical and inguinal nodes from a fetal pig of this size as consisting of a few lymphocytes a- round a blood capillary. She also found, in these nodes, lymphatic cords composed of an arteriole surrounded by lym- phocytes. The cords were bounded by sinuses. Lymphatic cords were seen as dense concentrations of lymphocytes, with- out clearly definable sinuses. Observations on one specimen of mesenteric node suggested that it was more advanced at this stage than the cervical node as described by Sabin (1905) and an inguinal node observed during this study. This was attributed to the greater development of the nodules and the absence of the sinuses around the lymphatic cords in the mesenteric node. Lack of definition in the latter was pro- bably due to occlusion of their lumina by primitive reticular cells. This was suggested by observations made on a mesen- teric node from a two day old pig and a popliteal node from a six day old animal (fig. 16). Obliteration of the sinuses occurred to some extent in lymph nodes of other Species, but particularly in the pig nodesa In the rest of the cortex a few small sinuses and blood capillaries were present. The lymphocytes were not as heavi- ly concentrated as they were in nodes from older animals. 12 Two kinds of cells predominated in the cortex, namely, reticular cells and lymphocytes. Cowdry (192%) referred to the former as cells of the embryonic reticular syncytium. From these cells the reticulo-endothelial cells or macro- phages were formed as well as the cells of the lymphocytic series. Reticular cells were stellate with a pale cytoplasm investing large nuclei with a sparse amount of chromatin (fig. 3). They surrounded the reticular fibers, but it is still open to question as to whether reticular fibers owe their origin to these cells. The cells were partially ob- scured by the great numbers of lymphocytes found in the cor- tex. At this stage large and medium sized lymphocytes pre- dominated. Occasionally a lymphocyte undergoing mitosis occurred. Other cells frequently found in the cortex were myelocytes, metamyelocytes, and mature cells of both the neu- trophilic and eosinophilic series. Macrophages were seen occasionally, as were monocytes, plasma cells, and megakaryo- cytes (fig. 3). The medulla was distinguished by the presence of few lymphocytes. Consequently, the primitive reticular cells were prominent. Sinuses of various sizes were present, the average diameter of the smallest was about ten micra while the largest were approxim.tely 45 micra in diameter. The large sinuses which anastomosed to form the efferent lymph yessels were evident in some sections (fig. 11). OCCaSional blood capillaries were found rather uniformly distributed in the medulla. In addition to the primitive reticular cells Figure 3 Mesenteric node from a 15 cm fetus H. and E. stain 1100 X A. Recticular cell nucleus B. Megakaryocyte Figure 3 Mesenteric node from a 15 cm fetus H. and E. stain 1100 x A. Recticular cell nucleds B. Megakaryocyte l3 and lymphocytes, some cells of the neutrophilic and cosine- philic series were found. Sabin (1905) stated that embryos of 22 cm in length and also larger embryos had many minute nodes adjacent to the larger nodes. These minute nodes may have consisted of as little as a tuft of blood Capillaries and a few lymphocytes. Clumps of lymphocytes and plasma cells were found adjacent to blood vessels in the connective tissue of the mesentery in the 15 cm pig fetus, and it was suggested that these were also developing nodes. 2. The mesenteric lymph node of the 21 centimeter pig Ifetus. The mesenteric lymph node of the 2; cm pig fetus re- ssembled the definitive nodr in its general structure. Modul- 3. and cortex could be recognized and the trabecular system vwith its efferent lymph vessel: UL; well established kfig. Eb). The plexus of lymphatics that Sabin (1905) described as smxrrounding the entire node of a 23 cm fetus was not yet 'evident. A thin but definite capsule had formed around parts of Inany of the nodes examined and it was virtually complete a- round one node. The capsule was composed of fine collage- xious fibers with few to many fibroblasts. These observations tvere in general agreement with those of Sabin (1905) who gstated that in nodes from 23 cm fetal pigs the capsule was ccnnplete except for those locations where a plexus of lymph VfBSSGlS was present on the border of the node. Only that system of trabeculae which carried the affer- Figure 4 Mesenteric node from a 22 cm fetus H. and E, stein 90 X A. Trabeculae with afferent vessels Figure 4 Mesenteric node from a 22 cm fetus H. and E, stain 90 x ‘ A. Trabeculae wfth afferent vessels Iv 1% ent lymphatics was seen (fig. h). It showed a considerable advance in development over the previously described node in which the trabeculae consisted largely of closely packed fi- broblasts. In the node from the 21 em fetus the trabeculae were formed of fine connective tissue fibers. Isolated lym- phocytes were common within the trabeculae. Both blood and lymph vessels were contained within the large trabeculae. Valves to control the direction of the flow of lymph were seen in some afferent veSsels. Fine fibers of connective tissue branched from the trabeculae in groups to enter the gaarenchyma and often a sinus surrounded part or all of the txrabeculum. The sinuses were also traversed in a few places lazy reticular cells. The cortex of the node at this stage showed very little cliange from the cortex of the mesenteric node in the 15 cm feetus. However, the lymph nodules were more numerous and trieir borders were more clearly defined. Chievitz (lbbl) (lescribed connective tissue fibers surrounding the nodule in Inesenteric nodes from fetal pigs of sixteen to eighteen centi- Ineters. Connective tissue fibers investing lymph nodules 1vere not observed in nodes from fetal animals during the course of this study. With the exception of eosinophils, leucocytes of the gzwunflocytic series were not often seen. Eosinophils were furequently a common constituent of the cortex of pig lymph Ingéies. Blood capillaries were numerous and macrophages and Inoziocytes were fairly abundant in both cortex and medulla. 15 It was apparent that the greatest difference between the mesenteric lymph nodes of the 21 cm pig fetus and the 15 em pig fetus lay in the develOpment of the stroma rather than the parenchyma. 3. Tpp mesenteric lympp node 2; i two-da -pld pig. Un- der the microscope the general appearance of the node which was examined resembled nodes in the fetal condition more than it resembled nodes from older animals. Within the node the sinuses around the trabeculae were Wide, and frequently a plexus of small lymphatics was found Ilear the capsule (fig. 5). The presence of this plexus was xaossibly the reason Ranvier (1695) described this tissue as (:avernous. The mesenteric node at this age was completely surround- ced by a very thin capsule of fine collagenous fibers. Blood \ressels passed through the capsule adjacent to both afferent arui efferent lymphatics. The connective tissue fibers of 'the trabeculae were still very fine and many young fibroblast ruiclei were present. Occasional smooth muscle cells and elastic fibers were also present. Figure six illustrates this node. In the cortex some of the nouules now showed a small secondary nodule. Many nodules were larger than those ob— :;erved in the fetus. The largest nodule measured in the two deay’old node had a diameter of about 300 micra. In some ar’eas of the cortex the lymphocytes were packed as closely 515 they were in nodes from older animals, but in others the Figure 5 Mesenteric node from a 2 day old pig. H. and E. stain 155 X A. Plexus of lymphatics B. Capsule Figure 5 Mesenteric node from a 2 day old pig. H. and E. stain 155 X A. Plexus of lymphatics B. Capsule '- 5 vAr ' ‘ p2" . ,_ . Figure 6 mesenteric node from a 2 day old pig H. and E. stain 180 X A. Afferent vessel with valve B. Cortex jigure 6 mesenteric node from a 2 day old pig H. and E. stain 180 X A. Afferent vessel with valve B. Cortex 1 l ‘l . V . . . l 1 . ‘ '9 I . 