Ultrastructural changes in rat liver sinusoids during prolonged normothermic and hypothermic ischemia in vitro

By means of electron microscopic analysis of liver fragments incubated in an air-tight wrapping (in vitro ischemia), the following facts have been established with regard to the development of signs of irreversible damage in cells from the sinusoidal wall compared with hepatocytes. With normothermic (37 degrees C) in vitro ischemia, signs of irreversible damage appeared in cells of the sinusoidal wall at a a much earlier stage than in hepatocytes (60-90 min and 90 min-2 h respectively). With in vitro ischemia in the cold (4 degrees C), these differences were even more marked; irreversible cell damage was apparent after between 24 and 36 h incubation in endothelial cells, whereas in hepatocytes flocculent densities followed by other signs of irreversible damage were found only after 79 h incubation. These findings are discussed in relation to the 'no reflow' phenomenon after ischemia in general. The rule that changes in the vascular system following ischemia may well obscure the actual sensitivity of parenchymal cells is particularly applicable to the liver. Attempts to lengthen the period of ischemia which 'liver tissue' can stand for example, with a view to transplantation, attention should be focussed primarily on the events in the sinusoidal wall.     

Gluconeogenesis in the rat renal cortex after warm ischemia and hypothermic storage

Rat kidney cortex slices were tested for their gluconeogenic capacity after the kidney has been either subjected to warm ischemia or flushed with and stored in cold hyperosmotic media. Kidneys damaged by warm ischemia for up to 60 min did not lose their ability to convert pyruvate to glucose. However, they then rapidly lost this capacity so that after 2 hr of ischemia they were devoid of activity. This observation closely corresponded to survival of partially nephrectomized rats whose remaining kidney had been treated in a similar manner. Cortex slices obtained from kidneys flushed and stored in cold hyperosmotic media were found to lose most of their gluconeogenic capacity after 3 days of storage.     

A comparison of the metabolic effects of continuous hypothermic perfusion or oxygenated persufflation during hypothermic storage of rat liver

The metabolic consequences of supplying oxygen by two different modes were investigated. The effects of hypothermic liver preservation after cold hypoxic flush (Group I), oxygenated vascular persufflation (Group II), and continuous oxygenated perfusion (Group III) were compared. Adenine nucleotides were measured to assess energetics, and 1H nuclear magnetic resonance spectroscopy was employed to investigate other metabolic pathways. Energetics were maintained by both modes of oxygenation at 24 h. The mitochondrial redox state is indicated by the ratio of acetoacetate (Ace) and beta-hydroxybutyrate (betaHb). The detection of only betaHb or Ace in the hypoxic flush and perfused livers, respectively, suggested that the mitochondria of these livers were hyperreduced and hyperoxidized, respectively. In contrast, both components of the redox couple were detected in the persufflated livers, suggesting that persufflation may be a simple and effective method of maintaining hepatic energetics long-term while maintaining a more normal mitochondrial redox state.     

Quantitative analysis of mitochondrial flocculent densities in rat hepatocytes during normothermic and hypothermic ischemia in vitro

The development of flocculent densities in mitochondria as a sign of irreversible cell injury in rat hepatocytes has been studied by quantitative electron microscopy during in vitro ischemia under both normothermic (37 degrees C) and hypothermic (4 degrees C) conditions. At 37 degrees C flocculent densities first appear after 1 h ischemia; at this stage they are small in diameter (170 nm) and occur in only 8% of mitochondria. After 1.5 hour ischemia, flocculent densities increase in diameter (207 nm) and are seen in 37% of mitochondria. Death of the majority of hepatocytes seems to occur between 1.5 and 2 h ischemia since at this stage the percentage of mitochondria containing flocculent densities reaches a maximum (48%). However, flocculent densities continue to increase in size (to 337 nm diam.) up to between 2 and 4 h ischemia (the prenecrotic phase). In contrast, at 4 degrees C signs of ischemic damage to hepatocytes are considerably delayed. Flocculent densities of comparable size and frequency to those observed after 1 h ischemia at 37 degrees C are not seen till as late as 4 days at 4 degrees C. At the latter temperature, only after 7 days ischemia a substantial rise (to about 25%) in the proportion of mitochondria containing flocculent densities occurs. A further slow increase in size and in the percentage of mitochondria containing densities occurs up to 14 days ischemia at 4 degrees C. It is concluded that the development of flocculent densities may be used only as a parameter of irreversible damage in cells with a sufficient number of mitochondria, such as hepatocytes, under normothermic conditions. With ischemia at 4 degrees C, possibly due to a different protein denaturation pattern, the development of flocculent densities is of much less value as an indication of irreversible cell damage and cannot, therefore, be considered as a reliable sign of cellular damage in organs stored at 4 degrees C for transplantation purposes.     

Ultrastructural changes during cholestasis induced by chlorpromazine in the isolated perfused rat liver.

Addition of cholestatic doses of chlorpromazine-HC1 to the perfusate of isolated rat livers produces widespread changes in hepatocyte membrane structure. These findings include a marked increase in intrasinusoidal cytoplasmic bullae, appearance of intracellular vacuoles within hepatocytes at both sinusoidal and biliary poles, dilation of bile canaliculi and evagination of canalicular diverticuli, and the formation of myeloid bodies within hepatocytes. These findings obtained in the bile acid depleted perfused liver may result from physiochemical interactions between chlorpromazine or its metabolites and lipid-protein components of cell membranes, consistent with chlorpromazine's properties as a cationic detergent. They occur independently of the vasoconstrictive effects of chlorpromazine and suggest that chlorpromazine may produce cholestasis by altering hepatocyte membrane function.     

