Pathways of lymph flow to and from the medulla of lymph

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1 J. Anat. (1987), 155, pp With 19 figures Printed in Great Britain Pathways of lymph flow to and from the medulla of lymph nodes in sheep TREVOR J. HEATH AND HUGH J. SPALDING School of Veterinary Science, University of Queensland, St Lucia, Queensland 4067, Australia (Accepted 24 February 1987) INTRODUCTION The sheep is often used for studies on the lymphatic system, but little information has been available on the pathways taken by lymph through lymph nodes in sheep. Most of the information on lymph pathways has been derived from rats and rabbits, but it is now clear that significant differences in the functional morphology of lymph nodes exist between species (Kurokawa & Ogata, 1980; Sainte-Marie, Peng & Belisle, 1982; Heath & Brandon, 1983; Heath, Kerlin & Spalding, 1986). For example, rat lymph nodes apparently can be divided into 'physiological compartments', each of which is associated with a single afferent lymphatic (Sainte-Marie et al. 1982), but these are not evident in sheep (Heath et al. 1986). In a previous paper, we described the afferent pathways within the popliteal lymph node in sheep (Heath et al. 1986). In this paper, we describe the lymph pathways to and from the medulla in sheep lymph nodes. MATERIALS AND METHODS Twenty mature merino ewes and wethers, which had not been subject to any deliberate immunorogical stimulation, were used. Methods described by Heath & Brandon (1983) and Heath et al. (1986) were used for anaesthesia and surgery and for making, clearing and photographing Microfil (Canton Biomedical Products Inc., Boulder, Colorado) casts of lymphatic vessels and sinuses from the following lymph nodes: popliteal (9 nodes), iliofemoral (8), superficial inguinal (4), medial iliac (3), medial retropharyngeal (1). Eight lymph nodes were fixed by perfusion through an afferent lymphatic or by immersion of 1 mm slices in fixative and tissue from these was studied either by scanning or transmission electron microscopy (Heath et al. 1986). In some cases, structures were located and identified by light microscopy. RESULTS Lymph sinuses Studies with light and electron microscopy and with Microfil casts revealed two main pathways to convey lymph through the cortex to the medullary sinuses (Fig. 1): afferent lymphatics which penetrated the node within trabeculae, and sinuses which were continuous with the subcapsular sinus, and which surrounded the trabeculae (Heath et al. 1986). These trabeculae, together with their lymph pathways, often branched as they approached the medulla.

2 178 T. J. HEATH AND H. J. SPALDING Penetrating Hilus Fig. 1. Diagrammatic representation of the lymph pathways within an iliofemoral lymph node. Afferent lymphatics approach the dorsal surface of the node and divide into terminal afferents which either enter the subcapsular sinus ( ), or penetrate within trabeculae. Lymph reaches the sinuses of the medulla (----) from these penetrating afferents, or through invaginations of the subcapsular sinus around the trabeculae. Tubular sinuses (.) within the cortex also join medullary sinuses. Initial efferent lymphatics arise over a large area of hilus. Most have their origin in the hilar subcapsular sinus or adjacent medullary sinuses. The initial efferents join together as they pass across the hilar surface, forming two efferent vessels which carry lymph towards the lumbar trunks. Tubular sinuses The cortex also contained a system of tubular sinuses (Figs. 1-5). These were independent of the trabeculae, although some of them were closely associated with trabecular sinuses over part of their course. Some of the tubular sinuses were in the superficial cortex, and these were often more or less parallel to the capsule. Others penetrated the cortex either to join medullary sinuses, or to become continuous with a network of tubular sinuses in the deep cortex (Fig. 2). These tubular sinuses in the deep cortex, which were often adjacent to venules and other blood vessels (Fig. 5), joined medullary sinuses at the corticomedullary border (Fig. 4). Figs Illustrations of tubular sinuses. Fig. 2. Photograph of a superficial inguinal lymph node which received an injection of Microfil through a single afferent lymphatic. In the lower half of the Figure, Microfil can be seen within a network of tubular sinuses which extend beyond the limits of the cast of the subcapsular sinus (S). A, branches of afferent lymphatic. x 7. Fig. 3. Transmission electron micrograph of a tubular sinus from an iliofemoral lymph node which was fixed by perfusion through an afferent lymphatic. A few small lymphocytes remain in the sinus lumen. x Fig. 4. Photograph of a Microfil cast of sinuses within an iliofemoral lymph node showing a tubular sinus passing through the cortex. Branches leave this sinus at points indicated by arrows, and join medullary sinuses (M). The Figure also shows a terminal afferent lymphatic (A) joining the subcapsular sinus (S); a trabecular sinus (T) invaginated from the subcapsular sinus and surrounding a penetrating afferent lymphatic (P); other trabecular sinuses (*) joining medullary sinuses (M). x 50. Fig. 5. Light micrograph of a 1 psm section of deep cortical tissue, showing a tubular sinus (outlined by arrows) which is packed with small lymphocytes and is close to two small blood vessels (B). x 700.

