REGULAR ARTICLE

Histology of hemal nodes of the water buffalo (Bos bubalus)

Mohamed Zidan & Reinhard Pabst

Received: 26 November 2009 /Accepted: 4 March 2010 /Published online: 13 April 2010# Springer-Verlag 2010

Abstract Hemal nodes are independent lymphoid organsfound in various mammals but are ignored by mostimmunologists. They seem to play a role in defense againstblood-borne infections in some species. The structure of thehemal node has been described in various species but, sofar, not in the water buffalo. Specimens were obtained fromten clinically healthy male animals (five calves: 2–3 monthsold; five bulls: 2–8 years old). Six hemal nodes wereobtained from each animal from the mesenteric andperirectal region. The samples were studied by light andtransmission electron microscopy. The hemal nodes arebean-shaped or spherical, with one hilus through which thehilus arteries and nerves enter the node and from whichveins and lymphatics leave it. The buffalo hemal node has athin capsule of connective tissue and a few smooth musclecells. Trabeculae extend from the capsule partially dividingthe parenchyma. Subcapsular and trabecular blood sinusesare present. The parenchyma is composed of irregularlymphoid cords rich in erythrocytes, macrophages, andplasma cells and is separated by blood sinuses of variablesize engorged with blood. These blood sinuses drain intothe trabecular sinuses and then into the subcapsular sinus.

In calves, the size of the lymphoid cords is larger than thatin adult bulls. Buffalo hemal nodes can be classified astypical hemal nodes, because they are definitely differentfrom hemolymph nodes in other species. They may play arole in filtering the blood.

Keywords Hemal nodes .Microscopy . Electronmicroscopy . Potential functions .Water buffalo, Bosbubalus

Introduction

In textbooks of immunology (e.g., Murphy et al. 2008; Paul2008) or the “Encyclopedia of Immunology” (Delves andRoitt 1998), neither hemal nodes nor hemolymph nodes arementioned, with the result that these organs are not knownto many immunologists. In contrast, in Gray’s Anatomy,hemal nodes are described within a short paragraph(Williams 1995). Hemal nodes are independent lymphaticorgans located within the blood circulation of variousmammalian species and also in some birds and are capableof performing hemopoietic and immunological functions(Turner 1969). Gibbes (1884) has described the hemalnodes in man as a structure resembling lymph nodes butwith blood in the sinusoids instead of the lymph. Thisstructure was named the hemolymph node by Robertson(1890). Andreasen and Gottlieb (1946), Turner (1969,1971), Hogg et al. (1982), and Abu-Hijleh and Scothorne(1996) have examined the hemal nodes of the rat.Al-Bagdadi et al. (1986) and Gargiulo et al. (1987) havedescribed the hemal nodes of sheep. Winqvist (1954),Ceccarelli et al. (1986), Cerutti et al. (1998), and Ceruttiand Guerrero (2001) have studied bovine hemal nodes. Thehemal nodes of goats have been documented by Ezeasor

M. ZidanDepartment of Histology and Cytology,Faculty of Veterinary Medicine, Alexandria University,Alexandria, Egypt

R. PabstInstitute of Immunomorphology, Hannover Medical School,Hannover, Germany

R. Pabst (*)Center of Anatomy, Immunomorphology 4160,Hannover Medical School,Carl-Neuberg-Strasse 1,30625 Hannover, Germanye-mail: pabst.reinhard@mh-hannover.de

Cell Tissue Res (2010) 340:491–496DOI 10.1007/s00441-010-0962-z

and Singh (1988, 1990) and Ezeasor et al. (1989). Recently,Zidan and Pabst (2004) have described the hemolymphnode of the dromedary camel. Singh (1959) has describedthe structure of the hemal nodes of the buffalo calf in ashort paper without giving the age or numbers of calves orthe number of hemal nodes studied. Thus, no detaileddescription of the hemal nodes of the water buffalo (Bosbubalus) is available.

