Electron Microscopic Cytochemical Studies of Anionic Sites in the Rat Spleen

FULL PAPER Anatomy

Hiromi UEDA1), Kazushige TAKEHANA1), EERDUNCHAOLU1), Kenji IWASA1), Osamu FUJIMORI2) and Shoichi SHIMADA2)

1)Department of Veterinary Anatomy, School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu, Hokkaido 069–8501 and 2)2nd Department of Anatomy, Nagoya City University Medical School, Nagoya 467–8601, Japan

(Received 31 May 2000/Accepted 29 November 2000)

ABSTRACT. The distribution patterns of the intensity of negative charge on the free surfaces (glycocalyx of the plasma membrane) of endothelial cells (ECs) in blood vessels and reticular cells (RCs) in the splenic cord of the rat spleen were studied by an electron microscopic cytochemical method using polyethyleneimine (PEI) as a cationic probe. Spleens from adult male rats were perfusion-fixed with 0.5% glutaraldehyde – 4 % paraformaldehyde containing 0.05% cetylpyridinium chloride and then perfused with 0.5% PEI at pH 7.4. On the free surfaces (glycocalyx of the plasma membrane) of the ECs examined, distinct PEI-positive reactions were observed in blood vessels, such as trabecular arteries, central arteries, arterial capillaries, pulp veins and trabecular veins. These PEI-positive electron-dense substances in the trabecular arteries, central arteries, and trabecular veins took the shape of a band of 170–250 nm in thickness. On the other hand, the corresponding ultrastructure of the ECs lining the splenic sinuses and the RCs in the splenic cord showed exceedingly weak PEI reactions. The PEI-reactive deposits were significantly thinner than those in the above blood vessels. As the thickness of the electron-dense substances can be related to the density of the negative charge, these results suggest that there is a high intensity of negative charge on the free surfaces (glycocalyx of the plasma membrane) of ECs in blood vessels where blood cells and plasma pass into the red pulp or are discharged from the red pulp. In contrast, the splenic sinuses and RCs, which are the main components of the red pulp, contain weakly negative-charged sites. This may contribute to the microcirculation of the splenic blood vessels and elucidate the possible physiological functions of the spleen, such as blood storage. KEY WORDS: anionic site, cytochemistry, electron microscopy, rat, spleen. J. Vet. Med. Sci. 63(3): 287–291, 2001