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4 180 T. J. HEATH AND H. J. SPALDING

5 Lymph pathways of the lymph node medulla 181 S H Sl H 8-11 T h n. Fig. 8. Photograph of an iliofemoral lymph node which has been filled with Microfil through afferent lymphatics (A) Soe aia in o.t of the subcapsular sinus over the hilus (H), which is convex and covers the lateral and ventral C surfaces, i ps o he h s l -~~~~ -~- Figs. 8-1t1.The hilus and its margin. Fig. 8. Photograph of an iliofemoral lymph node which has beenefiled with Microfil through afferent lymphatics (A). Some of the terminal afferents enter the node near the hilar margin (arrows). The cast of the subcapsular sinus over the hilus (H) which is convex and covers the lateral and ventral surfaces, is perforated by numerous small holes. Initial efferent vessels emerge from the hilus then pass over the hilar surface. They join with other small vessels before leaving at an acute angle and combining to form two large vessels (E). x 5. Fig. 9. Light micrograph of a section through the medulla beneath the hilus of an iliofemoral node. The subcapsular sinus (S) is continuous with the medullary sinuses between medullary cords (M), and is traversed by reticular cell processes and small trabeculae (T). C, capsule. x 130. Fig. 10. Light micrograph of a section through the hilar margin of a medial iliac node. A single trabeculum (T) separates the cortex on the left from the medulla on the right. The capsule (C) is thicker, and the subcapsular sinus (S) more obvious, over the cortex than over the medulla. x 60. Fig. 11. Light micrograph of a section through the hilar margin of a popliteal node. Some trabeculae (arrows) run parallel to the node surface and give the appearance there of a second capsule. M, medulla; C, capsule; Co, cortex; S, subcapsular sinus. x 120. Figs Medullary cords and sinuses. Fig. 6. Scanning electron micrograph of medullary sinuses (S) which are crossed by numerous reticular processes (R). M, medullary cord. x 800. Fig. 7. Transmission electron micrograph of a medullary cord and its adjacent sinus (S). The sinus is lined by elongated cells which have many lysosome-like bodies (arrows) in their cytoplasm. Underlying these cells are scattered bundles of collagen fibres (F) and a discontinuous layer of perisinusal cell processes (P). A large mononuclear cell (MC) is traversing the sinus lining. R, reticular process; V, venule. x 4400.