In all mammals studied previously, the hemal nodes havebeen found to have a general structure with some speciesvariations (Dellman and Brown 1987). They are covered bya thin capsule of connective tissue, with a hilus in whichblood vessels are detectable. A subcapsular blood sinus andlymphatic nodules are present below the capsule. Areticular meshwork extends through the interior, its inter-stices containing a large number of free blood cells, manymacrophages, lymphocytes, and plasma cells (Gargiulo etal. 1987). Weller (1938) has concluded that two types ofnodes occur: hemal nodes containing blood-filled sinusoidsonly, which are peculiar to ruminants (Singh 1959; Dellmanand Brown 1987), and hemolymph nodes containing bothblood and lymph sinusoids, which occur in other mammals.Zidan and Pabst (2004) have concluded that the dromedarycamel has hemolymph nodes that are organized in an outercortex with lymphoid follicles and an inner medulla oflymphoid cords. The present work aims to describe thestructure of the hemal nodes of the water buffalo, bycombining various techniques, as a basis for understandingthe function of hemal nodes.

Materials and methods

Fresh specimens were obtained from ten clinically healthymale animals (five buffalo calves: 2–3 months old; fivebuffalo bulls: 2–8 years old). The animals were slaughteredfor human consumption in the abattoir of the Faculty ofAgriculture, Alexandria University, Alexandria, Egypt.Each animal was delivered to the abattoir by a differentfarmer.

Six hemal nodes were obtained from each animal fromthe mesenteric and perirectal region. Each hemal node wasexamined macroscopically and dissected for the followingstudies:

1. Small samples were fixed in 10% phosphate-bufferedformaldehyde and processed for paraffin sectioning.Sections (4 µm) were prepared and stained by usingMayer’s hematoxylin and eosin (H&E) stain, Gomori’ssilver impregnation stain, and the Crossman trichromestain.

2. Fresh specimens, about 1 mm3 in size, were fixedimmediately in 4% phosphate-buffered glutaraldehyde

pH 7.4 for 2 h at 4°C. After postfixation in a 1% solutionof phosphate-buffered osmium tetroxide at 4°C, speci-mens were dehydrated in ascending grades of ethanoland embedded in Epon (Serva, Heidelberg, Germany)according to standard protocols. Semithin sections(1 µm) were prepared, stained with toluidine blue, andexamined by light microscopy. Suitable areas forelectron-microscopic examination were selected. Fromthese areas, ultrathin sections (50–70 nm) were cut witha diamond knife and stained with uranyl acetatefollowed by lead citrate. Sections were observed with aZeiss EM10 electron microscope (Zeiss, Oberkochen,Germany) at 80 KVs.

Results

The hemal nodes of the buffalo were brown to dark-red, bean-shaped or spherical structures of variable size embedded in thefat of the mesentery in the abdomen and in the perirectal fat.No differences were seen in the basic structure of the hemalnodes between the individual animals or between mesentericand perirectal nodes. Each node had one hilus through whicharteries and nerves entered the node and fromwhich veins andlymphatics left it. The hemal node consisted of a thin capsuleand fine trabeculae enclosing the parenchyma (Fig. 1). Thiscapsule was composed mainly of connective tissue and a fewsmooth muscles.

The subcapsular sinus was always engorged with bloodand lined with endothelial cells. Collagen and reticular fibersextended through the lumen of the sinus (Fig. 2a). A fewtrabeculae extended from the capsule partially dividing theparenchyma. Trabecular sinuses surrounded the trabeculaeand were continuous with the subcapsular sinus. Thesesinuses were lined with endothelial cells resting on thebasement membrane and collagen fibers, and some macro-phages were observed inside them (Figs. 2b, 3).

The parenchyma was supported by a fine network ofreticular fibers and reticular cells and was composed ofirregular lymphoid cords of dispersed lymphocytes plusmany free erythrocytes and macrophages. Other blood cells,platelets, plasma cells, and mast cells were also seen withinthe cords. The macrophages were observed at variousstages of phagocytosis of erythrocytes (Fig. 3).

