The old rats showed more serious liver injury than young rats during hepatic IR. Partial (70%) liver arterial/portal venous blood was interrupted for 40 minutes in 3-month- and 22-month-old rats. Blood and liver samples were harvested at 24 h after reperfusion. (a) H&E and TUNEL (green) staining of the liver. (b) Liver histological scores. (c) Percentage of necrotic areas. (d) Percentage of TUNEL-positive cells. (e) Serum ALT. Rat hepatocytes were isolated, and the hypoxia/reoxygenation (H/R) model was performed in hepatocytes by exposing to hypoxia condition (94% N₂, 5% CO₂, and 1% O₂) at 37°C in glucose/FBS-free RPMI-1640 medium. One hour later, the hepatocytes returned to normal culture conditions. The sham group was treated with normal medium without hypoxia. The subsequent experiments were performed at 8 h after reoxygenation. (f, g) Flow cytometry analysis of hepatocyte apoptotic percentage. ; ; and .