Enhanced Role for Apoptosis in a Hepatic Ischaemia-Reperfusion Injury Model

Background:Prior to reperfusion, a variable period of ischaemia occurs in transplanted donor organs There is growing evidence that apoptosis contributes to the ischaemia-reperfusion injury conundrum that follows, but its extent has not been precisely quantified. Understanding its extent and effects can help find ways to mitigate or counteract such an injury in the liver. We therefore evaluated the extent of apoptosis semi-quantitatively from the expression of Caspase-3 and M30 in a porcine ex-vivo hepatic ischaemia-reperfusion injury model and correlated the timing of expression of M30 antibody with that of Caspase-3.Method:Livers were harvested from three healthy white Landrace Cross pigs and perfused for six hours using oxygenated extracorporeal circuits with heparinized autologous blood at 380C as described in our previous work [1]. A 2-hour delay prior to perfusion represented the period of ischaemia. All the livers were uniformly perfused. Three biopsies were taken for each of 7 time points (total 21); before dissection of the liver (BD) of the livers, after dissection (AD), after connection (AC) to the circuit, at one hour perfused (1hrP) & non-perfused (1hrNP), at 4 hours perfused (4hrP) and non-perfused (4hrNP). Samples were stained with standard Haematoxylin–Eosin (HE) and immunohistochemically with Caspase-3 and M30 monoclonal antibodies. Positive cells were quantified using an ocular grid and expressed as number per square area. Cells positive for Caspase-3 & M30 antibodies were counted on the whole section. Data were expressed as median number per square millimetre. Statistical analysis was done using AVOVA and t-test.Results: After four hours of perfusion, multiple areas of perfusion defects appeared macroscopically in all 3 livers. HE staining confirmed that these non-perfused areas were ischaemic. There was a gradual increase in Caspase-3 expression over time in all samples. There was no statistically significant difference in the Capase-3 and M30 between the 9 specimens at 1hrP (P=0.06) but there was a highly significant difference between Caspase-3 and M30 at 1hrNP (0.001). Caspase-3 expression peaked at 4hrP (P=0.001) and 4hrNP (P=0.03). A similar pattern was noted with M30 which peaked at 1hrP (P=0.001) and maintained a non-significant expression at 4hrP (P=0.07) and 4hNP (P=0.1).Conclusion: The significant expression of Caspase-3 and M30 confirms that apoptosis plays an important part in tissue loss in this model of liver ischemia-reperfusion injury. Inhibiting both Caspase-3 and M30 might be useful in alleviating hepatic reperfusion injury.?