The cords were separated by blood sinusoids engorgedwith concentrated blood cells (Fig. 4). The diameter of thesinuses and their blood content was variable. The size ofthe blood sinuses increased at the expense of the lymphoidcords with age. The blood sinusoids of the parenchymawere connected to the subcapsular sinus by trabecularsinuses, and some macrophages were observed inside them.The few lymphatic sinuses were characterized by a low

492 Cell Tissue Res (2010) 340:491–496

lymphocyte content and the presence of some erythrocytes(Fig. 4).

A few small primary lymphoid follicles were found insome hemal nodes. These follicles were located peripher-ally, close to the subcapsular sinus, and were supported byspare reticular fibers (Fig. 5). Germinal centers were rarelyobserved and were small in diameter without a clearcorona. The germinal centers contained lymphocytes,lymphoblasts, dividing lymphoblasts, and plasma cells(Fig. 5). No afferent lymphatics or high endothelial venuleswere observed.

Discussion

The present study shows that the hemal nodes of the buffaloconsist of a cavernous capsule of connective tissue and

smooth muscles enclosing several blood sinuses, which aretightly packed with erythrocytes. The blood sinuses arepartitioned by lymphoid cords rich in various blood cells.The hemal nodes of the buffalo possess a histologicalfeature comparable to the splenic red pulp, i.e., both areformed by lymphoid cords separated by blood sinuses. Asimilar structure has been described in goat hemal nodes(Ezeasor and Singh 1988). The degree of hemoconcentra-tion observed in the sinuses of buffalo hemal nodes issimilar to that demonstrated in the spleen (Blue and Weiss1981) and goat hemal nodes (Ezeasor and Singh 1988).This might depend on the contraction of the smoothmuscles of the capsule, e.g., is possibly induced by thefixation (Blue and Weiss 1981). Hemoconcentration wouldresult in production of plasma that has to be drained by thelymph. The lymphatics have hardly been seen, probablybecause of the collapse of the lumen after the slaughter of

Fig. 2 a The thin capsule (arrowhead) is formed by collagen fibersand a few smooth muscles. Collagen bundles (arrows) originate fromthe capsule and cross the subcapsular sinus (S), which is engorgedwith blood. Crossman trichrome. b The trabecular sinuses are linedwith endothelial cells (arrowheads) and engorged with blood. Macro-phages (arrows) are found inside the trabecular sinuses (T trabecula).Toluidine blue

Fig. 1 a Basic structure of a hemal node, which is spherical with athin capsule (arrowheads). A subcapsular sinus (S) and trabecularsinuses (arrows) are prominent. Crossman trichrome. b Semithinsection showing the hilus (H), hilar artery (A), and hilar vein (V).Numerous blood sinuses (S) engorged with concentrated blood arefound in the hilar region. Toluidine blue

Cell Tissue Res (2010) 340:491–496 493

Fig. 4 a The parenchyma is composed of irregular lymphoid cords(C) of diffused lymphocytes separated by blood sinuses (S) engorgedwith concentrated blood. Crossman trichrome. b A semithin section ofa buffalo hemal node showing a blood sinus lined with endothelialcells (arrowheads) and containing macrophages (arrow) in addition toother blood cells (B concentrated free blood). Toluidine blue. c Asemithin section of a buffalo hemal node showing lymph sinuses (L)containing a few lymphocytes and some erythrocytes. Toluidine blue

Fig. 3 a A reticular cell (R) extends several processes (arrows)separating erythrocytes (E) from each other. b The pseudopodia(arrow) of a macrophage (M) embrace erythrocytes (E) beforephagocytosis. c An erythrocyte (E) has been taken up by amacrophage (M) by phagocytosis (arrow platelet, P plasma cell)

494 Cell Tissue Res (2010) 340:491–496

the animals. The observed lymphatic sinuses characteristicallycontain few lymphocytes and much lymph originating fromthe plasma separated as a result of hemoconcentration.