16 density of these cells was no more than that found in fetal mesenteric nodes. Many eosinophilic metamyelocytes and eo- sinophils were seen as well as a few neutrophils, neutrophilic metamyelocytes, macrophages and monocytes. The eosinophilic cells were seen singly or in groups of up to four cells. The medulla made up a very small portion of the peri— phery of the nOue. It showed little change from the medulla of the 21 cm fetal node. The plexus of lymphatics near the capsule when present penetrated the medulla as well as the cortex. Examination of serial sections of one mesenteric node from a two day old pig disclosed the presence of two groups of afferent vessels entering it at two separate locations on the surface of the node. Each group consisted of two or three vessels. Further, this node had two systems of tra- beculae which Carried the vessels into the node. Thus it might be assumed that the node was formed by the fusion of two smaller nodes, and that each small node originally had only one set of afferent lymphatics and one system of tra- beculae. This was also observed in a popliteal node from a two day old pig (fig. 15).Sabin (1905) stated that fusion of nodes occurred in the embryo after the capsule had formed and that the capsules of the small nodes became trabeculae in the fused node. Trautmann (1946) Stated that the age at which fusion begins had not been reported, but that nodes in the beginning stages of fusion as well as completely fused nodes were found in the nine month old pig.“ He also stated 1? that the process of fusion continued until all traces of the capsules have disappeared and the parenchyma of the nodes had fused. As stated before, the mesenteric nooes from a 21 cm fe- tus showed no indications of fusion. As a fetus of this size was approximately 100 days old and as the period of gestation of the pig was approximately llh days it may be suggested that intrauterine fusion of mesenteric lymph nodes occurred late in pregnancy. Indications of the trabeculum that ori- ginally could have Deen a portion of the fused nodes were not found. Thus it may be stated that the description of fusion given by Sabin (1905) was incomplete because observa- tions made in this study indicate that the fused capsules did form a trabeculum, but only in the earlier stages of fusion. 4. $22 mesenteric node 9; g gixrda —glg pig. The ne- senteric node of a six—day-old pig which was examined showed some evidence of change when compared with the mesenteric node from the two day old animal. The capsule was about the same as that of the younger animal, but the connective tissue fibers'of the trabeculae were less delicate in nodes from the older animals. A few lymphocytes were seen in the tra- beculae. The sinuses adjacent to trabeculae were less pro- minent in nodes from older animals, and a subcapsular Sinus was frequently present around at least part of each node. The sinuses near the periphery of the nodes from younger animals were not seen in sections made of noues from a six lo day old pig. Little change had taken place in the cortex. Cells of the neutrophilic series were seen, and those of the eosino- philic series were very common. Monocytes and macrophages were also present. The medulla was more readily distinguish- ed at this stage than in the stages previously described, because of c stronger develOpment of the primitive reticular cell network, out it still formed only a small portion of the periphery of the node. 5. The mesenteric Lymph 229E g; a three-week-old pig. A mesenteric node from a pig three weeks old showed evidence of continued quion. It was provided with three groups of afferent vessuls. Between these groups of vessels the pa- renchyma was crossed from side to side by narrow bands or regions characterized by many sinuses and small blood vessels (figs. 7 and b). According to the description given by Trautmann (1926) these bands were indicative of the stage of fusion which follows resorption of the capsule. The capsule showed a few short trabeculae which pro- jected into the cortex. One of the trabeculae was seen to connect with a branch of the trabecular system which carried the afferent vessels. The larger trabeculae at this stage had a considerable quantity of smooth muscle and their col- lagenous fibers were heavier than those seen in the earlier stages. Here and there blood vessels were seen passing through the capsule into the parenchyma, particularly where the cap- y-v figue/ Mesenteric node from a three weeks old pig H. and E. stain 75 X A o i'iedullf \ l ‘ B. Cortex 1 C. Line of fusion with lymph sinuses and blood vessels. Figure 8 1‘! Enlarged vi;w of a portion of Fig. l 2;?) x I" mgue/ Mesenteric node from a three weeks old pig H. and E. stain 75 X A. 4edulle B. Cortex I n ' ' v. . 1 C. Line of IuSion with lymph Sinuses and uloou vessels. A Figure e l‘.‘ Enlarged View cf a portion of F4; s; '53:, 0". . _» twi- .s \&\‘}\:VA‘hm‘ ' 2-4‘ at ‘0 51‘?- a. ‘4“?- @W h "(l e m ‘r. ‘"‘1' s m" ,fia .d- l9 sule was adjacent to medullary tissue. This suggests that the major blood supply to the node entered through the cap- sule adjacent to the medulla rather than with the afferent lymphatics and through the trabecular system. This was in agreement with a statement made by Chievitz (1881), who re- ported that in the pig node the blood supply to the nodules entered the node at the place where the efferent vessels left the node. Baum (1911) stated that the nodules in lymph nodes of the domestic animals were surrounded by concentric layers of reticular fibers. Richter (1902) reported that in the pig lymph node these fibers formed the boundary of the nodule. Goldkuhl (1927) and Hellman (1930) described these fibers in the pig node as having the appearance of a capsule which sur- rounded the nodule. In the present study sections of the mesenteric node from a three week old pig were subjected to silver precipitation stains to ascertain if the nodules were surrounded by a Capsule-like concentration of reticular fi- bers. Examination of these preparations disclosed that this structure had not yet developed. Baum and Hille (l90t) de- scribed the nodules of the mesenteric and other nodes of a three to four week old pig as being clearer and more numerous than those in younger animals, but without the appearance they had in older animals. Observations made during the present study were in agreement with their description. The various cells of the leucocytic series present in mesenteric nodes of the previously described stages were al- 20 so seen in both the cortex and medulla of mesenteric nodes from a three weeks old animal. Cells of the eosinophilic series were the most numerous of the leucocytes seen. 6. The mesenteric lymph node of a four—month—old n'g. —————_—————_ The nodes from two pigs four months old which were examined as histological preparations showed evidence of continued fusion. However, the mesenteric node had not become a con— glomerate of smaller nodes as had those from the popliteal and other regions (fig. 17). The capsule remained thin, but the trabecular system which carried the afferent vessels was well developed (fig. 9). Trabeculae