Sinusoids in the liver lobule ( Figure 1) are invested with a unique type of endothelial lining (Figure 2) consisting of endothelial cells with flattened processes perforated by small fenestrae of about 0.1 pm in size ( Figure 3). The open fenestrae lack a diaphragm and are arranged in groups to which the name "sieve plates" was given (1). A basal lamina is lacking and for that reason the fenestrae constitute an open connection between the sinusoidal lumen and the space of Disse. It is thought that the major part of the transport and exchange of fluid, solutes and particles between the blood and the space of Disse occurs through these open fenestrae. As a result, obvious filtration effects can be expected when particles of about the size of the fenestrae arrive in the liver. Another unique feature of liver sinusoidal endothelial cells is the high endocytotic capacity, which, together with the fenestrae and the absence of a basal lamina, makes them unique in the body (2). To the best of our knowledge, this particular type of endothelial cell is not found in any other organ. The distinction between endothelial cells and three other types of sinusoidal cells, i.e., Kupffer, fat-storing and pit cells, has repeatedly been described and reviewed (3, 4).Fenestrae were first observed following perfusion fixation (1); fixation is highly critical in the preservation and visualization of endothelial fenestrae, and this is valid for the sinusoidal area as a whole. No sophisticated apparatus is ileeded for perfusion fixation of the liver, but successful perfusion fixation is critically dependent on fast and well-dosed anesthesia of relatively stress-free

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.

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We have investigated living and glutaraldehyde fixed liver endothelial cells (LEC) by atomic force microscopy (AFM). In living cells, resolution is generally poor compared with what we have found with the same cells after fixation. LECs possess arrays of pores or fenestrae in their membrane, grouped in so-called sieve plates. In living cells these sieve plates could not be resolved by AFM. However, they could be picked up easily in fixed cells. The size of these fenestrae is around 200 nm. The difference in resolution can be explained by the difference in softness in both cases, as measured by taking and analyzing force curves. Typical values of the elastic modulus are around 2 kPa for the living cell and more than 100 kPa for the fixed cell.The atomic force microscope (AFM), invented in 1986 [1], has found an increasing number of applications in studying biological specimens. Since it can easily be operated in a liquid environment [2, 3], especially under physiological conditions, it is well suited to studying biological processes. Examples are the activation of platelets [4], the detection of enzymatic activity [5], the measurement of rupture forces between specific ligand-receptor pairs [6], and the observation of the activity of RNA polymerase [7] and of exocytotic events in living cells [8]. Despite the fact that resolution can be very high on inorganic crystals like calcite [9] and even on two-dimensional protein arrays [10], resolution is rather poor on living cells. The earliest investigations demonstrated a lateral resolution of 50-100 nm [11][12][13]. However, no substantial improvement was achieved in the meantime. As has been demonstrated with gelatin films [14], the obtainable resolution depends on the elastic modulus and can be estimated by the contact area between tip and sample. Taking a given loading force, which will finally depend on the sensitivity of the instrument, the contact area can be calculated by employing a simple model such as the Hertz model [15,16]. The elastic modulus of living cells ranges roughly between 1 kPa and 100 kPa [17], which results in a contact area of * To whom correspondence should be addressed more than 100 nm at the softest areas of the cell [18]. However, when fixing cells with glutaraldehyde the resolution can be improved drastically, as has been demonstrated by Braet et al. [19,20].The chemistry involved in the process of fixation by using aldehydes is complex and not well understood yet [21,22]. To date, aldehydes have been widely used as fixatives and are known to react with the amine groups (NH 2 ) of proteins. In the case of glutaraldehyde, the fixation action is reinforced by the fact that it is a dialdehyde and can cross-link proteins covalently, therefore adding strength to the cells. Whatever the mechanism of fixation is, glutaraldehyde preserves excellent surface details in biological samples [23].Liver sinusoidal endothelial cells (LEC) possess delicate fine structural details of well-known dimensions, such as fenestrae (∼ 200 nm in diameter) which ar...

Pit cells--a new type of cell first described here and so named because they contain highly characteristic granules--are situated in the wall of rat liver sinusoids, and have hyaloplasmic pseudopodia intermingling with the microvilli of the parenchymal cells. The characteristic granules are mainly situated at one side of the nucleus, the other side showing organelle-free hyaloplasm. Pit cells are also found in portal tracts and in granuloma-like cellular aggregates. They also occur in rat peripheral blood, although there are morphological differences between cells in these two sites. Pit cells can be regarded as regular inhabitants of the sinusoidal wall, and therefore belong to the series of sinusoidal cells, i.e., the endothelial (Wisse, 1972), Kupffer (Widmann et al., 1972; Wisse and Daems, 1970; Wisse, 1974a, b), and fat-storing cells (Ito, 1973). Pit cells do not phagocytose and do not react to a great number of experimental conditions, to which endothelial and Kupffer cells do react (Wisse, 1972, 1974b). Mitosis has been observed in a pit cell. The function of pit cells remains obscure, but an endocrine function is suggested by the morphology of their highly characteristic granules.