The size of the blood sinuses increases with ageassociated with a reduction in the size of the lymphoidcords. This indicates increases in the storage capacity of theblood sinuses and reduced immunological activity (Ezeasorand Singh 1988). The artery enters the hemal node as thehilar artery and ramifies into smaller branches opening intoblood sinusoids. These sinusoids are drained by thesubcapsular sinus, which collects the blood and is drainedby the hilar vein. No clear evidence has been found as towhether the circulation is open or closed. Sakita et al.(1997) suggest that the hemal nodes possess functionallyopen blood vessels in the intermediate sinus area, and thismay be the situation in the buffalo, because of the presenceof numerous free erythrocytes in the lymphoid cords.

The reticular network extends throughout the hemalnodes forming the structural backbone. In agreement withGargiulo et al. (1987), the large number of free blood cellsseems to collect in the subcapsular sinus by flowingthrough the reticular meshwork aided by the particularalignment of some of the reticular cells in channel-likepassageways.

According to the classification of the hemal node byWeller (1938), the organ that we have examined in thebuffalo is a hemal node, as it possesses many blood sinuses,with rare lymphatic sinuses and only a few afferentlymphatics. Thus, it fulfills the criteria of a hemal node,similar to that in cattle and sheep (Singh 1959; Dellman andBrown 1987), but different from hemolymph nodes, whichare known in rats (Andreasen and Gottlieb 1946; Tilney1971; Nopajaroonsri et al. 1974), goats (Ezeasor and Singh1988), and camels (Zidan and Pabst 2004).

This study shows the presence of characteristically fewprimary and secondary follicles in the hemal nodes ofbuffalo. The presence of secondary follicles is indicative ofthe role of hemal nodes in antibody production, as in ovineand bovine hemal nodes (Ceccarelli et al. 1986). Anotherpossible function is the storage of blood inside the sinuses,but because of the small size of the nodes relative to thebody weight of the water buffalo, this will not be of greatrelevance.

Macrophages involved in erythrophagocytosis have beenobserved in the blood sinuses and the lymphoid cords. Thisis a clear indication that buffalo hemal nodes are involvedin the phagocytosis of old or defective blood cells from thecirculation, as described for bovine (Winqvist 1954) andgoat (Ezeasor and Singh 1988) hemal nodes. Anotherimportant function of macrophages might be to filter outparticles circulating in the blood.

Folse et al. (1971) and Ezeasor and Singh (1988) havereported that bovine and caprine hemal nodes, respectively,

Fig. 5 a The hemal node of the buffalo has a few small lymphoidfollicles (LF) close to the subcapsular sinus (S). Note the parenchyma(P). Crossman trichrome. b A lymphoid follicle (LF) lying close to thesubcapsular sinus (S) and supported by a few reticular fibers. Gomori'ssilver impregnation. c A small germinal center (GC) without a clearcorona. It contains lymphocytes, lymphoblasts, dividing lymphoblasts(arrow), and plasma cells (arrowheads). Erythrocytes (E) are found inthe surrounding parenchyma. Toluidine blue

Cell Tissue Res (2010) 340:491–496 495

contain prominent megakaryocytes and are involved inthrombopoiesis. However, we have detected no megakaryo-cytes in the buffalo hemal node.

The hemal nodes of buffalo, with their numerous bloodsinusoids engorged with blood and the direct contactbetween immune cells and the blood, may have a similarhematological and immuonological function to the spleen,such as defense against blood-borne infection and theclearance of damaged and aging erythrocytes. Hemal nodesshould be included in the list of secondary lymphoid organswhen the structural basis of the immune reactions isdescribed and should be brought to the attention ofstudents. The differences in the secondary lymphoid organs,e.g., no lymph nodes in birds but hemal nodes similar tothese organs in some mammals, point to the need forcomparative structural and functional studies of these basiclymphoid organs in order to define evolutionary steps inimmunology.

Acknowledgement The technical assistance of G. Preiss and M.Peter and the revision of the English text by S. Fryk are gratefullyacknowledged.

