Effect of normothermic ("Warm") hypoxia on oxidative stress and metabolism in isolated/perfused rat liver: histochemical and biochemical evaluations.

It is generally believed that the damage induced by “warm” ischemia is negligible with respect to that caused by reperfusion and that Reactive Oxygen Species (ROS) are the main effectors of tissue damage (Le Masters & Thurman, 1993). In order to check the presumed poor response and ROS production of the various liver cell populations to lack of oxygen and nutrients, we used histochemical and biochemical techniques to document the response of isolated rat liver perfused with N2- or O2-saturated medium at 37°C for up to 2 hours. We analyzed the perfusate for LDH activity released from dead cells and for thiobarbituric acid reactive species (TBARS) derived from oxidative stress and measured bile flow as index of liver metabolism. Assays were made every 10 min. Two significant time points of tissue damage (40 and 70 min) were selected to perform the histochemical assays. These comprised the demonstration of: (a) ROS production, with Kerver et al. (1997) Mn2+-Co3+-DAB technique; (b) the putative diaphorase activity of Nitric Oxide Synthase (NOS), with a technique improved in our laboratory (Freitas et al., 2002); and (c) the activity of several enzyme activities linked to liver metabolism, namely Lactate Dehydrogenase (LDH), Succinate Dehydrogenase (SDH), Alkaline Phosphatase (AlkPh), Purine Nucleoside Phosphorylase (PNP) and Xanthine Oxidoreductase (XOR; comprising both the reductase and the oxidase forms of the enzyme), with standardized techniques (Van Noorden & Frederiks). Trypan Blue uptake was used as indicator of cell death. A scoring method was used to compare semi-quantitatively the response of hepatocytes, sinusoidal and biliary cells. Normoxic perfusion had negligible effects. By contrast, warm hypoxia caused lack of bile flow, hepatocyte staining by Trypan Blue in the midzone, alteration of the activity of all enzymes, increased ROS production by hepatocytes and sinusoidal cells (in keeping with high TBARS concentration in the perfusate) and release of LDH to the perfusate (in keeping with LDH loss from mid-zonal hepatocytes). ROS appeared to be produced mostly as by-products of the mitochondrial electron transfer chain in hepatocytes and during (auto)phagocytosis in sinusoidal cells and pericentral hepatocytes. NOS activity in Ito cells appeared to be correlated to sinusoid dilatation and PNP activity to be a sensitive marker of damage to sinusoidal and bile duct cells. In conclusion, our results suggest that the vulnerability of the liver to warm hypoxia is lobular zone and cell type-dependent and that damage to the parenchyma is more serious than that expected from biochemical data alone. (Funds from FAR-UniPV; MIUR-COFIN 1999, 2001)