which originated at the capsule were rarely found, but a few were seen in the medulla where fusion was occurring and others were seen where corti- cal tissue was in contact with the capsule. Thus sections from mesenteric nodes had the appearance of consisting large- ly of parenchyma with a small quantity of stroma. Richter (1902) classified the lymph nodes of the pig according to the region from which the node was obtained and the development of the connective tissue septal system. He placed the mesenteric node, as well as some other nodes which received lymph from the viscera of the abdominal cavity, in the group which had the least stroma. Trautmann (1926) stat- ed that the quantity of stroma observed in sections was de- termined by which plane of the node the section was taken from, and not the region from which the node was taken. He further Stated that since hila were absent it was impossible to orient pig nodes in such a way that sections could be cut Mesenteric noee from L H. and E. stain 75 X area with heavy stroma Same node as Fig. 9. H. and E. stain 75 A Area with light stroma ‘igure 9 a month old pig Figure l0 i'flu-LWX _... as al in the same plane from two or more nodes. The present study indicated that Trautmann (1926) was correct in both state- ments. Further, there was considerable variation in the quantity of stroma at different levels in the same node. When the section under examination was cu- at a level where afferent vessels entered the node the heavy trabeculae which carried the vessels were evident, and the section showed a strong development of stroma (fig. 9). Examinations of sec- tions above and below this level showed only the smaller branches of these trabeculae and consequently the stroma appeared poorly developed (fig. 10). As stated above trabeculae that originated at the cap- sule were rarely seen in mesenteric nodes. However, examina— tion of popliteal nodes disclosed the presence, in some re- gions, of many trabeculae originating at the capsule which in this node was frequently adjacent to cortical tissue. These regions appeared very much like the cortex of a typi- cal mammalian node because the trabeculae divided the cor- tex into alveoli and frequently joined the trabecular system which carried afferent vessels. Further, the subcapsular sinus was confluent with sinuses adjacent to the trabeculae as it was in the typiCal node (fig. 17). These trabeculae gave the popliteal node the appearance of having a much stronger development of stroma than the mesenteric node. Thus it must be concluded that there was some justification for Richter's classification. although examination of pop— liteal nodes also disclosed areas in the interior of the '42 node with few trabeculae the present study suggesced that the mesenteric node had less stromal development than the popliteal node. Bundles of elastic fibers were present in both the cap— sule and trabeculae of mesenteric nodes from a four months old pig, but their occurrence was sporadic (fig. 18). Both had many heavy collagenous fibers. In the capsule blood cap- illaries and pre-and post capillary vessels were occasionally seen. Probably some of these larger vessels were entering or leaving the parenchyma. Lymphocytic infiltration of the trabeculae, a characteristic of nodes from adult pigs, was common. In the cortex most of the nodules had a thin dark stain- ing border of closely packed lymphocytes, and a pale stain- ing secondary nodule or germinal center. Mature lymphocytes were not nearly as numerous in the secondary nodule, thus the primitive reticular cells were more evident. Lympho- blastic cells with pale staining nuclei and a few cells in mitosis also were present in the secondary nodules. at this stage the largest nodules seen were approximately #00 micra in diameter. The medulla formed a thin layer around most of the peri- phery, although cortical tissue was adjacent to the capsule at a few places (fig. 17). In some regions the medulla widened and extended well into the interior of the node, as was char cteriSLic of nodes from adult pigs (fig. 17). Iso- J. lated areas of medulla surrounded by corticcl tissue were 23 seen occasionally at this stage. This also was characteris- tic of nodes from adult pigs. Isolated areas of medulla have been described by numerous authors as transverse sec- tions of medulla which project into the cortex from the periphery. Observations made during the present study sup- port this view. Cells of the leucocytic series seen in mesenteric nodes from four months old pigs were apparently largely confined to the medulla, and cells of the eosinophilic series were not numerous. .7. The mesenteric lymph node of the five—month~old pig. .—-—.-——————_ Two mesenteric nodes from one animal were studied, but no structural differences were noted from the nodes from four months old pigs, except in the development of the nodules. Nodules seen in mesenteric nodes of the five months old pig were less numerous and rarely showed secondary nodules al- though they were almost as clearly differentiated from the surrounding cortical tissue. Secondary nodules have been described by various authors as being centers of lymphocyto- poietic activity, phagocytic centers or reaction centers to noxious substances with an accompaning increase of lymphocyte production. It Was not the intent of this work to inquire into this subject, but merely to suggest that the develop- ment of nodules and secondary nodules was not closely relat- ed to the age of the animal. Figure ll, which illustrates the mode of formation of efferent lymphatics, was prepared from a mesenteric node from a five months old animal. Figure 11 Mesenteric node from a five month old pig. H. and E. stain 135 X A. Origin of an efferent lymph vessel B. Efferent lymph vessel. 0 rd a be, ,fifies . «2%. “firms... . “flat“ 9...}. «$5: . c \. flung-”fly.“ . an". 3...“ .. Io Wit. 5.3mm.“ WWWMV—Vfi .2 V l u n 21.)! p . .Nm, .fiafidfis. Figure 11 Mesenteric node from a five month old pig. H. and E. stain 135 x n. Origin of an efferent lymph vessel B. Efferent lymph vessel \" 2% t. T.e mesenteric lymph node of a seven-month-old pig. The mesenteric lymph node of a seven-month-old pig, when viewed as a histological preparation, showed little structural difference from mesenteric nodes of four and five months old animals. One distinct difference found between ‘the nodes which were examined was in the development of the elastic tissue. In nodes from the older animal a thin lay- er of elastic fibers was observed in much of the capsule. In the remaining portion of the capsule elastic fibers were seen occasionally either as single fibers or in groups. Maximow and Bloom (1957} stated that the elastic tissue lay- er of lymph node capsules was located near the inner edge of the capsule. However, in the nodes examined in the present study the elastic tissue layer, when present, was found in the middle of the capsule as well as near the outer edge (figs. 12 and 18). In the trabeculae elastic fibers were exhibited more frequently than in nodes from younger animals, but much of the trabecular system was without them. Occa- sionally a suggestion of a small network of these fibers was seen in the parenchyma, but when present they were found to be either single fibers or a.small net-like formation. Sin- gle fibers in the parenchyma probably were continuations of fibers from the trabeculae and were connected with the reti- culum. Goldkuhl (1927) reported the presence of small aggre— gations of lymphoid tissue within the substance of the tra— beculae. Further, they were continuous with the rest of