Original Paper Acta Anatómica 1992; 143:41—47 Department of Anatomy. Abel Salazar Institute for the Biomedical Sciences, and Metallurgic and Material Sciences Center of the Engineering Faculty. University of Porto, Portugal: Department of Anatomy. Faculty of Medicine. University of Agostinho Neto. Luanda. Angola Key Words Scanning electron microscopy Arterial capillaries Popliteal lymph node Tracheobronchial lymph node Paratrachéal lymph node Blood Vasculature of the Lymph Node in the Dog: Anatomical Evidence for Participation of Extrahilar Arterial Vessels in the Blood Supply of the Cortex Abstract The organization of the arterial vessels of dog lymph nodes (LN) was studied using methods of visualization of the vasculature by systemic injection of differ ent tracers (colloidal carbon. Micropaque® resin and methylmethacrylate) fol lowed by observation of the samples by light microscopy (after clearing of the thick sections of LN) or scanning electron microscopy (corrosion casts). LN from all of the three groups of nodes studied (tracheobronchial, paratracheal and popliteal) showed an extensive network of arterial vessels encircling the cap sule of the organ. We found that branches of these capsular arteries penetrated deeply into the cortical domain of LN. The capsule-originating vessels appeared to have a significant participation in the blood supply of the LN parenchyma at the cortical domains of the organs. Our findings are in contrast with current views on the angiology of the LN that consider that virtually all of the arterial capillaries of the LN parenchyma come from hilar arteries. We propose, there fore. that important segments of the LN cortex receive their blood supply from capsular arteries rather than from hilar vessels. Introduction The lymph nodes (LN) play a central role in host defence of mammals against foreign infectious agents and particles [ 1-5]. These lymphoid organs contain all the cellu lar elements of the immune system and are capable of trig Received: June 15. 1991 Accepted: August 20. 1991 gering specific responses directed at antigenic components. The LN vasculature shows characteristic anatomical arrangements that are known to modulate the physiology of the organ, namely lymphocyte traffic and recirculation [6,7]. Current prevalent views on the micro-anatomy of the LN vascular system consider that virtually all arteries enter Nuno Rodrigues Grande. MD, Phi) Professor and Chairman. Department of Anatomy Abel Salazar Institute for the Biomedical Sciences P-4000 Porto (Portugal) © 1992 Kargcr A G . Basel 0001-5180/92/1431-0041 S 2.75/0 Downloaded by: Vanderbilt University Library 220.127.116.11 - 10/25/2017 12:17:49 PM A.C. Salvador, a , b A.S. Pereira" C.M. de Sdc N.R. Grande1 Materials and Methods Animals Nine male and female aduli mongrel dogs were used in this study. The animals were housed in individual kennels, fed standard commer cial dog food and had unrestrained access to water. The dogs were anaesthetized by intravenous injection of sodium pentobarbital (-40 mg/kgof weight), the abdominal cavity was surgically exposed and the animals were bled by cutting the inferior cava vein. Before death of the animals, a cannula was introduced into the abdominal aorta, and the vascular system of the animals was washed with warm saline. This was immediately followed by injection of different tracers (see below) into the arterial system of the dogs to visualize LN vessels. Injection o f Colloidal Carbon and Micropaque Colloidal carbon (Pelikan China ink: 30% in gelatin) or 5% gelati nated Micropaque were injected either in the external iliac arteries (to visualize the popliteal LN) or in the upper portion of the abdominal aorta (to visualize the tracheobronchial and paratrachcal LN). Injection o f Methylmethacrylate Methylmethacrylate (MMA: tensol cement No. 70; ICL Chemical and Polimcrs Ltd.. Darwin. Lanes, UK) was injected into the distal portion of the femoral arteries (to visualize the popliteal LN) or in the thoracic aorta at the origin of the bronchial arteries (to visualize the tracheobronchial and paratrachcal LN). Fig. 1,2. General views of whole vascular casts of a popliteal (1) and a tracheobronchial (2) LN from dogs perfused with MMA resin. The nodes show a high density of vessels coating all of the outer sur face of organ in a hair-like fashion. 