c-kit immunoreactive interstitial cells of Cajal in the human small and large intestine.

c-kit immunohistochemistry was performed on unfixed frozen sections of human small (duodenum, jejunum, and ileum) and large intestine (ascending, transverse, descending, and sigmoid colon). The c-kit immunoreactive cells in the muscularis externa of the intestinal wall were identified as interstitial cells of Cajal (ICC) and mast cells. ICC were identified by their morphology, localization, and organization based on previous light and electron microscopic studies. In the small intestine, ICC were located primarily in relation to the myenteric plexus of Auerbach, but also in septa between circular muscle lamellae. In the large intestine, ICC were seen in relation to Auerbach's plexus, but also and in great numbers in the circular muscle layer and in teniae of the longitudinal muscle layer. The morphology of the ICC was similar in the small and large intestine, but the pattern of distribution was obviously different. c-kit immunoreactive mast cells were found predominantly in the inner part of the circular muscle layer. The anti-c-kit method is found to be an easy and reliable method to study at least most of the interstitial cells of Cajal and thereby contribute to further normal and pathological studies.

Histological evaluation of the zonation of colloidal gold uptake by the rat liver.

The distribution and endocytotic function of Kupffer cells in the rat liver were studied after administration of fibrinogen stabilized colloidal gold suspensions either by injection directly into the circulatory system of anaesthetized rats or by application to the isolated perfused liver. After exposure to gold particles the livers were perfused with fixative and studied using several microscopic techniques. Gold was predominantly endocytosed by a highly active population of Kupffer cells surrounding the portal spaces resulting in distinct dark patterns around the terminal portal veins. In cross-sections of lobules the pattern appeared as incomplete networks composed of dark triangular areas with distinct borderlines towards light areas concentric with the terminal hepatic veins (central veins). The light areas contained few and relatively inactive small Kupffer cells. A wide variation of conditions gave essentially the same uptake pattern compatible with the concept of microcirculatory zones concentric with the terminal hepatic veins (Lamers et al., 1989; Quistorff and Rømert, 1989), but contradicting the traditional view of microcirculatory zones advanced by Rappaport et al. (1954). Since the same pattern developed during conditions of anoxia, it seems that oxygen is not the stimulus for the developmental distribution of Kupffer cells with high endocytotic activity. In vivo and perfusion experiments gave identical patterns, but a higher endocytotic activity of endothelial cells was found in perfused isolated livers.

Ultrastructural and morphometric study of hepatocytes from near-term minipig fetuses exposed to ethanol in vivo.

Hepatocytes of near-term minipig fetuses were studied after their mothers had received a daily addition of 3 g/kg body weight of ethanol to the ordinary sufficient fodder during the last half of pregnancy. The ethanol-exposed hepatocytes were evaluated ultrastructurally and morphometrically, and compared with hepatocytes of unexposed fetuses. The present results show that hepatocytes of near-term fetuses, exposed to a high concentration of ethanol for a long period exhibit neither qualitative nor quantitative changes. This is in contrast to maternal hepatocytes which show an adaptation of cellular components to ethanol by developing increased volume densities of mitochondria and smooth endoplasmic reticulum and a decreased volume density of glycogen.

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

It 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).

High zone-selectivity of cell permeabilization following digitonin-pulse perfusion of rat liver. A re-interpretation of the microcirculatory zones.

The 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.

Changes of secretory ameloblasts in mini-pig fetuses exposed to ethanol in vivo.

After addition of ethanol to the ordinary fodder of pregnant mini-pigs, ultrastructural changes were found in secretory ameloblasts from tooth germs of their mid-term fetuses. Compared with controls, many mitochondria showed abnormal shape and size, and some exhibited deposition of paracrystalline material in the matrix. An abnormal deposition of stippled material intercellularly was also observed. These changes were interpreted as signs of an abnormal secretory function.

Quantitative study of hepatocytes from minipigs and minipig fetuses exposed to ethanol in vivo.