the Figure 12 Mesenteric node from a 7 months old pig. w. and V.G. stain 600 X A. Adipose cells outside of capsule Figure 12 Mesenteric node from a 7 months old pig. W. and V.G. stain 600 x A. Adipose cells outside of capsule 25 lymphoid tissue. In the present study these structures were not seen in mesenteric nodes from pigs less than seven months old. In the capsule capillaries and slightly larger blood vessels were frequently seen. Since the larger vessels were frequently adjacent to the inner and outer surface of the capsule, it is suggested that they conveyed blood to and from the parenchyma of the node as well as to the tissues of the capsule. Figure 13 shows a portion of medulla of this node and a small artery in the medulla. Examination of the parenchyma showed that cells of both the neutrophilic and eosinophilic series were occasionally present. Ihese cells as well as monocytes seemed to occur more frequently in the medulla than in the cortex. Some no- dules were partly surrounded by a few short fibers. Pigs reached sexual maturity when they became four to six months of age, but full maturity was not attained until eighteen to twenty-four months. Few important structural changes were detected in mesenteric nodes of pigs from four to seven months of age. This suggests that at sexual ma- turity mesenteric lymph nodes exhibited most of the charac- teristics they possessed in adult animals. 9. The mesenteric lymph node of the three-year-old pig. The capsule of the mesenteric lymph node of a three-year—old pig was reported by many authors to be thinner than the cap- sule of nodes from the other domestic animals. In the pre- sent study the thickness of the capsule was seen to vary I f I. L' .i: E . I Figure 13 Same node as Fig. 12. H. and E. stain 000 X 26 considerably, in part due to the presence of numerous blood vessels. As described above these frequently passed through the capsule into the parenchyma of the node. Examination of sections showed that the principal constituent of the capsule was heavy collagenous fibers. Some smooth muscle tissue was present, but its distribution was erratic. This ‘ was in agreement with the findings of various authors. Elas- ' tic fibers were seen to form either an irregular layer or a ; network in the capsule (figs. 12 and 10). They were not necessarily more numerous in the vicinity of blood vessels as reported by Engelmann (1907). In the region of the medulla, where efferent vessels left the node the capsule sent short branches into the sub- stance of the medulla. Most of these branches were contin- ued in the formation of the walls of the efferent vessels within tie node (fig. ll). at the periphery of the node the capsule was continuous with the trabecular system that carried the afferent vessels. As stated above, trabeculae which branch from the capsule, except where afferent vessels entered, were rarely present in mesenteric lymph nodes of the pig. a subcapsular sinus waS'or was not present, but in either case reticular fibers which extend from the reticular network of the parenchyma formed the only important connection between the Capsule and the parenchyna. However, some collagenous and elastic fibers branched from the capsule to enter the parenchyma. The preScnce of lymphocyte- that had infiltrated the 27 C2 Figure 17 Popliteal node from a 3 week-old pig. Ho and E. Stain 30 X Illustrates the lobulated and conglomerate appearance and peripheral cortex typical pf popliteal nodes. A. Cortex B. Medulla a C. Carbon particles in afferent vessel following injection of India ink into leg below the level of the node. i i? 'rere usxall_y 1a' 35 promyelocytes and the‘later stages of the granulocytic series of cells werc frequently seen in this node, only onc plasma cell was seen during a Careful examination. #. The popliteal lymph node from a six-week-gld pig. The sections of popliteal nodes from a six-week—old animal that were examined did not exhibit any definite indications of fusion, however it was possible that fusion was occurring in other portions of the node. Most of the capsule Was heavy, but it varied considerably in thickness. The trabe- culae were well developed and those that Carried the affer- ent lymph vessels often showed groups of smooth muscle cells. In many regions trabeculae that branched from the capsule divided the cortex into the ampullae that might be consider- ed charat er istie of popliteal nodes. However, where the ampullae were aLcent the popliteal noie coul; not be distin- guished from nodes fFOL ot.:r regians W11 .17). The occur- rence of elastic fibers was Sporadic, but where present they .. . 3.2V. I. - - , A..-__ . ‘ .. .l -. . L‘JUVY l.)_.' L115, .1.l".‘.(;d31..u.L_. ll; LLu-ll_l‘_l\’u. (“Q They U'J; ;_,:-_; in both Warencfivua and stroma. In som section; cxu;_incd most of Jhc cortex “.3 Lith- out nodules, Lut in others nodules mere COMMVU. a few nc- dules sxhiuited indic.tions cf ; c..3‘;e—like structure form- ed by . few short fibers. 5. [he towiite.l lymih 1mo_e 1L the thrtt-menth-old pig. __-.. .——— —-.-—'—- ~— The hiSbOlO_1C.l preparctichs cf the popliteal node from a three-month-olu pig th.t -erc examined showed evidence of ex- .Q h tensive fusicn iLvelving sevesel nodes and Various stage 0 36 Both the gross end microsc0pic appeerence of the node was that of e conglomerate of smaller nodes. The Capsule showed u great varietion in thickness be- tween two sides of the node as seen in the section. On one side it was what might be conSiuered as normal thickness for a poplitetl node. 0n the opposite side of the node where fusion was occurring the capsule has much thinner in some cases. However, there was no evidence to indicate thet the occurrence of fusion ufld thickness of the espsule were re- lated. The cupsule wts in contsct with .dipose tissue in most locstions on each side. Bectuse of its lurge size it wss impos.ible to mount sections of the entire conglomerate that formed this node, out ex minution of the available 800-. tions showed thut the thick capsule probably surrounded the entire conglomerstc, or a portion of it, ind that the thin cupsule branched from it and surrounded the individual nodes. This structural feature WeS not noted durinv th gross ex- ;nination of the node. The trsbcculde were not as heev, es some that Lee been seen in other pepliteal nodes, but their erangemcnt, in re- lation to the distribution of cortex end medulla was impor- tent in interpreting the structure of otLer poplitecl nodes and some nodes from >ther regions. In portion 0: s. node the presence w;‘ noted of a region hlth u perip.erul cortei somewhtt divided into schOll. This region surround- ed the medulla which ir turn surreunded trebtculse which carried efierent lymphatics. _" w.s determined Ch.t this 37 type of structure was frequently present, but when section- ed in a different plane it was not readily recognized. It was also determined that this structure was radially sym- metrical and that if it was sectioned in a transverse plane the trabeculae'appeared similar to the afferent trabeculae, but were surrounded by medulla. The subcapsular and trabe- cular sinuses were usually well developed. In addition to the type of structure described in the paragraph above, the pepliteal nodes also were formed of the type of structure more typical of pig lymph nodes. This struCture had a central cortex surrounded by medulla, but the thickness of the medulla varied tremendously and in places was absent so that the cortex was adjacent to the capsule. In this type the presence of a subcapsular sinus was not at all consistent although various authors have stat- ed