1 Notice that several large arterial vessels are scattered around all of the node surface without a precise indication of the positioning of the Itilus. The vessels shown in the lower half of figure l are depicted in higher magnification in figure 6. x 10. the node through the hilus and that the blood supply of the organ’s parenchyma comes from branches of hilar arteries [8-12], According to this view, the few arterial vessels that reach the LN independently of the hilar arteries are directed to the capsule and have no or very limited partici pation in the blood supply of the lymphoid tissue. In this work we have investigated the micro-anatomy of the arterial blood vasculature of dog LN. We integrated information retrieved by several methods of filling the lumen of the vessels: resin casts of the LN arteries, sections of nodes perfused with gelatinated colloidal carbon or Micropaque® resin, and scanning electron microscopy of resin casts. We show here that LN have elaborated net works of extrahilar arteries. The striking development and the topography of these arteries suggest that extrahilar arteries may form an independent blood supply system of the cortical domain of LN, rather than being restricted just to the capsular tissue of the node. 42 Processing o f LN All nodes were surgically removed and processed according to the method used to trace their blood vasculature. The LN injected with gelatinated colloidal carbon were fixed in 10% forntol. The organs were either sectioned into 1-mm layers and xylol cleared or serially fro zen sectioned (with widths of 60. 150 and 300 pm) and cleared in xylol. The tissue sections were mounted on glass slides anil studied by light microscopy. Routine histological sections were also made and stained with hacmatoxylin and eosin. The nodes injected with MMA were digested in HCI and washed in water with the help of sonication. The MMA samples were then mounted on metal plates to be further treated for scanning electron microscopy observation. Scanning Electron Microscopy The MMA casts were coated by Au/Pt under vacuum and studied in a JEOL ISM-35C scanning electron microscope. The electron micrographs were derived from secondary electrons. For identifica tion of the vessels we followed Rhodin's criteria 113. I4| in what con cerns dimensions and general morphology of the vessels and those of Lametshwandtner et al. 115] regarding their anatomical features by scanning electron microscopy. Results We studied the micro-anatomy of the arterial vascula ture of the dog LN using two types of nodes: popliteal LN that contain mostly primary follicles, and tracheobronchial and paratracheal LN that predominantly present second ary follicles. Salvador/Pereira/de Si/Grande Lymph Node Blood Vasculature Downloaded by: Vanderbilt University Library 18.104.22.168 - 10/25/2017 12:17:49 PM 2 43 Downloaded by: Vanderbilt University Library 22.214.171.124 - 10/25/2017 12:17:49 PM (For legends, see p. 45.) 44 Salvador/Pereira/de Sa/Grande Downloaded by: Vanderbilt University Library 126.96.36.199 - 10/25/2017 12:17:49 PM Lymph Node Blood Vasculature Sections o f LN Perfused with Colloidal Carbon or Micropaque Scrutiny of these light microscopy preparations con firmed the observations derived from MMA corrosion casts and further illustrated the penetration of the LN cortex by branches of capsular arteries in tracheobronchial LN (fig. 7-9). The sections showed multiple examples of corti- Fig. 3-5. Scanning electron micrographs of vascular casts of dog LN perfused with MMA resin. The capsule is supplied by multiple medium-size arteries (top in 3) that, on reaching the outer surface of the node, originate an elaborated network of anastomotic vessels that encircle the surface of the nodes. The arrangement of this anastomotic arterial network is best seen in face views of the node surface (4. 5). 3 Arrowheads show penetration of the cortical domain of the node by branches coming from capsular arteries. 3 x 80. 4 x 105. 