In normal rat liver, Kupffer cells were unequivocally identified using peroxidase cytochemistry by light microscopy in semithin plastic sections. The Kupffer cell population was found to constitute 31% of the sinusoidal cells and by morphometry and serial sectioning, a mean absolute number of 14 to 20 X 10(6) Kupffer cells per g liver was calculated. The mean distribution of Kupffer cells in the liver lobules was 43% in the periportal, 28% in the midzonal and 29% in the central area of the lobule. Administration of latex particles labeled only 64% of all Kupffer cells, and in particular centrally located cells, showed a lower activity of latex uptake, even at overloading doses. Furthermore, the latter cells were of smaller size than periportal Kupffer cell profiles. The mean number and distribution of latex-labeled Kupffer cells did not change over a period of 3 months, indicating a long lifetime for these resident macrophages. This slow population turnover was supported by the observed small mitotic index, 0.06% after a 6 hr arrest by vinblastine, and by the small [3H]thymidine labeling index which did not change over a period of 3 weeks after administration of the label. It is proposed that the Kupffer cell population, under physiologic conditions, is a long-living and self-renewing population, the kinetics of which substantially differ from those of other sinusoidal cell types.

SummaryCritical point drying (CPD) is a common method of drying biological specimens for scanning electron microscopy (SEM). Drying by evaporation of hexamethyldisilazane (HMDS) has been described as a good alternative. This method, however, is infrequently used. Therefore, we reassessed HMDS drying. Cultured rat hepatic sinusoidal endothelial cells (LEC), possessing fragile fenestrae and sieve plates, were subjected to CPD and HMDS drying and evaluated in the scanning electron microscope, atomic force microscope (AFM) and transmission electron microscope (TEM). We observed no differences between the two methods regarding cellular ultrastructure. In contrast with CPD, HMDS drying takes only a few minutes, less effort, low costs for chemicals and requires no equipment. We conclude that HMDS-dried specimens have equal quality to CPD ones. Furthermore, the method also proved useful for drying whole-mount cells for TEM and AFM.

The hepatic sinusoid harbors 4 different cells: endothelial cells (100, 101), Kupffer cells (96,102,103), fat-storing cells (34,51,93), and pit cells (14, 107. 108 (80,81).

This article describes the cytoskeleton associated with fenestrae and sieve plates of rat liver sinusoidal endothelial cells. Fenestrae control the exchange between the blood and parenchymal cells. We present evidence indicating that several agents that change the fenestrae and sieve plates also cause changes in the cytoskeleton. Cultured liver endothelial cells (LECs) were slightly fixed and treated with cytoskeleton extraction buffer. Detergent-extracted whole mounts of cultured cells were prepared for either scanning electron microscopy (SEM) or transmission electron microscopy (TEM). Extracted cells show an integral intricate cytoskeleton; sieve plates and fenestrae are delineated by cytoskeleton elements. Fenestrae are surrounded by a filamentous, fenestrae-associated cytoskeleton with a mean filament thickness of 16 nm. Sieve plates are surrounded and delineated by microtubuli, which form a network together with additional branching cytoskeletal elements. The addition of ethanol to cultured cells enlarged the diameter for these fenestrae-associated cytoskeleton rings by 5%, whereas serotonin treatment reduced the diameter by 20%. These observations indicate that the fenestrae-associated cytoskeleton probably changes the size of fenestrae after different treatments. After treatment with cytochalasin B the number of fenestrae increased. However, cytochalasin B did not change the structure of the fenestrae-associated cytoskeleton ring, but disperses the microtubuli. In conclusion, LECs have a cytoskeleton that defines and supports sieve plates and fenestrae. Fenestrae-associated cytoskeleton is a dynamic structure and plays a role in maintaining and regulating the size of fenestrae after different treatments. Therefore, the fenestrae-associated cytoskeleton controls the important hepatic function of endothelial filtration.

Hepatic endothelial fenestrae are dynamic structures that act as a sieving barrier to control the extensive exchange of material between the blood and the liver parenchyma. Alterations in the number or diameter of fenestrae by drugs, hormones, toxins, and diseases can produce serious perturbations in liver function. Previous studies have shown that disassembly of actin by cytochalasin B or latrunculin A caused a remarkable increase in the number of fenestrae and established the importance of the actin cytoskeleton in the numerical dynamics of fenestrae. So far, however, no mechanism or structure has been described to explain the increase in the number of fenestrae. Using the new actin inhibitor misakinolide, we observed a new structure that appears to serve as a fenestrae-forming center in hepatic endothelial cells.