that it was present but occluded by compression or by free lymphocytes. In these nodes the trabecular sinuses carried lymph to the cortex, thus these sinuses were func- tionally equivalent to the subcapsular sinus of the typical mammalian node and thus a subcapsular sinus was not neces- Sary for these nodes to function. Some of the pepliteal nodes examined were composed of both types of structure and others consisted only of the type with the central cor- tex. As stated above in the description of the mesenteric node, the presence of the many trabeculae which surround the c.iveoli added to the impression that the popliteal node contained more stroma in relation to parenchyma than nodes 36 from some other regions. A few of the nodules seen in this node showed some short fibers as evidence of the capsule-like structure at their periphery. Tremendous numbers of the eosinophilic series of cells were seen in both the cortex and medulla. In addi- tion several neutrophilic myelocytes in mitosis were seen. 6. The popliteal lymph node in g four-month-old pig. Both the gross and microscopic examination of a popliteal lymph node from a four—month-old pig disclosed that it con- sisted of a conglomerate of smaller nodes. The microscopic examination showed that much fusion had occurred. There was a great variation in the size and number of trabeculae in different regions of the node. where the stroma was lighter it was comparable to that of the mesenteric node. Occasionally small branches of the trabeculae which carried the afferent lymph vessels were seen to join the capsule. Both types of structure described in the preCeeding section were present. The development of elastic fibers in the cap- sule was considerably greater than that observed in mesen- teric nodes from pigs of the same age. In the capsule of the popliteal node a network of elastic fibers formed a thin but almost complete layer which varied in thickness and in its position in the capsule (fig. lb). rhis elastic tissue layer was occasionally seen to branch and form two layers which then anastomosea. In the trabeculae elastic fibers were not numerous and were scattered, or they rarely formed a heavy layer. Prominent groups of smooth muscle cells were Figure 18 Popliteal node from a 4 months old pig. w. and V.G. stain 800 X Elastic fibers in the outer portion of the capsule. Figure 19 Same node as Fig. 18 H. and E. stain 90 X Clear lymph channel from sinus in cortex (A) to medulla (B). Figure 18 Popliteal node from a % months old pig. w. and V.G. stain 800 X Elastic fibers in the outer portion of the capsule. Figure 19 Same node as Fig. 18 H. and E. stain 90 X Clear lymph channel from sinus in cortex (A) to medulla (B). l;-.——....._......U. __ 39 seen oceasionally in the trabeculae. The subcapsular sinus was well developed and present around most of the sections that were observed, and in some areas it was also adjacent to the medulla. A dircct lymph channel from the afferent lymphatics directly to the medulla, but not to efferent vessels was observed in these sections (fig. 19). Groups of adipose cells were seen in a few places in the medullary tissue. The capsule-like structure around the nodules was seen very rarely and when present it was formed by a few short fibers. Except for the lymphoid cells, leucocytes were very rarely observed. No significant age changes between this node and the popliteal node from a three-month-old animal were noted. 7. The popliteal lymph node in a seven-month-old pig. The histological preparations of two seven-month-old pig lymph nodes that were examined were removed from three dif- ferent portions of the node. No significant differences were noted between these sections and those of the node from a four-month-old pig that was examined. Both Kinds of struc- tures described abOVe were observed in this node. The stro- ma was not heavy, but many trabeculae were soon which origi- nated from the capsule. Usually thCSe were seen only where the capsule was adjacent to the cortex. The subcapsular sinus was well developed and adjacent to both cortex and medulla except where efferent lymphatics were forming. The capsule-like structure of the nodules was not com- pletely present around any of the nodules that were examined, ’40 and usually it was not observed at all. Many eosinophils Were seen either in groups or singly, but some sections ex- amined contained very few. A few cells in mitosis were seen. in other respects this node did not differ from the node described in the section immediately above. D. THE PAROTID LYMPH NODE ‘ The parotid lymph node consisted of a group of nodes lying anterior to and in part medial to the parotid salivary gland. Cranially they were related to the posterior aspect of the ramus of the mandible and the masseter muscle. Plats- ma (l92b) stated that the afferent lymphatics of this node drain the surroundings, upper lip, rostrum, corners of the mouth, nose, front of the head and the anterior portion of the external ear. He further stated that the efferent ves— sels lead to the lateral retropharyngeal node. Observations were made during this study on the parotid node of five animals. Grossly the node appeared to be a cloSe conglomerate of small and large nodes. This was con- firmed by histologiCal studies. EXamination of serial sec- tions disclosed that the small nodes were frequently com- pletely fused at small areas of contact in such a way that they could be considered as lobes of one larger node. 1. The parotid lymph node of the al centimeter fetal pig. The parotid node from a a1 cm fetus showed no impor- tant differenCes irom the mesenteric node of the same age except for the following. The capsule of the parOtid node was thicker and clumps of elastic fibers were occasionally 41 Seen in some trabeculae. Evidence of an early stage of fu- sion was seen. A narrow band of medullary tissue penetrated by many small sinuses extended entirely across the node. According to Trautmann (1926) this was a stage of fusion. This observation confirmed the statement above (p. 17) that fusion occurred late in pregnancy. The node, at this age consisted of two closely adjacent nodes. This suggested that the formation of the conglomerate parotid node seen in older animals started forming before birth. 2. ghg parotid lyhhh node ;h g ghg—ghy-ng pig. No differences were seen between parotid lymph nodes from a one— day—old pig and the late fetal pig described above. 3. Ehh harotiu lyhph none ih fl Egg-ghy-glg pig. The parotid node from a two-day—old pig was much like the mesen- teric node from the same animal. This parotid node distinct- ly showed the conglomerate appearance of the parotid node of older pigs. Six lobes could be distinguished in one section. The parotid node also had a thicker capsule. The node of the two—day—old animal showed a concentration of lymphocytes comparable to that found in nodes from older pigs, and the stroma showed a much greater development than that found in the node of the one-day-old pig. The changes obserVeu between nodes in one and two-day- old animals were so great that it did not seem likely that they occurred in only one day. It seemed more plausable that such change; occurred during a period of several days. As these nodes were from two different inuiViuuals it could | +2 be concluded that differences were due, at least in part, to differences in the rate of growth of the individual animals, rather than to the difference in age of the animals in days. However, the change from the appearance in the fetal state to that of the older pig had occurred within two days after birth in all nodes examined. 