The free surfaces of vascular endothelial cells (ECs) have face adhesion receptors [26]. Glycoconjugates in the endo- anionic or negatively charged sites. The negative charge thelium lining the blood vessels in the rat spleen have been associated with the free cell surfaces is considered to be an studied by electron microscopic cytochemical analyses [22, important determinant in a variety of the endothelial func- 24]. These analyses have shown that the spleen contains a tions [5]. Endothelial anions are known to be involved in variety of the glycoconjugates on the free surfaces of the the regulation of the transit of proteins and cells from circu- ECs, such as heparan sulfate, chondroitin sulfate and sialic lation, in the prevention of mural thrombus formation, and acid residues. Anionic sites on the free surfaces of the ECs in the localization of cytokines and growth factors on the are thought to play essential roles in effective splenic circu- endothelial free surfaces [10, 11, 19, 21]. Particularly, sialic lation of blood cells [24], as the glycoconjugates have strong acid residues, which usually occupy the terminal position of negative charges by virtue of their carboxylic and sulfate oligosaccharide chains in a variety of glycoconjugates, con- groups [12]. However, little is known about the precise dis- tribute greatly to the negative charge on the free surfaces of tribution patterns of intensity of the negative charge on the the vascular ECs and are known to promote unimpeded cir- free surfaces of ECs in the splenic blood vessels. culation between endothelial surfaces and blood cells [2, 9]. In the present study, attempts were made to perform In mammals, the spleen is a highly vascularized organ cytochemical analyses of the anionic sites on the free sur- and is unique in that most of the small terminal arterioles in faces (glycocalyx of the plasma membrane) of ECs in the the red pulp open into the reticular meshwork of the splenic splenic blood vessels and of reticular cells (RCs) in the cord. Blood cells and plasma flowing in the arterioles enter splenic cord by means of electron microscopy using poly- the splenic cord and then reenter the vascular system ethyleneimine (PEI). In addition, we discussed the physi- through the interendothelial slits of the splenic sinuses after ological mechanism of the anionic sites on the free surfaces percolating within the meshwork [7, 27]. In view of these (glycocalyx of the plasma membrane) of these cells. characteristic morphological features of the mammalian spleen, several electron microscopic studies have been con- MATERIALS AND METHODS ducted on the splenic blood vessels, especially with regard to their distribution patterns and fine structures [1, 3, 8, 14]. Eight-week-old male rats (Fischer strain) were anesthe- Cell-surface glycoconjugates in various tissues are known tized with Nembutal (pentobarbital; Dabbott, Japan) and to play essential roles in the maintenance of the peritoneal perfused through the thoracic aortae, first with Ringer’s cavity through prevention of peritoneal adhesion [15, 16], solution and then with a fixative of 0.5% glutaraldehyde and control of cell adhesion and migration [4, 13] and cell sur- 4% paraformaldehyde containing 0.05% cetylpyridinium 288 H. UEDA ET AL. chloride in 0.1 M cacodylate buffer (pH 7.4) for 10 min. on the neighboring RCs in the splenic cord (Fig. 5). The Following fixation, the animals were perfused with 0.1 M former was distinct thick layer, whereas the latter was elec- cacodylate buffer (pH 7.4) alone for 2–3 min to wash out the tron-dense layer comparable in thickness to that of the layer fixative. To label of anionic sites, the animals were perfused on the ECs lining the splenic sinuses (Figs. 5-inset A, 5-inset with 0.5% PEI (mol. wt 1800, Wako Chemicals, Tokyo, B). In the splenic cord, the electron-dense deposits on the Japan) in 0.1 M cacodylate buffer (pH 7.4) for 10 min. After free surfaces (glycocalyx of the plasma membrane) of the perfusion, spleens were removed, cut into cubes of 1 mm RCs formed a distinctly thin layer similar to those on the and then immersed in a mixture of 0.1% glutaraldehyde and neighboring RCs at the termination of the arterial blood ves- 2% phosphotungstic acid in 0.1 M cacodylate buffer (pH sels. 7.4) at 4°C for 30 min. The tissue blocks were rinsed in the Figure 6 shows a schematic representation of the same buffer and postfixed with 2% osmium tetroxide in 0.1 cytochemical characteristics of the free surfaces (glycocalyx M cacodylate buffer (pH 7.4) at 4°C for 2 hr. They were of the plasma membrane) of ECs in the blood vessels and dehydrated in a graded series of ethanol and embedded in RCs in the splenic cord of the rat spleen determined in the Epon 812 [6]. Ultrathin sections with thickness of 80–100 present study. nm were prepared, stained briefly with 2% methanolic ura- nyl acetate and lead citrate, and examined under a JEOL- DISCUSSION 1210 electron microscope. The free surfaces of ECs in blood vessels are coated with RESULTS a glycoconjugate layer that contains a negative charge due partially to the presence of carboxylic and sulfate groups. In the rat spleen, distinctly PEI-positive reactions were Previous histochemical experiments demonstrated that the observed on the free surfaces (glycocalyx of the plasma layer in the arteries is 250–300 nm in thickness [20]. In the membrane) of ECs in all splenic blood vessels examined present study, the thickness of distinctly PEI-positive elec- and of RCs in the splenic cord. However, the reaction prod- tron-dense layer on the free surfaces (glycocalyx of the ucts deposited on these free cell surfaces formed the PEI- plasma membrane) of ECs in arterioles of the rat spleen positive electron-dense layer, which showed characteristic ranged from 200 to 250 nm. As PEI has been widely used to variations in thickness and shape among the different seg- label anionic sites on the cell surface or basal lamina [6, 17, ments of the splenic blood vessels (Figs. 1–5). 28], we believe that the thickness of the electron-dense layer On the free surfaces (glycocalyx of the plasma mem- is related to the intensity of the negative charge. Thus, tra- brane) of the ECs, the PEI-positive electron-dense layer was becular arteries, central arteries, arterial capillaries and tra- 200–250 nm in thickness in trabecular and central arteries becular veins have high densities of negative charge on the and 170–220 nm in thickness in trabecular veins. In these free surfaces (glycocalyx of the plasma membrane) of ECs, blood vessels, the electron-dense layer on the free cell sur- whereas the corresponding ultrastructure of the ECs lining faces (glycocalyx of the plasma membrane) was in the form splenic sinuses and the RCs in the splenic cord contains of a thick band (Figs. 1-inset, 4-inset). In arterial capillaries, weakly negative-charged sites. the electron-dense layer on the free surfaces (glycocalyx of Examination of the ECs and RCs revealed that the PEI- the plasma membrane) of the ECs varied in thickness (150– positive electron-dense layer showed various profiles. In 200 nm) (Fig. 5-inset A). On the other hand, the PEI-posi- the trabecular arteries, central arteries and trabecular veins, tive electron-dense layer in pulp veins was of variable thick- the electron-dense layer was uniform in thickness, whereas ness and significantly thinner than that of the above blood in the arterial capillaries and pulp veins the layer was of var- vessel ECs (Fig. 3). The thickness of the layer was 70–110 ious thickness. In the ECs of the splenic sinuses and RCs of nm (Fig. 3-inset). In contrast, the electron-dense layer on the splenic cord, the electron-dense layer was thin, although the free surfaces (glycocalyx of the plasma membrane) of dot-like deposits were frequently observed on the free sur- the ECs lining splenic sinuses was very thin (Fig. 2). How- faces (glycocalyx of the plasma membrane) of these cells. ever, dot-like deposits, which were 40–60 nm in thickness, Our results showed that the PEI-reactive products that were were frequently observed on the free surfaces (glycocalyx of accumulated on the free cell surfaces (glycocalyx of the the plasma membrane) of the ECs of the splenic sinuses plasma membrane) tend to form spot-shaped precipitates, as (Figs. 2, 2-inset). In the present study, a terminal segment of the densities of the negative charge gradually diminish with arterial blood vessels was observed in the red pulp. At the branching from the arterioles to the splenic sinuses. Thus, termination in the red pulp, the electron-dense layer was of ECs in the splenic blood vessels, which have high densities different thicknesses on the ECs in the arterial capillary and of negative charge on the free surfaces (glycocalyx of the

