Annexin V Assay-proven Anti-apoptotic Effect of Ascorbic Acid 2-glucoside after Cold Ischemia/Reperfusion Injury in Rat Liver Transplantation

Controversy exists over whether the predominant cell death of hepatocytes is due to apoptosis or necrosis after ischemia/reperfusion injury. In this study we investigated the predominant cell death of hepatocytes after cold ischemia/reperfusion injury using the Annexin V-based assay, and evaluated the anti-apoptotic effect of ascorbic acid 2-glucoside (AA-2G) added to the University of Wisconsin solution (UW solution) in rat liver transplantation. The retrieved liver was preserved in 4°C UW solution for 24 h, and then transplanted orthotopically to the syngeneic Wistar recipient. The animals were divided into 2 groups, a control group (n = 10), in which liver grafts were preserved in UW solution (4°C), and an AA-2G group (n = 10), in which liver grafts were preserved in UW solution (4°C) with AA-2G (100 ug/ml). The serum AST level 4 h after reperfusion in the control group was significantly suppressed in the AA-2G group, and the bile production of the liver graft in the AA-2G group was well recovered. The mean survival time in the AA-2G group was significantly improved compared with that in the control group. Annexin-V and Propidium iodide staining 4 h after reperfusion showed a significantly higher percentage of viable hepatocytes in the AA-2G group compared with the control group (93.4 ± 2.0 vs. 80.3 ± 2.1%, P< 0.05). In the control group, the main cell death of hepatocytes was apoptosis (early apoptosis: 10.0 ± 4.7%, late apoptosis: 6.4 ± 1.7%). The addition of AA-2G to the UW solution significantly inhibited both early and late apoptotic cell death 4 h after reperfusion (early apoptosis: 0.98 ± 0.88%, late apoptosis: 2.2 ± 1.1%). The expression of caspase 9 in the immunostaining of the liver graft was suppressed in the AA-2G group compared with in the control group. Our study using the Annexin V-based assay provided evidence that the predominant cell death of hepatocytes was apoptosis after 24 h cold ischemia/reperfusion injury in rat liver transplantation. The addition of AA-2G to the UW solution attenuated 24 h cold ischemia/reperfusion injury by inhibiting the apoptosis of hepatocytes.