4. Thg parotid lymph node in a four-month-old pig. The parotid lymph node from a four-nonth-old pig in its gross ap- pearance seemed to be a conglomerate. This observation was confirmed by microscopic examination. In proportion to pa- renchyma little stroma was seen even though very small tra- beculae extended from the capsule into both cortex and medul— la. The Capsule was not heavy and a subcapsular sinus was present around most of the node adjacent to both cortex and medulla. Two stages of fusion were observed in the sections that were examined. The general structure of the node show— ed a large cortex much of which was surrounded by a narrow medulla. A few of the many nodules were surrounded by a well developed ring of collagenous fibers. Eosinophils were rarely seen. E. THE PHARYNGEAL LYMPH NODES Platsma (1926) described two lymph nodes in the region of the pharynx of the pig, but Somers (1951)_and Sisson and Grossman (1953) described only one. In all cases these nodes were found on both the right and left sides. The node term— ed suprapharyngeal by Sisson and Grossman (1953) in the pig and by Somers (1951) was the Same as the node that Platsma raw)”; {It‘ll}... «vial. its _.Lt§wrutrih§..l,...sx. . . (1928) named medial retropharyngeal. Platsma's description of the location of the node he called medial retropharyngeal. was similar to the description of the parapharyngeal node in the horse described by Sisson and Grossman (1953). According to Platsma (1928) the lateral retropharyngeal node received lymph from its surroundings, part of the ear, the posterior region of the head, skin, shoulder joint, tongue, tonsils and rostrum or snout as well as from the mandibular, parotid, and cranial cervical lymph nodes. Its efferent vessels carried lymph to the superficial cervical node. The medial retropharyngeal node drained the conchae, tongue, tonsils, larynx, pharynx and in some cases the super- ficial cervical lymph node. Its efferent lymphatics formed the tracheal lymph duct. For this study one node from each of these pharyngeal regions was removed from one pig. No structural or histolo- giCal differences were noted so these were treated as one node. 1. The alienate}. mm: 9929: 9f. 2 £93?.-M-.01_d £3- The pharyngeal lymph nodes of a four—month—old pig, except for their lobulated appearance showed no important.differ- ences from the mesenteric node in a pig of the same age. F. THE SUPERFICIAL CERVICAL LYMPH NODE Sisson ;nd Grossman (1953) used the term prescapular for this lymph node and gave the term superficial cervical as a synonym. They stated that it was located at the an- terior border of the anterior deep pectoral muscle, and that 44 it drained lymph from the neck, chest, shoulder, and arm. Somers (1951) stated that the prescapular node was located in a.deposit of adipose tissue slightly dorsal and medial to the shoulder joint and that several of these nodes form- ed a chain of partially or almost completely fused nodes. As a synonym for prescapular Somers used the term posterior superficial cervical, but did not clearly state that another superficial cervical node was present. The above named au— thors agreed in their descriptions of the areas drained by this node. Further they agreed that it emptied into the posterior cervical node. However, Somers stated that in some individuals it drained into the tracheal duct, and in some cases the efferent lymphatics of this node on the left side lead directly into the thoracic duct. Platsma (l92o) described a superficial cervical node and a middle superfi- cial cervical node. Neither of his descriptions agreed with Somers (1951) or Sisson and Grossman (1953). However Platsma (1928) reported that the middle superficial node received lymph only from the anterior limb and that the superficial cervical node drained the posterior-region of the head, the neck and shoulder, and certain other lymph nodes. 1. The superficial cervical lymph node from a four- month-old pig. The superficial cervical lymph node from a four-month-old pig exhibited several stages of fusion. Five separate nodes partially fused into one conglomerate node were distinguished in the sections examined. The trabeculae were developed to about the same extent as those of the 11.5 mesenteric node from a four-month-old pig, but the capsule in the one node studied frequently was thicker in the super- ficial cervical node. Small trabeculae were seen occasional— ly projecticng into the medulla from the capsule as well as those found where efferent lymphatics were forming. Elastic fibers were present in the stroma in approximately the same quantity and distribution as they were seen in the mesen— teric node at this age. However, in one portion of the cap— sule of the superficial cervical node elastic fibers formed a very thin layer. This layer was in the middle of the cap- sule. This was contrary to the statements of authors who reported that the elastic layer of the capsule was closer to the inner edge (see above p. 2%). In this respect it re- sembled the elastic tissue layer of the capsule of the mesen- teric node from a seven-month-old pig. In those locations :here fusion of nodes was in the earliest stage, i.e. where the two capsules had fused, no differences were seen in the quantity of distribution of elastic fibers from that seen in the capsule which surrounded the whole node. A few plasma cells were seen in sinuses which were ad- jacent to the medulla. In other respects this node resem- bled the meSenteric node of the two animals of the same age that were examined. This node was an excellent example of the difficulty or even futility of the attempts by various authors to classify pig lymph nodes according to the relative proportions of proportions of cortex stroma and parenchyma, or the relative #6 and medulla. Many variations of these proportions were ob- served in the sections examined. 2. ”1e superficial cervical lymph node from a seven- a—ou— .— month- ld pig. The sections of a superficial cervical lymph node from a seven-month-old pig that were examined did not show the evidences of extensive fusion that were seen in the node from the younger animal. The capsule varied considera- bly in thickness, but it was thicker than the capsule of the mesenteric node from the same pig. In some trabeculae more smooth muscle cells were observed than were seen in the mesenteric node. As these observations were made on only one node from each region they may not be significant. The superficial cervical node at this age exhibited some small trabeculae which traversed the medulla and connected with branches of the trabecular system which carried the afferent lymphatics. However, their development was not as extensive as that observed in popliteal nodes. These trabeculae were not seen in mesenteric nodes from pigs older than three we ch 5 . i - The cortex and medulla of the superficial cervical node did not exhibit any particular differences from those of a mesenteric node from the same animal with the following ex- ception. Most of the larger nodules, when viewed in histo— logical sections, were seen to be partially or completely surrounded by a few heavy collagenous fibers which formed the capsule—like structure discussed above in the section on the mesenteric node of a three weeks old pig (fig. 20). Figure 20 Superficial cervical node from a 7 months old pig. H. and E. stain 185 X Portion of a nodule and the adjacent cortex illus- trating the collagenous fibers which partially surround the nodule. r v i l 1 I i , ~ \ w | , \ It was suggested that this structure be termed the nodular capsule. This nouuiar capsule was more extensive in the superficial cervical node of a seven-month-old pig than that shown by mesenteric nodes from pigs of any age including the I node from a pig of three years of age. Examination of this structure in the superficial cer‘ical nodes suggested that the nodular capsule did not completely surround the nodule and that the lymphoid tissue of the nodule and the surround- ing cortex were continuous in at least one area on the peri- phery of the nodule. Reticular and collagenous fibers were ‘rarely seen within the nodule. This resulted in the circle of one or several fibers that formed the nodular capsule serving as an attachment for fibers in the surrounding cor- tex. This tended to make this capsule more conspicious in sections stained with precipitated silver than those stained by the usual histological stains.