Marginal liver grafts characterization: 2) In situ evaluation of oxidative stress in liver steatosis induced by MCD

Background and aim: Reclaiming fatty livers for transplantation could decrease donor shortage. We demonstrated that obese Zucker rat livers were damaged by cold-storage and better preserved by sub-normothermic machine perfusion (Vairetti et al., 2009). We presently evaluate the model of steatosis induced by Methionine-Choline deficient diet (MCD) in rat liver, respect to an isocaloric diet (control group). We report in situ, biochemical and molecular indices of oxidative stress. Methods: Wistar rats received MCD diet up to 9 weeks. Lipid peroxidation was assessed measuring thiobarbituric acidreactive substances (TBARS), ROS production was visualized with DAB–Mn2+–Co2+ histochemical reaction, protein peroxidation by immunodetection of carbonyl group adducts, glutathione (GSH) concentration with an enzymatic method and Mn-SOD expression with real-time PCR, respectively. Results and conclusion: In MCD-treated rats hepatic TBARS were enhanced respect to control animals, from the 2nd until the 8th week, with a small decrease at the 9th week. The GSH levels, lower compared to those of control animals, decreased from the 1st to the 3rd week and stabilized henceforth. Conversely, the Mn- SOD expression trend was to increase starting from the 1st week in MCD rats. ROS production was very intense in periportal areas, decaying towards centrolobular region, both in control and in MCD rats. In control animals, ROS zonation is consistent with higher mitochondrial activity in the periportal region, especially in perisinusoidal areas; peroxidized proteins were indeed detected only in this domain. By contrast, InMCD animals the ROS reaction regarded the whole lobule and was concentrated mainly in the cytoplasm surrounding lipid vacuoles; these were also strongly positive for peroxidized proteins (ascribed to coat proteins of lipid droplets). Our data indicate that the ROS produced in the liver of MCD-treated animals overcome the antioxidant defenses especially after the 3rd week. Supported by Fondazione Cariplo, grant n◦ 2011-0439.