The effect of ethanol on hepatocytes from pregnant minipigs and their half-term fetuses was studied with the aid of morphometric methods. In the pregnant minipigs the hepatocytes of the ethanol-treated animals showed a significant increase in the volume density of mitochondria, autophagic vacuoles, Golgi complexes and smooth endoplasmic reticulum, and a significant decrease of glycogen. In the half-term fetuses the hepatocytes of ethanol-exposed animals showed no significant change in the volume density of mitochondria, peroxisomes, autophagic vacuoles, Golgi complexes, smooth endoplasmic reticulum or glycogen, and no significant change in the surface density of granular endoplasmic cisternae. The present investigation indicates that in the maternal hepatocyte certain cytoplasmic components are quantitatively changed by ethanol, whereas the volume and surface densities of identical components in the fetal hepatocyte are unaffected. The significance of these results is discussed.

Tetracycline-induced changes in hepatocytes of mini-pigs and mini-pig foetuses as revealed by electron microscopy.

The effect of chlortetracycline on hepatocytes from pregnant mini-pigs and their half-term foetuses was studied after oral administration of 1-3 g daily for 6 days. Liver tissue from tetracycline-treated mini-pigs and their foetuses was immersion fixed, and hepatocytes were evaluated by qualitative and quantitative electron microscopy and compared to hepatocytes of untreated controls. After exposure to tetracycline the hepatocytes of the pregnant mini-pig showed a significant increase in the volume density of mitochondria and autophagic vacuoles. Also a significant decrease in the volume density of glycogen was observed. The hepatocytes of the tetracycline-exposed half-term foetuses showed profound morphological changes of mitochondria. Many mitochondria were of an abnormal shape and an increased size. In most mitochondria the matrix contained a paracrystalline material which had a characteristic prismatic shape with a regular internal lattice structure. Many mitochondria also exhibited numerous short stacked cristae or long longitudinally oriented cristae. No significant change in the volume density of mitochondria was demonstrated. Also no significant change in the volume density of glycogen was observed. In conclusion it can be stated that small doses of chlortetracycline administered orally for a few days provoke morphological changes in both maternal and foetal hepatocytes. While the changes in maternal hepatocytes may reflect metabolic changes, the distinct morphological changes of mitochondria in foetal hepatocytes probably indicate cellular injury.

Electron microscopic demonstration of non-mineralized and hypomineralized areas in dentin and cementum by silver methenamine staining of collagen.

An electron microscopic study on silver methenamine staining of hard dental tissues was made on a material that comprised human permanent teeth and primary tooth germs from human and porcine fetuses. It was demonstrated that silver-stained material consisted of collagen fibrils. In predentin and precementum all collagen fibrils were stained, while collagen fibrils of dentin and cementum were unstained except for some fibrils of minor special areas such as Owen's contour lines, interglobular dentin, Tomes's granular layer, and in cementum small "interglobular-like" areas. It is concluded that silver methenamine visualizes collagen fibrils of hypo- and unmineralized areas in dental hard tissues and therefore may be used to demonstrate abnormal patterns of mineralization. Finally variations of silver methenamine stainability in relation to differences in material and methods were studied and discussed.

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.

Ethanol-induced changes of granular endoplasmic reticulum in hepatocytes of mini-pig foetuses.

The present study describes the occurrence of oblong or oval membranous aggregations of the endoplasmic reticulum in the cytoplasm of hepatocytes from half-term mini-pig foetuses, whose mothers in addition to their ordinary sufficient fodder have been given ethanol in amounts comparable to those consumed by human alcoholics. The structure of the aggregations and their relation to the granular endoplasmic reticulum indicate that they represent a change of the granular endoplasmic reticulum, induced by ethanol. The oval aggregations show resemblances to concentric lamellar bodies described in hepatocytes altered by disease or by experimental procedures not involving ethanol. It is concluded that the change of granular endoplasmic reticulum is probably a sign of an injurious effect of ethanol on the foetal hepatocyte.