(fig. 23). Frequently some fibers of the nodular capsule were also attached to adjacent trabeculae. G. THE MANDIBULAR LYMPH NODE Platsma (1928), Sisson and Grossman (1953), and Somers (1951) stated that the mandibular or submaxillary lymph node of the pig was located in the mandibular space adjacent to the anterior portion of the mandibular salivary gland. Sis— son and Grossman (1953) stated that two mandibular nodes were often present on each side, one large and one small. The afferent lymphatics carried lymph from the surroundings and from most of the structures which formed the anterior 4t portion of the head. according to Platsma (1928) the effer- ent lymph vessels entered the cranial cervical, lateral re- tropharyngeal or superficial cervical node. However, Somers (1951) stated that they led to the atlantal node. In its gross appearance the mandibular node did not appear to be a conglomerate of smaller nodes. Only one node from this region was collected and examined. 1. The mandibular lymph node in the four—month-old pig. Examination of preparations of the mandibular lymph node from a four-month-old pig showed that it was an individual node and not a conglomerate of smaller nodes. This was also noted by Trautmann (1926). The stroma was not heavy nor pre- sent in proportionately large quantities. However, the sec— tions examined were not taken from a location near the en- trance of the afferent vessels and their heavy trabeculae. The capsule was variable in thickness, but it was never very thick. Trautmann (1926) stated that the capsule of non—fused nodes was stronger where blood vessels entered, but this was not cbserved. In the sections that were examined, the cor- tex was central. a subcapsular sinus was present around the node except where efferent lymph vessels were forming. As the cortex was completely surrounded by medulla this sinus was adjaCent to medulla. The nouular capsule was absent or poorly developed when present. a few plasma cells and only a few eosinophils were 56011. N we f %9 H. THE BdONCHIAL LYMPH NODE Platsma (192e) stated that the bronchial lymph node was located at or near the bifurcation of the trachea. Sisson and Grossman (1953) agreed with Platsma's statement and fur- ther stated that an additional bronchial node was located at the apical bronchus of the right lung. Somers (1951) agreed %5 with the statements of the authors named above. Furthermore, fig: these authors agreed that this node drained lymph from the lungs, heart and esophagus, and that the efferent vessels - ;‘ carried lymph to the mediastinal nodes. Somers (1951) fur— ther stated that the bronchial node occurred in groups of two or four on each side. ; :1 1. The bronchial lymph node in ; three-week-old pig. 1 The bronchial lymph node from a three-week-old pig did not show any evidences of fusion in the sections exemined. Trautmann‘(l92e) reported that little fusion occurred in the bronchial node. The capsule of the specimen examined was not particularly thin, but it was formed of delicate fibers. Only a few sm_ll trabeculae were seen and these were also formed of deliCate fibers. No subcapsular sinus was ob- served. The node consisted of cortex with a thin rim of medulla along one edge. A few nodules were present. They were distinct and all of those observed were surrounded in part by a fine nodular capsule. VSome eosinophils were observed. 2. The bronchial lymph node in a five—month-old pig. The bronchial lymph node from a five-month-old pig was small 50 1nrhen compared with the large conglomerate nodes from some ca‘ther regions such as the popliteal and the parotid. It 1AT€JS also small when compared with a node that showed exten- £;:ive evidences of fusion such as the mesenteric node. In 't:Ine sections of this bronchial node that were examined no ezleidence of fusion was seen. The general structure of the 1:1(3de showed a central cortex with a thin rim of medullary ‘tissue. The capsule varied in thickness from thin to fairly '13 Buick. Very few trabeculae were seen in the sections exam- :i—Jrled. The trabeculae were not thick and the collagenous fi- 'C3lears which were their principle constituents were fine. 143:1.astic fibers were rarely seen in the stroma or the paren— C3ikuyma. A well developed subcapsular sinus was present under IIl<:>st of the capsule. A few nodules were seen in the cortex and all of these ‘“Tere in part surrounded by the few connective tissue fibers 'Vvhich formed the nodular capsule. A few cells of the eo- ssinophilic series and a very few plasma cells were seen. I. THE LUMBAR LYMPh NODE The lumbar lymph nodes comprised a row of nodes adja- cent to the aorta in the sublumbar region. These were a part of 3 ch in which included the renal and internal iliac nodes. Platsma (1926) named the lumbar nodes the lumbar aortic nodes. He stated that the afferent vessels arose only from thu skin of the lumbar region and possibly the dorsal parietal peritoneum, and that the efferent vessels led to the pelvic or lumbar lymphatic trunk. Somers (1951) :5'tated that the lumbar nodes received lymph from the dorsal :lgumbar region and the upper ventral abdominal wall. In ad- cflgition he stated that they received lymph from most of the J:1<3des located further posteriorly, thus most of the lymph :1T:rom the posterior part of the body would pass through the 21.14mbar nodes. He also stated that the efferent lymphatics :1.‘vidence of fusion in the histological sections from one SEjpecimen that was examineds however, it could not positive- :1_:y be stated that fusion had not occurred as the node was 53knilic cells were seen, but one plasma cell and a cell of ‘tiliie basophilic series were seen. J - THE DEEP INGUli‘mL LYMPH NODE Platsma (192b) stated that the deep inguinal node of ‘t3lfle pig was located in the pelvic cavity adjacent to the ex— tSesernal iliac artery. According to Sisson and Grossman (1953) ie‘t: was most ventral of several nodes situated along this EiSIPtery. Somers (1951) did not describe this node in the <3>1? the deep inguinal node drained lymph from the deep por- ilixum of the rear limb, part of the parietal peritoneum, the ESkin of the lumbar region, the superficial inguinal node, Eind sometimes the popliteal and Sacral hypogastric nodes. TDhe efferent lymphatics went to the pelvic lymphatic trunk or occasionally to the medial iliac, or renal node. 1. The deep inguinal lymph node in phe 21 centimeter pig fetus. Ihe deep inguinal lymph node from one a1 cm fe- tal pig, when viewed as a histological preparation, did not appear to diiier greatly in structure from a mesenteric node from the same fetus. Very-little stroma was Seen in this node. The capsule was very thin and the few trabeCalae that were seen were , t... iii! If Tilt. 21:3. ,. trnrudeysz It... Frank; air ,. 