R De Zanger

Drying cells for SEM, AFM and TEM by hexamethyldisilazane: a study on hepatic endothelial cells.

Critical 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.

Cellular and subcellular distribution of injected lipopolysaccharide in rat liver and its inactivation by bile salts

The cellular and subcellular distribution of biologically tritiated Salmonella abortus equi lipopolysaccharide (LPS) was studied at different time intervals after intravenous injection in rats. At 1 min after injection of LPS via the portal vein label was present over Kupffer cell phagosomes. Between 30 min and 7 days after injection, silver grains were mainly associated with phagosomes and lysosomes and occasionally with the membrane of Kupffer cells. A few parenchymal cells were labeled at 5 min in their mitochondria, cell membrane and the periphery of the cell. Radioactivity was also present in the rough endoplasmic reticulum (from 15 min), fat droplets and the nucleus (from 3 h) up to 7 days. Sinusoidal endothelial and fat-storing cells were never labeled. In conclusion, both Kupffer cells and parenchymal cells play a role in the uptake of LPS by the liver. The uptake and processing of endotoxin is rapid, since label is found early after administration and radioactivity is detected in the bile within 1 h. This radioactivity represents non-detoxified LPS, since it is lethal for galactosamine-sensitised mice after extraction with hot phenol/water. However, in the presence of bile salts, the LPS is non-lethal and not capable of clotting the limulus amebocyte lysate. LPS injection causes bile flow reduction within 45 min.

Comparative atomic force and scanning electron microscopy: an investigation on fenestrated endothelial cells in vitro

Rat liver sinusoidal endothelial cells (LEC) contain fenestrae, which are clustered in sieve plates. Fenestrae control the exchange of fluids, solutes and particles between the sinusoidal blood and the space of Disse, which at its back side is flanked by the microvillous surface of the parenchymal cells. The surface of LEC can optimally be imaged by scanning electron microscopy (SEM), and SEM images can be used to study dynamic changes in fenestrae by comparing fixed specimens subjected to different experimental conditions. Unfortunately, the SEM allows only investigation of fixed, dried and coated specimens. Recently, the use of atomic force microscopy (AFM) was introduced for analysing the cell surface, independent of complicated preparation techniques. We used the AFM for the investigation of cultured LEC surfaces and the study of morphological changes of fenestrae. SEM served as a conventional reference.

Early detection of cytotoxic events between hepatic natural killer cells and colon carcinoma cells as probed with the atomic force microscope

The atomic force microscope (AFM) is a powerful tool to investigate surface and submembranous structures of living cells under physiological conditions at high resolution. These properties enabled us to study the interaction between live hepatic natural killer (NK) cells, also called pit cells, and colon carcinoma cells in vitro by AFM. In addition, the staining for filamentous actin and DNA was performed and served as a reference, because actin and nuclear observations at the light microscopic level during the cytotoxic interaction between these two cell types have been presented earlier. In this study, we collected evidence that conjugation of hepatic NK cells with CC531s colon carcinoma cells results in a decreased binding of CC531s cells to the substratum as probed with the AFM in contact mode as early as 10 min after cell contact (n = 11). To avoid the lateral forces and smearing artefacts of contact mode AFM, non-contact imaging was performed on hepatic NK/CC531s cell conjugates, resulting in identical observations (n = 3). In contrast, the first cytotoxic signs, as determined with the nuclear staining dye Hoechst 33342, could be observed 3 h after the start of the co-culture. This study illustrates that the AFM can be used to probe early cytotoxic effects of effector to target cell contact in nearby physiological conditions. Other routine cytotoxicity tests detect the first cytotoxic effects after 1.5-3 h co-incubation at the earliest.

Tracing DiO-labelled tumour cells in liver sections by confocal laser scanning microscopy.

Investigating rare cellular events is facilitated by studying thick sections with confocal laser scanning microscopy (CLSM). Localization of cells in tissue sections can be done by immunolabelling or by fluorescent labelling of cells prior to intravenous administration. Immunolabelling is technically complicated because of the preservation of antigens during fixation and the problems associated with the penetration of the antibodies. In this study, an alternative and simple approach for the labelling of cells in vitro with the fluorescent probe DiO and its subsequent application in vivo will be outlined. The method was applied to trace DiO‐labelled colon carcinoma cells (CC531s) in 100 µm thick liver sections. In vitro and in vivo experiments revealed that DiO‐labelling of CC531s cells had no cytotoxic or antiproliferative effect and the cells preserved their susceptibility towards hepatic NK cells or Kupffer cells. In addition, DiO remained stable for at least 72 h in the living cell. DiO‐labelled CC531s cells could be traced all over the tissue depth and anti‐metastatic events such as phagocytosis of tumour cells by Kupffer cells could be easily observed. In situ staining with propidium iodide (nucleic acids) or rhodamine‐phalloidin (filamentous actin) resulted in additional tissue information. The data presented improved the understanding of the possible effects of the vital fluorescent probe DiO on cell function and provided a limit of confidence for CLSM imaging of DiO‐labelled cells in tissue sections.

Endothelial Cells of the Hepatic Sinusoids: A Review

Hepatic sinusoidal endothelial cells differ from other endothelial cells. They possess open fenestrae that are grouped in sieve plates and lack a basal lamina. Fenestrae measure about 150nm, occur at a frequency of 9–13/mm2, and occupy 6%– 8% of the endothelial surface (porosity). These filter characteristics determine the exchange between the blood and the parenchymal cells, and are influencing the transport of lipoproteins including cholesterol and vitamin A. Forced sieving and endothelial massage are thought to enhance the passage of lipoproteins and to refresh the fluids in the space of Disse. Alcohol, nicotine, and other agents affect fenestrae in different ways and contribute either to fatty liver or hyperlipidemia. Furthermore, sinusoidal endothelial cells have: 1) a large capacity for pinocytosis via coated pits and macropinocytotic vesicles, 2) a variety of adhesion molecules, and receptors (scavenger, hyaluronan, collagen, Fc), and 3) a large capacity to digest material by numerous lysosomes. Endothelial cells secrete a limited amount of cytokines, but are vulnerable to products released by Kupffer cells, as in the case of endotoxemia or liver preservation. Capillarization of sinusoids includes the loss of fenestrae and the formation of a basal lamina and occurs in pathological conditions.