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.

Swelling of mitochondria in immersion-fixed liver tissue. Effect of various fixatives and of delayed fixation.

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).

Morphology of a simple ameloblastoma related to the human enamel organ.

The ultrastructure of a simple ameloblastoma was investigated. The epithelial cells of the tumor could be divided into peripheral cells of varying height, and dark and light central cells. The morphology was correlated to that of the human enamel organ. The low peripheral cells were very similar to the external enamel epithelium cells. The central cells had a certain resemblance to the stellate reticulum and stratum intermedium cells. The high peripheral cells had no counterparts in the enamel organ. Unlike the enamel organ the ameloblastoma showed extremely few and small gap junctions.

Ultrastructure of the human enamel organ. I. External enamel epithelium, stellate reticulum, and stratum intermedium.

The fine structure of external enamel epithelium, stellate reticulum and stratum intermedium in primary tooth germs (bell stage) from four human foetuses was investigated. Characteristically, the cells of the differentiated external enamel epithelium, stellate reticulum and stratum intermedium exhibit many free ribosomes, few rough endoplasmic reticulum cisterns, well-developed Golgi complexes, many coated and smooth vesicles, often in relation to the cell membranes, and many bundles of tonofilaments. The cells are connected by numerous desmosomes and gap junctions. A parallel differentiation of stratum intermedium - external enamel epithelium, and the ameloblast layer is demonstrated. The morphology of the cells of the three layers indicates that these have secretory, transport and supporting functions.

Ultrastructure of the human enamel organ. II. Internal enamel epithelium, preameloblasts, and secretory ameloblasts.

The fine structure of internal enamel epithelium, preameloblasts and secretory ameloblasts in primary tooth germs (bell stage) from four human foetuses was investigated. The characteristics of the differentiation of internal enamel epithelium via preameloblasts to secretory ameloblasts are described. The internal enamel epithelium consists of a row of low differentiated prismatic cells separated from the dental papilla by a distinct even basal lamina. In the preameloblasts the rough endoplasmic reticulum cisterns and mitochondria increase in number, the Golgi complexes become extensive and take up a distal position, and secretory granules are formed. Furthermore, the basal lamina is removed by coated vesicles, and proximally and distally in the cells a complex of zonulae adhaerentes, terminal webs and gap junctions is formed. The secretory ameloblasts make up a layer of highly differentiated cells demonstrating typical merocrine secretion.

Improved utilization of tissue blocks for electron microscopy. Effect of various concentrations of formaldehyde and glutaraldehyde.

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.

Fine structure of the human secretory ameloblast.

The fine structure of secretory ameloblasts in primary tooth germs from a human fetus was investigated. Various aldehyde fixatives produced varying qualities of preservation. The morphology of the best preserved ameloblasts differed from that earlier described, mainly in the following respects: the rough endoplasmic cisternae were lamellated and regularly arranged, the mitochondria were of regular shape and size, and the ground substance was dense and homogenous. The extensive Golgi complexes, the mitochondria and the rough endoplasmic cisternae in the apical (distal) part of the ameloblast were longitudinally arranged.

The influence of fetal circulation on liver cell ultrastructure.

This study shows that autolytic changes in liver tissue from mini-pig fetuses are less extensive if, after removal of the uterus, the fetal circulation is left anatomically intact until excision and fixation. It must be concluded that, if it is not possible to remove and fix fetal material immediately, the fetus should be kept in the intact amniotic cavity with an intact umbilical cord.

Argyrophilic cells in the larynx of the guinea-pig demonstrated by the method of Grimelius.

Argyrophilic cells with branching processes are shown in both the surface epithelium and in the glands in the larynx of adult guinea-pigs using the Grimelius silver technique after GPA fixation. When Bouin's fixative or neutral formalin were used as fixatives argrophilic cells could not be identified.