53 gaznall and delicate. No evidences of fusion were seen in ‘t313e sections examined. A few lymph sinuses were seen at 'tzlue periphery and more than a few in the parenchyma, but the 1:1eetwork of peripheral sinuses as described by Sabin (1905) eras not seen. A valve was observed in an efferent lymphatic- <::L1_ose to the node. Although other authors had stated that "7WE11ves were usually found close to the node in efferent ves— ES 6215 they were not seen in any of the nodes described above. It was not possible to make a clear distinction between .t:}:1e cortex and medulla although it was possible in the mesen— tS (Eerie node at this age. As in the mesenteric node a few Eslluall nodules were seen. The cellular constituents of the 1;’Elrenchyma were like those seen in the mesenteric node. 2. The deep inguinal lymph node in a four-month-old _————-—-——-———— 12glg, This node from a four-month-old pig was large and ex- €lrnination of histologicel sections disclosed evidence that flit was formed by the fusion of several smaller nodes. The <:apsule Varied in thickness from thin to heavy. The trabe- culae were usually heavy and they were observed to be more Innnerous in the cortical tissue than they were in the same tissue in mesenteric and other nodes. The node resembled the popliteal in this respect. Elastic fibers were seen occasionally in both the parenchyma and stroma. A subcap- sular sinus was present in places adjacent to both cortex and medulla. The general structure also resembled the pop- liteal node as both the central and peripheral cortex types were seen. The presence of nodular capsules was rare. In slides 3;. of one portion of the node that were examined no nodules I we‘re seen. The cortical tissue consisted only of a thick tube—like Structure around a trabeculum. These were scatter— ed throughout the medulla. which formed most of the node in the sections examined. This structure probably was a varia- tion of the central cortex type. No eosinophils were seen. a K - THE SUPERFICIAL INGUINAL LYMPH NODE The superficial or external inguinal lymph node was t ermed the supramammary node in the female because of its 3 I 1" elation to the posterior mammary gland. No important dis- ! <-‘-J:-epencies were found in the descriptions of this node given b3; Sisson and Grossman (1953), Somers (1951), and Platsma , \ C 1928). They occurred as subcutaneous groups at and adja- ‘ Cent to the external inguinal ring. The afferent lymphatics drained the external genitals and the slain of the surround- ing area and rear limb. In addition Platsma (192C) reported that they drained a portion of the peritoneum and the deeper portions of the upper rear limb. l. The superficial inguinal lmph node in a three-week- gld pig. The superficial inguinal node from a three-week- old pig resembled the popliteal nodes from the pigs of the same age in three respects. Proportionally large quantities of stroma were seen, most nodules were adjacent to a trabe- culum, and considerable fusion had occurred. On the other hand, the general structure W;S the central cortical type. Because of the extensive fusion that had occurred the general gs'truetural type was obscured but it clearly was not the peri- 3;Lheral cortical type. A subcapsular sinus was present a- ;:?<3und most of the node in the sections that were examined. .IEZIastic fibers were seen more frequently than in other nodes :f?3rom pigs of this age. However, they were not often seen EEIJCMi examination of nodes from several animals might indi— Czteate that this situation was exceptional. Large blood vessels were seen entering and leaving the Ilaueedulla of the node near the locations of the efferent ves- ES (2315. This was observed in other nodes also. -Where pre- 53 (:ent the nodular capsule was formed of one or two layers of hLreparations. Goldkuhl (1927) injected dye directly into Tzhe node and demonstrated that it left through efferent ves- ssels which did not originate at a hilus. Valves prevented Jeeverse flow of dye from the afferent vessels. Both methods could be criticized because the pressure used could concei— ‘vably have induCCd artifacts'in the reticular tissues of the {‘1 nodal sinuses. ihe methods described below were less likely __ ,1 to onus; artifacts and were sulficient to delineate the path- ways taken by the lymph. Three techniques were used to study tLe circulation of lymph through the node. Two of these involved injection of materials into the p'g end the third consisted of examina- tion of sections to ascertain the position of the valves. [-1 - Subcautaneous and intramuscular injections of liv ng 1‘ pigs with an aqueous suspension of India ink or of e. U1 .13.,1, .....1. -Swatfiflxsirhifwhaa.» iii... ... .a , 1}“ . 0’ ,‘ "5“ jpereent aqueous trypan blue were used for localization of :phagocytie cells within lymph nodes. Regions of the nodes 1Nhich exhibited the phagocytized materials served as means (of identification of passages. Thus some of the regions of izne node through which lymph had passed were demonstrated. ESubcutaneous injections permitted the injected material to 130 carried to the node by normal lymph flow, thus artifacts Taicre not likely to be produced. In another xperiment 0.5 ]:>ercent aqueous trypan blue was injected into the su mucosa <:>f the small intestine of a pig immediately subsequent to Citeath by exsanguination. Injection of materials was discon— ‘tzinued when the lacteals in the mesentery exhibited an abun- Cflance of dye. As the pressure used to inject dye into the Esubmucosa was not directed against the nodal tissues it was Ibelieved that no artifacts were induced. The node adjacent ‘to the injected area, subsequent to fixation, dehydration and staining was used for study of lymph channels in nodes deep in the body. As stated above the third technique used was, the ex- amination of sections that exhibited valves. These valves had the same structure as thOSe that occur in the veins. They were found in pairs on opposite sides of the vessel with one edge attached to the wall of the vessel and one edge free. The direction of lymph flow past the valves was from the attaeted end toward and beyond the free end. Thus by examination of the valve the direction of lymph flow could could be determined. ‘29 The injection of trypan blue into tlm gut wall demon- strated the pathway of lymph through mesenteric nodes. Al— though little phagocytosis of dye materials had occurred within the node there were sufficient accumulations of dye to make evident the channels through which it flowed. In all sections examined the adjacent tissue was well stained in areas where the dye had passed through. frypan blue could be seen in the afferent lymphatics within the trabe- culae (fig. 21). It could also be seen in the sinuses adja- cent to the smaller trabeculae that lead to the medulla. Very little was seen in the cortical tis ue. On the o ther hand? large areas of the medulla were uniformly stained in- dicating that lymph had diffused widely in the medulla. fivi— dence of dye was also seen in some efferent vessels In section: of popliteal nodes following subcutaneous injection of In L ink suspensions phagocytosis had occurred in the cortex adjacent to the s n see around the trabeculae. No carbon particles were seen elsewhere in the cer‘eY or in 4.4. the medulla, exoctt in afferent vessels (fi 5. l7 and 22). x C‘} .‘ ihe evidence cited above sliows that lynuh was transport— 3 L! ed deep into buC node through afferent vessels which were located in trabeculae, and_whicL drained into sinuses m‘j- CI.) cent to the t‘abeeulae. ‘heso si;us\: continued as far as tho nedulla althou