6 182 T. J. HEATH AND H. J. SPALDING

7 Lymph pathways of the lymph node medulla 183 se-~~,. h~16 Fig. 16. Light micrograph of a section from an iliofemoral lymph node showing two efferent lymphatics (E) within a recess in the hilar surface. The origin of one of these vessels (arrow), and a valve near the origin, can be seen. The recess also contains several small blood vessels. E', an efferent lymphatic on the hilar surface. x 30. Fig. 17. Light micrograph of a section from a medial iliac lymph node showing efferent lymphatic vessels (E) within the substance of the medulla. V, valve; A, small artery. x 120. Tubular sinuses were mainly a feature of the cortex, but some appeared to extend a short distance into the medulla before joining medullary sinuses. They could be distinguished from other sinuses by their tubular outline and a paucity of reticular fibres in the lumen (Fig. 3). They appeared to be up to 60,um in diameter in nodes which had been perfused with fixative through afferent lymphatics, and up to 50 um in tissues fixed by immersion. In tissue fixed by immersion the tubular sinuses were packed with cells (Fig. 5) - mostly lymphocytes about 6 4csm in diameter with some macrophages - and so were less readily visible than in tissue which had been fixed by perfusion. The sinus wall appeared to be continuous (Fig. 3), except where cells were migrating across it. Medullary sinuses These sinuses contained numerous reticular processes which were continuous with the lining cells (Figs. 6, 7). The lining cells had a sparse amount of chromatin in their nucleus, and their cytoplasm contained numerous lysosome-like bodies and pino- Figs The origins of efferent lymphatics (E). Fig. 12. Scanning electron micrograph showing an initial efferent lymphatic arising within an indentation in the hilar surface. Medullary sinuses are continuous with the lymphatic vessel at points indicated by arrows. V, valve cusps; M, medullary cords. x 140. Fig. 13. Detail of the origin of the efferent lymphatic shown in Fig. 6, but taken at a different angle to show apertures (arrows) in the wall of the vessel. x 530. Fig. 14. Scanning electron micrograph showing an initial efferent lymphatic arising just beneath the capsule (C). Medullary sinuses are continuous with the lymphatic vessel at points indicated by arrows. M, medullary cords. x 170. Fig. 15. Light micrograph of a 1 gm section showing the origin of an initial efferent lymphatic just beneath the capsule (C). Medullary sinuses (S) are continuous with the lymphatic vessel at the point indicated by the arrow. M, medullary cord. x 180.

8 184 T. J. HEATH AND H. J. SPALDING g L :~~~~~~~~~~~~~~ ::.-.. :..,it ~~~~~~~~~~~ a Fig. 18. Scanning electron micrograph of four efferent lymphatics on the hilar surface of an iliofemoral lymph node. The vessel to the right which is cut longitudinally, illustrates a close association between the capsule and the vessel wall (*). Valves (V) are shown in this vessel, and at the point of entry of an initial efferent (arrow). M, medulla; C, capsule. x 40. Fig. 19. Photograph of a Microfil cast of the hilus of a popliteal lymph node, showing efferent lymphatics emerging from the node surface and joining together within the hilar depression. Numerous small holes are present in the cast of the subcapsular sinus (S) within the hilus and at its margin. V, valve. x 14. cytotic vesicles. Some also contained phagosomes, apparently containing red blood cell fragments. The lining cells appeared to form a continuous layer, except where cells were migrating between medullary cords and sinuses (Fig. 7), or between the deep cortical parenchyma and medullary sinuses at the cortex/medulla interface. They were surrounded by a discontinuous layer of perisinusal cells (Fig. 7). These cells were also elongated, but differed from lining cells in often lacking junctional complexes between adjacent cells, and containing many fewer cytoplasmic inclusions. Scattered bundles of collagen fibres were present between the lining cells and the perisinusal cells. Hilus The hilus was defined as that part of the node surface adjacent to medullary tissue. It varied between nodes from a deep, fat-filled depression as in many popliteal nodes