5 x 120. Fig. 6. Scanning electron micrograph of vascular cast of dog popliteal LN perfused with MMA resin. This is a higher magnification of an area of the same node shown in figure I. The large arteries seen on the right side of the figure penetrate deeply into the node: they may correspond to hilar vessels. Notice, however, that in figure 1 several other arteries, with similar large calibres, enter the node in other areas of its surface. x70. cal regions that appeared to receive their blood supply from branches of capsule-associated arteries (arrowheads in fig.7-9). We found that elements from all of the three groups of dog LN presented some areas of their cortical domains receiving their arterial vessels directly from capsu lar arteries. Discussion Current views on the organization of the arterial supply of the LN consider that virtually all of the node arteries enter the organ through its lulus [8-12]. It is. therefore, assumed that cortical and medullary regions of the organ are irrigated by branches of the hilar vessels. Only small calibre vessels would pass through the lulus and go directly to the LN capsule; these vessels are considered not to have a significant role in the arterial blood supply of the LN parenchyma [12. 16-18). In this report we offer anatomical evidence that in differ ent groups of LN of the dog. the capsular arteries make up a well-developed surface vascular network. These vessels were so numerous that it was difficult to identify precisely which arteries did enter the nodes through the hilar invagi nation. Some of the capsular vessels penetrated deeply into the LN cortex. This topography indicated that they contrib uted to the blood supply of at least part of the cortical region and could play a significant role, albeit not com pletely understood as yet. in the circulation physiology of the LN parenchyma. It has been shown before that the total occlusion of the hilar blood supply of the popliteal LN did not result in interruption of the physiological activity of the LN , This surprising result was interpreted as an indication of the capacity of the lymphatic supply of the LN to take over by itself all the nutritional needs of the lymphoid organ. Our anatomical data suggest an alternative explanation: the occlusion of the hilar arteries may not result in the com plete interruption of all of the arterial blood supply of the node, since, in our preparations, it was clear that the corti cal tissue received extrahilar vessels which originated from capsular arteries. We suggest, therefore, that the arterial branches coming from capsular vessels may have a signif icant participation in the blood supply of the LN paren chyma. In conclusion, we propose that the parenchyma of the LN receives its arterial blood supply from two vascular systems, the hilar and the capsular arteries. We believe that the plasticity of these two arterial systems of LN deserves to be investigated in well-defined pathophysiological events of the organ. 45 Downloaded by: Vanderbilt University Library 188.8.131.52 - 10/25/2017 12:17:49 PM MMA Resin Casts o f LN Vessels These preparations offered surface views of the LN and of their peripheral arterial blood supply. Because of the high viscosity of this resin. MMA perfusion stopped at the level of arterial capillaries, and the casts, therefore, did not include any venous vessels. In low-magnification views of these corrosion casts, we found an abundant number of arterial vessels around the outer surface of all of the LN studied (fig. 1.2). These vessels formed a continuous hair like coating that completely encircled the LN. The striking development of this peripheral vascular layer did not allow an easy identification of the hilar pole in a good number of LN by observing a concentration of arteries in the hilar region of the nodes (fig. 1.2). Higher-magnification views of the MMA casts were obtained by scanning electron microscopy (fig.3. 4). Here, it was clear that the predom inant direction of the arterial vessels seen on the node sur face was parallel to the capsule (fig. 3-5). The capsular arte rial vessels made up a very elaborated network that was best seen in face views (fig.4, 5). In several areas of the extrahilar regions of the LN surface, vessels originating from this encircling arterial network penetrated the capsu lar layer and entered deeply into the cortical domain of the organ (see arrowheads in fig.3 and compare fig.6 with its lower magnification illustrated in fig. I). 