References

Abu-Hijleh MF, Scothorne RJ (1996) Studies on haemolymph nodes.IV. Comparison of the route of entry of carbon particles intoparathymic nodes after intravenous and intraperitoneal injection.J Anat 188:565–573

Al-Bagdadi FK, Seger CL, Titkemeyer CW, Archibal LF (1986)Ultrastructural morphology of plasma cells in normal ovinehemal lymph nodes. Anat Histol Embryol 15:344–354

Andreasen E, Gottlieb O (1946) The hemolymph node of the rat. BiolMed 19:3–27

Blue J, Weiss L (1981) Vascular pathways in nonsinusal red pulp. Anelectron microscope study of the cat spleen. Am J Anat 161:135–168

Ceccarelli P, Gargiulo AM, Fagiulo O, Pedini V (1986) Cytochemicalidentification of lymphocytes and other mononuclear cells inovine and bovine hemal nodes. Comp Immun Microbiol InfectDis 9:297–302

Cerutti P, Guerrero F (2001) Identification of positive cells tointerleukin-4 in bovine haemal nodes. Anat Histol Embryol30:219–223

Cerutti P, Marcaccini A, Guerrero F (1998) A scanning andimmunohistochemical study in bovine haemal node. Anat HistolEmbryol 27:387–392

Dellman H-D, Brown EM (1987) Textbook of veterinary histology.Lea & Febiger, Philadelphia

Delves PJ, Roitt IM (1998) Encyclopedia of immunology, 2nd edn.Academic Press, San Diego

Ezeasor DN, Singh A (1988) Histology of the caprine hemal node.Acta Anat 133:16–23

Ezeasor DN, Singh A (1990) Morphologic features of lymph vesselsin caprine hemal nodes. Am J Vet Res 51:1139–1143

Ezeasor DN, Singh A, Sims DE (1989) Erythrophagocytosis in thecaprine hemal node. Acta Anat 134:341–345

Folse DS, Beathard GA, Marshall RB, Fish JC, Sarles HE, RemmersAR, Ritzmann SE (1971) Characterization of the bovine hemalnode. J Reticuloendothel Soc 10:461–481

Gargiulo AM, Ceccarelli P, Pedini V (1987) Architecture of sheephemal nodes. Res Vet Sci 42:280–286

Gibbes H (1884) On some structures found in the connective tissuebetween the renal artery and vein in the human subject. Q JMicrosc Sci 24:186–190

Hogg CM, Reid O, Scothorne RJ (1982) Studies on hemolymphnodes. III. Renal lymph as a major source of erythrocytes in therenal hemolymph node of rats. J Anat (Lond) 135:291–299

Murphy K, Travers P, Walport M (eds) (2008) Janeway’s immunobi-ology, 7th edn. Garland Science, New York

Nopajaroonsri C, Luk SC, Simon GT (1974) The structure of the hemalnode—a light, transmission and scanning electron microscopestudy. J Ultrastruct Res 48:325–341

Paul WE (2008) Fundamental immunology, 6th edn. Lippencott,Williams and Wilkins, Philadelphia

Robertson WF (1890) The prevertebral haemolymph glands. LancetII:1152–1154

Sakita K, Fujino M, Koshikawa T, Ohmiya N, Ohbayashi M, Asai J(1997) Structure and function of the hemolymph node in rats.Nagoya J Med Sci 60:129–137

Singh A (1959) On the microscopic structure of the haemal nodes ofbuffalo calves. Br Vet J 115:271–275

Tilney NL (1971) Patterns of lymphatics drainage in the adultlaboratory rat. J Anat 109:369–383

Turner DR (1969) The vascular tree of the hemal node in the rat. JAnat 104:481–493

Turner DR (1971) The reticulo-endothelial components of the hemalnode. A light and electron microscopic study. J Anat 108:13–22

Weller CV (1938) The hemolymph nodes. In: Downey VE (ed)Handbook of hematology. Harber, New York, pp 1759–1787

Williams PL (1995) Gray’s anatomy. Churchill Livingstone, NewYork

Winqvist G (1954) The bovine hemal nodes. Acta Anat 22:108–112Zidan M, Pabst R (2004) Histological, histochemical and immuno-

histochemical study of the hemal nodes of the dromedary camel.Anat Histol Embryol 33:284–289

496 Cell Tissue Res (2010) 340:491–496