Fig. 1. A trabecular artery stained with PEI. × 14,000. The free surface of the endothelial cell (EC) is stained strongly (arrowheads). Inset: A higher magnification of the free surface of an endothelial cell. × 75,000. Fig. 2. A splenic sinus stained with PEI. × 12,000. The free surface of the EC is stained weakly (arrowheads). Inset: A higher magnification of the free surface of an endothelial cell. × 75,000. Fig. 3. A pulp vein stained with PEI. × 14,000. The free surface of the EC is stained moderately (arrowheads). Inset: A higher magnification of the free surface of an endothelial cell. × 75,000. ANIONIC SITES IN RAT SPLEEN 289

Fig. 4. A trabecular vein stained with PEI. × 7,000. The free surface of the EC is stained strongly (arrowheads). Inset: A higher magnification of the free surface of an endothelial cell. × 75,000. Fig. 5. Terminal segment of an arterial capillary in the red pulp stained with PEI. × 4,500. The free surface of the EC in the arterial capillary is stained strongly (arrowheads), whereas that of the reticular cell (RC) exhibits weakly positive reactions (arrows). Inset A: A higher magnification of the free surface of an endothelial cell of the arterial capillary. × 75,000. Inset B: A higher magnification of the free surface of a reticular cell in a splenic cord. × 75,000. 290 H. UEDA ET AL.

Fig. 6. Diagram showing the relationship between the blood circulation of the spleen and anionic sites on the cell free surface. A high density of negative charge on the free surface of the EC is recognized in blood vessels where blood cells and plasma pass into the red pulp or are discharged from the red pulp (red). In contrast, the splenic sinuses and the reticular cells, which are the main components of rep pulp, contain weakly negative- charged sites (blue). TA: trabecular artery, CA: central artery, AC: arterial capillary, SS: splenic sinus, PV: pulp vein, TV: trabecular vein, RC: reticular cell, WP: white pulp, RP: red pulp. plasma membrane), are thought to be covered with a layer for the splenic sinuses to possess weakly negative-charged rich in glycoconjugates. On the other hand, ECs in the sites on the free surfaces of the ECs for the effective passage splenic sinuses and RCs in the splenic cord possessed of red blood cells through the walls of the sinus. It was also weakly negative-charged sites on the free surfaces (glyco- proposed that the weakly negative-charged surfaces of both calyx of the plasma membrane), where the glycoconjugates the ECs in the splenic sinuses and RCs in the splenic cord were distributed sparsely. In view of the microcirculation of seem to reflect a storage function of the spleen [23, 24]. The the rat spleen, we have come to the conclusion that there is results obtained in the present study support these notions high densities of negative charge on the free surfaces and contribute to the elucidation of the role of anionic sites (glycocalyx of the plasma membrane) of ECs in blood ves- on the free surfaces (glycocalyx of the plasma membrane) of sels where blood cells and plasma pass into the red pulp or ECs lining the splenic sinuses and RCs in the splenic cord. are discharged from the red pulp. In contrast, the ECs in the The results of the present cytochemical analysis suggest splenic sinuses and RCs, which are the main components of that anionic sites on the free surfaces (glycocalyx of the the red pulp, possess weakly negative-charged sites on the plasma membrane) of ECs in the blood vessels and RCs in free surfaces (glycocalyx of the plasma membrane). How- the splenic cord are closely related to the microcirculation of ever, the mechanism underlying the difference between the blood vessels in the spleen and a series of splenic physiolog- levels of intensities of the negative charge on the free sur- ical mechanisms, such as blood storage. faces (glycocalyx of the plasma membrane) of the former vessels and the latter remains to be clarified. The negative REFERENCES charge on the free surfaces of vascular ECs has been reported to play a role in the promotion of unimpeded circu- 1. Abe, M., Takehana, K., Iwasa, K. and Hiraga, T. 1989. Scan- lation of ECs by mutual repulsion between the surface of the ning electron microscopic studies on the red pulp of the mink endothelium and red blood cells [2], as the red blood cells spleen. J. Vet. Med. Sci. 51: 775–781. carry a net negative electrical surface charge under normal 2. Born, G.V.R. and Palinski, W. 1985. Unusually high concen- trations of sialic acids on the surface of vascular endothelia. Br. physiological conditions [18, 25]. In the former blood ves- J. Exp. Pathol. 66: 543–549. sels, therefore, the negative charge on the free surfaces may 3. Chen, L.T. and Weiss, L. 1972. Electron microscopy of the red be related to the electrostatic repulsion of circulating blood pulp of human spleen. Am. J. Anat. 134: 425–458. cells. In previous reports, we suggested that it is important 4. Couchman, J.R. and Woods, A. 1993. Structure and biology of ANIONIC SITES IN RAT SPLEEN 291

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