Electron microscopic cytochemical studies of anionic sites in the rat spleen

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.