Paul Rømert

High zone-selectivity of cell permeabilization following digitonin-pulse perfusion of rat liver

SummaryThe plasma membrane permeabilization obtained by exposure of hepatocytes to digitonin is utilized in the so-called digitonin-pulse perfusion of rat liver (Quistorff and Grunnet 1987). Brief pulses of digitonin applied with antegrade and retrograde perfusion of the liver caused selective elution of cytosolic enzymes and metabolites from the periportal and the perivenous zone of the same liver. In the present study a light microscopical examination of the liver fixed immediately after the digitonin pulse confirmed the very high zonal selectivity of the method inferred from the marker enzyme pattern of the eluates: Only cells around the port of entry of digitonin were affected and the borderline between affected and non-affected cells was always sharp. The typical periportal lesion was triangular in shape, enclosing the portal space, while the perivenous lesion was roughly circular, concentric with the hepatic vein. Assuming that the digitonin lesion reflects the microcirculatory flow pattern these findings seem to be at variance with the acinar model of Rappaport (Rappaport et al. 1954). The lesion in the lobuli near the surface of the liver as reflected by the discoloration pattern observed on the surface was the same as the lesion of deeper lobuli. The conducting vessels of the liver were only insignificantly affected by digitonin. At the cellular level only the sinusoidal luminal surface of the hepatocytes was affected. The cytoplasmic matrix of the cells including glycogen appeared thinned. All cell types of the liver parenchyma seemed to be equally affected by the digitonin treatment.

Swelling of Mitochondria in Immersion-Fixed Liver Tissue

In order to study the occurrence of swollen and disrupted mitochondria in tissue preserved for electron microscopy by ordinary fixation methods, liver tissue from miniature pig fetuses was immersion-fixed in fixatives with various types and concentrations of fixing agents and vehicles. Also commercial and purified products have been tested, different fixation times and temperatures as well as the consequences of a short rinsing in buffer solutions prior to fixation. Furthermore, the significance of delayed fixation (autolysis) was studied. It was found that swollen and disrupted mitochondria occur predominantly in liver cells exposed to low concentrations of glutaraldehyde. It is shown that this phenomenon is a result of a specific effect of glutaraldehyde on the mitochondrial membranes. It is not accompanied by parallel changes of other organelles or nuclei, and it not provoked by other fixing agents, vehicles or by delayed fixation (autolysis).

Fixation of foetal pig liver for electron microscopy

SummaryLight and electron microscopic investigations of the ability of various fixatives to preserve foetal pig liver tissue (immersion fixed 5 min, 20 min, and 40 min after the death of the mother) gave the following results:1)Fixation with 4% formaldehyde gives, in all circumstances, an unacceptable tissue preservation.2)Fixation with 2.5% glutaraldehyde gives fair tissue preservation in a ca. 130 μ thick zone below the surface.3)Fixation with a fixative mixture containing 2% formaldehyde and 1.25% glutaraldehyde or 2% formaldehyde, 1% glutaraldehyde and 1% acrolein gives good tissue preservation in a ca. 500 μ thick zone under the surface.4)The addition of DMSO does not improve tissue preservation.5)Delayed fixation of liver tissue removed from the foetus after the uterus has been placed in an incubator for 40 min has only a slight effect on tissue preservation.

Alcohol-induced injury of mitochondria in hepatocytes of mini-pig fetuses

The present study describes the occurrence of abnormal mitochondria in the cytoplasm of hepatocytes from half-term mini-pig fetuses, whose mothers in addition to their ordinary fodder have received alcohol (ethanol) in daily amounts comparable to those consumed by human alcoholics. The abnormal mitochondria exhibit elongation and distortion, disorientation of cristae and the appearance of paracrystalline and electron-dense material in the matrix. These ultrastructural changes show striking similarities to the mitochondrial injuries seen initially in the hepatocytes of human alcoholics and probably reflect damage to the mitochondria caused by alcohol itself.

The early development of the median thyroid gland of the mouse

SummaryThe mesobranchial area and the median thyroid anlage of embryonic albino mice were investigated from the somite stage 4 to 40 (81/2–10 days of gestation). In stage I (5–25 somites), there is an unequal growth and differentiation of the epithelium in the floor of the pharynx, whereby a mesobranchial area with a stratified or pseudostratified epithelium is formed. This area is distinct from the remaining pharyngeal epithelium, among other things by an apical microfilament system in the superficial epithelial cells. It is found just basal to a row of plump cytoplasmic protrusions, which extend into the lumen of the pharynx. In stage II (26–40 somites), the cranial part (median thyroid anlage) of the mesobranchial area thickens in relation to the caudal part and grows down into the underlying mesenchyme. The filament system is concentrated in the superficial cell layer of the median thyroid anlage at the beginning of stage II and disappears during downgrowth.In both stages, but most pronounced in stage II, there is a population of 0.1–5 μ intracellular bodies, which occasionally contain the remains of organelles. The larger bodies, which often contain the remains of nuclei, are usually found peripherally while the smaller ones are more evenly distributed. Acid phosphatase can often be demonstrated histochemically in small bodies, while larger bodies are usually without reaction. Cells with pycnotic nuclei and/or degenerated cytoplasmic components are regularly found. Acid phosphatase can also be demonstrated in Golgi complexes and surrounding vesicles. Basal to the epithelium, bodies are occasionally found which may possibly have been extruded from that tissue.