46 Fig. 8, 9. Light micrographs of sections of paratrachéal (8) and popliteal (9) LN of the dog after arterial perfusion of the animal with the resin Micropaquc. Examples of penetration of the cortex of the two nixies by branches of capsular arteries are indicated by arrow heads. x 35. Salvador/Pereira/de S¿¡/Grande Lymph Node Blood Vasculature Downloaded by: Vanderbilt University Library 184.108.40.206 - 10/25/2017 12:17:49 PM Fig. 7. Light micrograph of xylol-cleared section of dog LN after arterial perfusion of the animal with colloidal carbon. A large artery coming from the capsule of the nixie penetrates and branches into the cortex of the organ (arrowheads at the top of the figure). The LN hilus is indicated by arrows. x30. Acknowledgements The authors thank Dr. Artur P. Aguas for help with the writing of the manuscript. Mr. Alfredo Ribeiro (Depart. Experimental Surgery. ICBAS) for surgical help and Mr. Jose' Aurelio for the photographic work. During the course of this research work. A.C'.S. was a recipient of a fellowship front the Calouste Gulbenkian Foundation (Lisbon. Portugal). This research work was supported by a research grant front the Junta Nacional de Investí garfio Científica e Tecnológica (Portu guese Research Council). References 7 Hay JB. Hobbs BB: The flow of blood to lymph nodes and its relation to lymphocyte traffic and the immune response. J Exp Med 1977:145:31-44. 8 Heath T. Brandon R. Fogarty SJP: The arte rial supply to lymph nodes in sheep. J Anat 1985:141:41-52. 9 Kowala MC. Schocfl GI: The popliteal lymph node of the mouse: Internal architecture, vas cular distribution and lymphatic supply. J Anat 1986:148:25-46. 1(1 Steeber DA. Erickson CM. llodde KC. Albrecht RM: Vascular changes in popliteal lymph nodes due to antigen challenge in nor mal and lethally irradiated mice. Scanning Electron Microsc 1987:1:831-839. 11 Weiss L: A Textbtxtk of Histology, ed 6. Balti more. Urban & Schwarzenberg. 1988. 12 Belisle C. Sainte-Maric G: Blood vascular net work of the rat lymph node: Tridimensional studies by light and scanning electron micros copy. A tit .1 Anat 1990:18:111-126. 13 Rhodin J: The ultrastructure of mammalian arterioles and precapillary sphincters. J Ultrastruct Res 1967:18:181-223. 14 Rhodin J: The ultrastructure of mammalian venous capillaries, venules and small collecting veins J Ultrastruct Res 1968:25:45-500. 15 Laniclshwandtner A. I.ametshwandtner U. Wciger T: Scanning electron microscopy of vascular corrosion casts - Techniques and applications. Scanning Electron Microsc 1984: 2:663-695. 16 Blau JN: A comparative study of microcircula tion h the guinea-pig thymus, lymph nodes and Peyer’s patches. Clin Exp Immunol 1977:27: 340-347. 17 Herman PG: Microcirculation of organized lymphoid tissue: in Hanson LA. Sltakib F (eds): Monogr Allergy. Basel. Karger. 1980. vol 16, pp 126-142. 18 Heath T. Brandon R: Lymphatic and blood vessels of the popliteal node in sheep. Anat Rec 1983;207:461-472. 19 Hall JG. Sinncl HD: The functioning in un anaesthetized sheep of the lymph node after the surgical removal of its blood supply. Br J Exp Pathol 1987:68:125-132. 47 Downloaded by: Vanderbilt University Library 220.127.116.11 - 10/25/2017 12:17:49 PM 1 Heinen E. Cormann N. Kinct-Dcnocl C: The lymph follicle: A hard nut to crack. Immunol Today 1988:9:240-241. 2 Szakal AK. Kosco MH. Tew JG: Microanat omy of lymphoid tissue during humoral im mune responses: Structure function relation ships. Annu Rev Immunol 1989;7:91-102. 3 van Rooijen N: The in situ’ immune response in lymph nodes: A review. Anat Roc 1987:218: 359-378. 4 Grande NR. Sii CM. Aguas AP. Carvalho E. Soares M: Time course and distribution of tungsten-laden macrophages in the hilar lymph nodes of the dog lung after experimental instil lation of calcium tungstate in the left apical bronchus of the dog. Lymphology 1990:23: 171-182. 5 Aguas AP. Grande NR. Carvalho E: Inflam matory macrophages in the dog contain high amounts of intravcsicular ferritin and are asso ciated with pouches of connective tissue fibers. Am J Anat 1991:190:89-96. 6 Gowans JL. Knight EJ: The route of recircula tion of lymphocytes in the rat. Proc R Soc Lond (Biol) 1964:159:257-282.