9 Lymph pathways of the lymph node medulla 185 (see Heath & Brandon, 1983), to a relatively smooth, flattened or convex part of the node surface, which was characteristic of the iliofemoral node (Fig. 8). The capsule was thinner at the hilus than over the cortex, and there was a distinct change in the subcapsular sinus where the cortex and medulla came together at the periphery of the hilus (Figs. 9-11). In Microfil preparations there were many holes in the casts of this sinus within the hilus (Fig. 8), and these were particularly evident at the hilar margin. It was deduced from histological sections that these perforations were due to large reticular processes, and to numerous small trabeculae (Fig. 11). At the hilar margin some of these trabeculae were parallel to the capsule and appeared to form the inner wall of the subcapsular sinus; they gave the impression of a second capsule at that point (Fig. 11). In some sections a single trabeculum, extending into the node for a millimetre or more, separated the cortical and medullary tissues (Fig. 10). The subcapsular sinus at the hilus was continuous with the medullary sinuses between medullary cords, and with initial efferent lymphatics (Figs. 9, 12-15). Efferent lymphatics The initial efferent lymphatics were as little as 60,m wide at their origin, and most were continuous with either the subcapsular sinus or adjacent medullary sinuses (Figs ). There was no indication of marked convergence of sinuses towards the origin of the efferent vessels, but in some cases adjacent sinuses were confluent with them through apertures 20-50,sm across, in the walls of the vessels (Figs. 13, 14). It was difficult to estimate the total number of initial efferent lymphatics, particularly in nodes with a deep fat-filled hilus, but we counted in some popliteal nodes. Most initial efferent lymphatics emerged from the surface of the node directly, but some arose from indentations up to 0 5 mm deep in the hilar wall (Fig. 12). Two other patterns were seen occasionally. In one, lymphatics left the node through recesses up to 2 mm long, which were continuous with the hilus and lined by capsule, and which contained blood vessels and connective tissue (Fig. 16). In the other pattern, efferent lymphatics, which were not surrounded by an invagination of the capsule or any significant amount of other connective tissue, passed through the medullary parenchyma before emerging at the hilus. These lymphatics were accompanied by small blood vessels (Fig. 17). The initial efferent lymphatics usually contained valves (Figs. 12, 16, 18, 19), and they followed a course that varied with the nature of the hilus. If the hilus was flat or relatively shallow, they generally coursed over the capsule (Figs. 1, 18), then left at an acute angle. Their walls often fused with the capsule, and in some cases small vessels connected them with sinuses in the adjacent medulla (Figs. 8, 18). If the hilus was deep, the efferent lymphatics usually left the node surface at an angle of between 30 and 90 (Fig. 19); these joined together in the hilar fat to form 1-3 efferent vessels which carried the lymph away from the region of the node. Some efferent lymphatics emerged near the margin of the hilus, and were less than 2 mm from where terminal afferent lymphatics entered the cortical subcapsular sinus (Fig.8; see also Heath & Brandon, 1983). However, efferent lymphatics did not connect with the subcapsular sinus overlying cortical tissue, nor did afferent lymphatics join with medullary sinuses. DISCUSSION There are several pathways by which lymph may flow to and from the medullary tissue in lymph nodes of sheep. Not all of these pathways are consistent with the 7 ANA 155

10 186 T. J. HEATH AND H. J. SPALDING traditional concept which involves lymph flowing from the subcapsular sinus to medullary sinuses along 'a few narrow sinuses' (Olah, Rohlich & T6ro, 1975) which 'run radially, often along trabeculae' (Weiss, 1984), and then out of the node through 1-3 efferent lymphatics at a hilus resembling that of a kidney (Raviola, 1975; Weiss, 1984). In sheep, the cortical trabeculae are each surrounded by an invagination of the subcapsular sinus, and lymph is transported to the medulla within these trabecular sinuses (Heath et al. 1986). Some lymph also flows directly to the medulla through afferent lymphatics which penetrate into the node within trabeculae (Heath et al. 1986). The medullary sinuses are also continuous with a network of tubular sinuses in the cortex. These sinuses, which we have referred to as 'cortical sinuses' (Heath & Kerlin, 1986; Heath et al. 1986), appear comparable to the 'lymph labyrinths' (He, 1985) and 'mudstreams' Soderstrom & Stenstrom (1969) described in rodents, and the 'tubular sinuses' (Kurokawa & Ogata, 1980) and 'paracortical sinuses' (Kelly, 1975) described in rabbits. However, Kelly (1975) found "no apparent connection between the paracortical and medullary sinuses", whereas in sheep many connections occur between the tubular and medullary sinuses. It seems likely that these tubular sinuses are important as pathways for lymphocyte transport. They are packed with small lymphocytes; they are closely associated with the venules through which lymphocyte recirculation occurs (Soderstrom & Stenstr6m, 1969; Kelly, 1975; Kurokawa & Ogata, 1980; He, 1985), and cells can be seen migrating across their walls. The paucity of reticular processes in the lumen could also be significant if these sinuses are a major route for the transport of either recirculating lymphocytes, or for those newly synthesised in cortical nodules. Tubular sinuses in sheep may also be important in the lateral spread of lymph constituents within the node. This possibility is supported by observations on the distribution of Microfil (Fig. 2), and carbon particles (Heath et al. 1986) over a wide area of the node after they had been introduced into a single afferent lymphatic. In this respect differences may exist between sheep and other animals. In rats for example, lymph from a single afferent lymphatic is apparently restricted to a 'physiological compartment' based on that lymphatic (Sainte-Marie et al. 1982). Furthermore,in rabbits the orientation of tubular cortical sinuses was shown by Kurokawa & Ogata (1980) to be generally at right angles to the node surface; in sheep, many are parallel to the surface. The walls of sinuses throughout the sheep lymph node have a more or less continuous layer of endothelial cells with scattered collagen fibres and a discontinuous layer of perisinusal cells. In each case, the wall seemed to be permeable to lymphocytes and other cells (cf. Heath et al. 1986), and gaps in the sinus lining were virtually all associated with migrating cells. It is difficult to be sure if this is the situation in all animals, as some contradictions appear in the literature (Moe, 1963; Forkert, Thliveris & Bertalanffy, 1977; Farr, Cho & De Bruyn, 1980), although some of these may be related to different methods of fixation (Farr et al. 1980; Heath et al. 1986). A question which has exercised workers for many years is whether lymph can flow along the subcapsular sinus around the periphery of the node to the efferent lymphatics, without entering the substance of the node (see Drinker, Field & Ward, 1934). This still does not seem to have been answered clearly. Perusal of illustrations in standard textbooks could lead one to the conclusion that no impediment exists to such lymph flow (Raviola, 1975; Weiss, 1984). In the rabbit, however, the subcapsular sinus apparently does not extend into the hilus (Kurokawa & Ogata, 1980).