Fine Structure of Hepatocytes from Mini-Pigs and Mini-Pig Fetuses Exposed to Alcohol (Ethanol) in vivo

The effect of alcohol on hepatocytes from pregnant mini-pigs and their half-term fetuses was studied after addition of 100-300 g ethanol daily to the ordinary sufficient fodder for about 20 days. Well-defined areas of liver tissue from ethanol-exposed mini-pigs and their fetuses were immersion fixed. The hepatocytes were evaluated ultrastructurally and compared to hepatocytes of non-treated control animals. After exposure to ethanol the hepatocytes of the pregnant mini-pig developed an extensive smooth endoplasmic reticulum, and showed an increased number of mitochondria, microbodies and autophagic vacuoles, extensive Golgi complexes with accumulation of secretion, and a reduction of glycogen. The hepatocytes of the half-term fetuses exhibited profound changes of mitochondria and endoplasmic reticulum after alcohol exposure. Many mitochondria showed abnormal shape and increased size, disorientation of cristae and accumulation of paracrystalline material. An increased number of autophagic vacuoles containing remnants of mitochondria were observed. The granular endoplasmic reticulum exhibited aggregations of endoplasmic cisternae which were well defined and not bounded by a membrane. Thus, the ultrastructural changes in the hepatocytes of the pregnant mini-pig seem to indicate an adaptation of these cells to ethanol by development of a microsomal or catalase ethanol-oxidizing system, while the hepatocytes of the mini-pig fetus in contrast show obvious signs of cellular injury.

Improved utilization of tissue blocks for electron microscopy

Liver tissue from miniature pig fetuses was immersion-fixed in fixative mixtures with various concentrations of formaldehyde and glutaraldehyde. The preservation quality of hepatocytes was evaluated ultrastructurally in a peripheral zone (30--130 micron below the surface) and a central zone (500 micron below the surface). In the peripheral zone the best preservation was obtained with a fixative mixture containing 2% formaldehyde and 2% glutaraldehyde and in the central zone with a fixative mixture containing 8% formaldehyde and 8% glutaraldehyde. It is concluded that a better utilization of fairly large tissue blocks for ultrastructural investigation can be obtained by division of the block and subsequent fixation in fixatives containing various concentrations of formaldehyde and glutaraldehyde.

Cytochemical demonstration of cholinesterase in follicle cells and parafollicular (C-) cells of the rat thyroid gland

SummaryThe localization of cholinesterase in rat thyroid gland has been studied light- and electron-microscopically after incubation in an acetyl-thiocholin medium.The enzyme, a pseudocholinesterase, is found in follicle cells as well as in parafollicular (C-) cells.The distribution and volume of cholinesterase-containing cells have been investigated under normal conditions by means of a point count method. The results indicate some constancy in the pattern of distribution and in volume.

Ultrastructural changes of liver parenchyma following digitonin-pulse perfusion of rat liver

SummaryIt has been shown that pulse perfusion of rat liver with a digitonin-containing medium results in a highly zonated hepatocyte permeabilization, allowing selective sampling of cytosolic constituents from periportal and perivenous (centrolobular) hepatocytes “in situ”. In the present paper we provide an ultrastructural evaluation of the perfusion method. Identical changes in hepatocytes from affected periportal and perivenous zones are found. Affected hepatocytes appear light (electron-lucent) in electron micrographs with a sharp transition to normal hepatocytes. The most conspicuous ultrastructural findings are: (1) transformation of the sinusoidal part of the light hepatocytes, the lipocyte processes and the endothelium of affected zones apparently unifying into a continuous layer dominated by disrupted plasma membranes and 7-nm filaments; (2) deposition of osmiophilic digitonin-cholesterol complexes along the sinusoidal plasma membranes of affected zones; and (3) reduction of the cytoplasmic matrix (cytosol) in the light hepatocytes, a dilation of the mitochondrial intermembrane space with a preserved mitochondrial matrix, and a dilation of cisternae of the granular endoplasmic reticulum. The ultrastructural findings are consistent with marker-enzyme activity measured in eluates from digitonin-perfused livers, except that lysosomes appear intact, apparently contrasting with the observed eluation of amyloglucosidase (Quistorff et al. 1985).

Origin of cholinesterase-containing follicle cells and parafollicular cells of the developing thyroid gland in the rat.

SummaryThe location of cholinesterase-containing cells in the thyroid gland and its precursors (median thyroid primordium and ultimobranchial bodies) has been investigated light-microscopically in rat embryos from the 13 to the 20th day of gestation.From the 13th to the 16th day of gestation the median thyroid primordium and the ultimobranchial bodies are distinct from each other. Cholinesterase-containing cells are found in both. On the 17th–18th day of gestation the reacting ultimobranchial cells spread into the median thyroid primordium where they take up a parafollicular position. At the 19th–20th day of gestation the distribution of cholinesterase-containing cells is as in the adult rat. The results seem to show that cholinesterase-containing follicular cells derive from the median thyroid primordium and cholinesterase-containing parafollicular cells from the ultimobranchial body.