We have analyzed the effect of ischemia-reperfusion on expression of hepatic Na+,K+-ATPase on bile canalicular (BCM) and basolateral membranes (BLM) in human liver allografts using confocal laser scanning microscopy imaging. Na+, K+-ATPase, an integral membrane enzyme, plays a key role in the physiology and structure of hepatocytes, where it maintains the electrochemical gradients for Na+ and K+ across the cell membrane. The concentrations of these ions as well as their gradients regulate the active transport across the plasma membrane for bile acid and water from sinusoidal to canalicular membranes. In addition, Na+,K+-ATPase is also involved in cellular structure because of its close relationship with submembrane microfilaments and its implication in tight junction assembly. Therefore, Na+,K+-ATPase appears as an indicator of tissue viability and hepatic functionality during liver transplantation. Its localization and its function in BCM are still controversial. As in previous studies, we found an enzyme expression in both BLM and BCM. We show that ischemia induced a decrease in Na+,K+-ATPase expression only in BCM. This result could be explained by the differences in biochemical membrane environment between basolateral and bile canalicular Na+,K+-ATPase. Membrane lipid fluidity, which is more elevated in BLM than in BCM, could protect the enzyme during ischemia. After reperfusion, Na+,K+-ATPase expression was strongly decreased in both BCM and BLM. This alteration following reperfusion is probably due to multiple factors: direct alteration of the enzyme catalytic subunit and modification of its environment and membrane lipid fluidity by free radicals and changes in ATP levels and ionic distribution. This important decrease in Na+,K+-ATPase expression of both BLM and BCM could disturb not only hepatic secretory function but also cellular volume and structure during the postoperative period.

We studied and quantified the effect of ischemia-reperfusion on hepatic F-actin on bile canalicular and basolateral membranes in human liver allografts by means of confocal laser scanning microscopy imaging. The phalloidin-FITC staining of F-actin was normal in liver hepatocytes before reperfusion but decreased significantly after reperfusion (by 25% of controls). These results indicate that hepatic F-actin alteration is produced during the reperfusion phase. This modification, probably induced by reactive oxygen species, could impair bile canalicular contraction and tight junction permeability and consequently bile secretion in the postoperative period.

We studied and quantified the effect of ischemia-reperfusion on expression of intercellular adhesion molecule-1 (ICAM-1) on sinusoidal endothelial cells and hepatocyte plasma membranes by means of confocal laser scanning microscopy imaging. We found that ischemia induced an increase in ICAM-1 expression on sinusoidal endothelial cells and hepatocytes. After reperfusion, ICAM-1 expression was increased on sinusoidal endothelial cells, whereas it was unmodified on hepatocytes. On the other hand, ICAM-1 expression was not correlated to ischemia-reperfusion liver injury. Therefore, hepatocellular ischemia-reperfusion injury could be induced by other causes than immune-mediated damages.

SUMMARYBecause diabetes causes alterations in hepatic membrane fatty acid content, these changes may affect the Na ϩ ,K ϩ -ATPase. In this study we documented the effects of streptozotocin (STZ)-induced diabetes on hepatic Na ϩ ,K ϩ -ATPase catalytic ␣ 1 -subunit and evaluated whether these changes could be normalized by fish oil supplementation. Two groups of diabetic rats received fish oil or olive oil supplementation. Both groups had a respective control group. We studied the localization of catalytic ␣ 1 -subunit on bile canalicular and basolateral membranes using immunocytochemical methods and confocal laser scanning microscopy, and the Na ϩ ,K ϩ -ATPase activity, membrane fluidity, and fatty acid composition on isolated hepatic membranes. A decrease in the ␣ 1 -subunit was observed with diabetes in the bile canalicular membranes, without changes in basolateral membranes. This decrease was partially prevented by dietary fish oil. Diabetes induces significant changes as documented by enzymatic Na ϩ ,K ϩ -ATPase activity, membrane fluidity, and fatty acid content, whereas little change in these parameters was observed after a fish oil diet. In conclusion, STZ-induced diabetes appears to modify bile canalicular membrane integrity and dietary fish oil partly prevents the diabetes-induced alterations.

We localized HCV C-100 protein in liver biopsies of 15 patients with chronic hepatitis C using immunohistochemistry. The results were compared to serum, tissue extract analysis of HCV RNA and in situ RT-PCR described in a previous study. HCV was detected in 80% of the sera tested, in 40% of the tissue extracts and in 80% and 60% of the tissue sections tested by immunohistochemistry and in situ RTPCR respectively. Compared to the serum positive cases, 83% and 67% of the cases were respectively positive with immunohistochemistry and in situ RT-PCR and 41% were positive with tissue extract detection. Compared to the tissue extract positive cases, 25% and 50% of the cases were respectively positive with immunohistochemistry and in situ RT-PCR. Finally, 75% of the cases positive by immunohistochemistry were also positive by in situ RT-PCR. These results underline the complementarity of the different methods for the precise diagnosis of hepatitis C