11 Lymph pathways of the lymph node medulla 187 In our experiments, Microfil preparations revealed some continuity between the subcapsular sinus over the cortex and that within the hilus. However, the margin of the hilus did contain a high concentration of trabecular and reticular processes, and it is considered that these may decrease the volume of the sinus and thus increase the resistance to lymph flow in this region. Furthermore, the trabeculae at the hilar margin may divert some lymph from the cortical subcapsular sinus along the trabecular sinus and into the depths of the node. There was no indication that the medullary sinuses in sheep converge towards efferefit lymphatics as described by Kurokawa & Ogata (1980) in rabbits, and by Weiss (1984). Instead, up to 100 or more initial efferent lymphatics, some as small as 60,cm across, emerge from the node at a large hilus. In this regard, the situation in sheep differs substantially from that represented in most publications on lymph nodes, namely 1-3 lymph vessels emerging from a single depression which resembles a renal hilus (Drinker et al. 1934; Sainte-Marie et al. 1982; Banks, 1986; Weiss, 1984). The arrangement of the initial efferent lymphatics in sheep implies that numerous alternative pathways must exist for lymph to flow through the medulla. This may help prevent blockage of lymph flow, especially when the node is responding to an infection, and the lymph contains products of tissue breakdown and a high concentration of cells and protein. It is not exactly clear how the pattern of lymph flow is influenced by the shape of the lymph node and of its hilus. In the popliteal node for example, the hilus often forms a deep depression with the medulla and cortex being wrapped around it. In this case, the afferent lymph enters at the periphery and flows in a generally centripetal direction. The iliofemoral node, on the other hand, has a flattened shape with lymph entering at the dorsal surface and then percolating in a generally ventral direction to the efferent vessels which emerge over the ventral surface (Heath & Kerlin, 1986). SUMMARY Medullary sinuses are continuous with penetrating afferent lymphatics, and with the trabecular and tubular sinuses which penetrate through the cortex. Tubular sinuses are often associated with blood vessels, especially in the deep cortex, and they appear to be important in the transport of lymphocytes. The subcapsular sinus is continuous over the cortex and the medulla, although trabeculae and reticular processes appear to restrict the flow of afferent lymph to the subcapsular sinus over the medulla. Lymph leaves the medulla through up to 100 or more initial efferent lymphatics, some only 60,m across. Almost all of these arise from sinuses adjacent to the capsule lining the hilus. Some efferents remain associated with the capsule for a short distance whereas others, especially in nodes with a deep hilar depression, leave immediately at an angle of We are grateful to Paul Addision for help with histology, and to Lynn Tolley and Dr John Hardy for help with electron microscopy. REFERENCES BANKS, W. J. (1986). Applied Veterinary Histology, 2nd ed. Baltimore and London: Williams & Wilkins. DRINKER, C. K., FIELD, M. E. & WARD, H. K. (1934). The filtering capacity of lymph nodes. Journal of Experimental Medicine 59, FARR, A. G., CHO, Y. & DE BRUYN, P. H. (1980). The structure of the sinus wall of the lymph node relative to its endocytic properties and transmural cell passage. American Journal of Anatomy 157,

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