Ultrastructural changes in the gallbladder mucous membrane during temporary experimental ischemia]

Temporary ischemia of the gall bladder was induced in rabbits by ligation of the gall bladder artery with silk. Histological examination revealed vascular disorders, such as hyperemia, blood stasis and focal hemorrhages. Electron microscopic studies showed the presence of increased number dark epithelial cells, expansion of intercellular area, loosening of the basal membrane and defects in it with invagination of the epithelial cells into the submucous layer. The most striking changes were discovered after a thrice-repeated 30-minute occlusion of the gall bladder artery. The degree of destructive changes proved to depend on the number of stimulated spasms (occlusions) and not on the duration of ischemia. This gives grounds to believe that multiple circulatory disorders participated in the complicated pathogenesis of cholecystitis.     

Ultrastructural changes in the rat thyroid gland during iodine deficiency

Thyroid ultrastructure changes were studied during the course of a low iodine diet in rats. At day 20, follicles were normal, but a number of them contained cells of higher density and with greatly elongated microvilli. Endoplasmic reticulum cisternae were frequently dilated. From day 20 until day 80, the most characteristic changes in the thyroid cells were the progressive accumulation of subapical peroxidase-positive exocytotic vesicles. After 80 days of the low iodine treatment, Golgi apparatuses were very active. Cell division could be observed. At this stage, exocytotic vesicles were generally very abundant. These data suggest that the remarkable accumulation of subapical exocytotic vesicles between day 20 and day 120 might represent an adaptation to the moderate and gradual increase in TSH stimulation that occurs in the conditions of low iodine diet.     

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

Summary 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).     

Ultrastructural changes in the liver of the sand lamprey,Lampetra reissneri,during sexual maturation

Ultrastructural changes of hepatocytes were examined in the sand lamprey,Lampetra reissneri, during various phases of the life cycle. In hepatocytes of ammocoetes, the rough endoplasmic reticulum was composed of short cisternae and the Golgi apparatus were scarcely developed, showing no sexual differences at this stage of life cycle. In hepatocytes of female lampreys at the metamorphic stages 4 to 5, the rough endoplasmic reticulum was developed to form long parallel cisternae and the Golgi apparatus were well-developed. The rough endoplasmic reticulum developed further to form stacks of long parallel cisternae extending over the cytoplasm in hepatocytes of females at the young adult stage, and became composed of both long parallel and vesicular cisternae in the cells of females at the adult stage. The Golgi apparatus were invariably welldeveloped in hepatocytes of young adult and adult females. No consipcuous development was observed in profiles of the rough endoplasmic reticulum and the Golgi apparatus in hepatocytes of males during and after metamorphosis. The ultrastructural changes of the rough endoplasmic reticulum and the Golgi apparatus observed in hepatocytes of female sand lampreys are considered to have an intimate relation to the activity of vitellogenin synthesis in the liver, and it is suggested that the hepatocytes begin to rapidly synthesize vitellogenin in the sand lamprey at the metamorphic stages 4 to 5.     

A histochemical study of changes in mitochondrial enzyme activities of rat liver after ischemia in vitro

Changes in the activity of three mitochondrial enzymes in rat liver after in vitro ischemia have been determined by enzyme histochemical methods. The changes were correlated with the appearance in the electron microscope of flocculent densities in the mitochondria indicative of irreversible cell injury. The flocculent densities were observed in rat liver after about 2 h of ischemia in vitro at 37 degrees C. At the same time the activity of glutamate dehydrogenase, localized in the mitochondrial matrix, started to decrease. However, the activities of succinate dehydrogenase localized in the inner membrane of mitochondria, as well as monoamine oxidase of the mitochondrial outer membrane did not change at that stage. It is concluded from the results of this study and those of others that flocculent densities are formed by denaturation of proteins of the mitochondrial matrix in which glutamate dehydrogenase takes part. It should be considered more as a sign than as the cause of cell death.     

Peroxidative injury of the mitochondrial respiratory chain during reperfusion of hypothermic rat liver

Mitochondrial dysfunction in ischemic liver has been demonstrated to be due to decrease in the intramitochondrial level of ATP and the subsequent disruption of the proton barrier of the inner membrane (Watanabe, F., Hashimoto, T. and Tagawa, K. (1985) J. Biochem. 97, 1229-1234). In this study, another injury process, impairment of the electron-transfer system, which occurred during reoxygenation of ischemic liver, was studied during reperfusion of cold preserved liver and during cold incubation of isolated rat-liver mitochondria. The sites of the respiratory chain that were sensitive to peroxidative damage were ubiquinone-cytochrome c oxidoreductase and NADH-ubiquinone oxidoreductase. These enzymic activities decreased with increase in lipid peroxidation. Incubation of submitochondrial particles with t-butyl hydroperoxide or with an NADPH-dependent peroxidation system decreased the enzymic activities of the electron-transport system. These data strongly suggested that lipid peroxidation during reoxygenation of ischemic liver impaired the electron-transfer system. Thus, mitochondria of ischemic liver suffer from two different types of injury: increase in proton permeability during anoxia, and decrease in enzymic activities of the electron-transport system during reoxygenation.