The Protective Effect Of Normothermic Mechanical Perfusion On Donor After Cardiac Death Of Rat Liver

ObjectiveThe aims of this study were to develop an ex vivo normothermic mechanical perfusion(NMP),and to compare the effect of different portal perfusion pressures on reducing hepatic injury from donor after cardiac death(DCD),and to assess the implication of ATF6 in liver retrieval from DCD rat livers with NMP and explore the effect of pharmacologic ATF6 activation on liver retrievalMethodsComparing different portal vein perfusion pressure on DCD liver injury repair,establish rat heart death model and normal temperature mechanical perfusion model.All the rat livers were subjected to 30 in situ warm ischemia after cardiac attest and thereafter stored for 8 h under cold preservation.Six livers were harvested and regarded as control(Group CS,n=6).In experimental groups,livers received an ex vivo dual NMP with oxygenated perfusion via hepatic artery for 2 h after cold storage.Hepatic injury was assessed and compared from the perfused livers with the full portal vein pressure(Group M1,n=6)and low portal vein pressure(Group M2,n=6).The evaluation parameters included perfusion flow,liver enzymes of the perfusate,pathological changes by HE staining and Suzuki histological criteria,expression of activation markers of polymorphonuclear neutrophils and macrophages,myeloperoxidase and CD68 by immunohistochemistry,and level of malondialdehyde(MDA)and activity of superoxide dismutase(SOD).When discussing the pharmacological effect of ATF6 activation in liver repair,the more suitable liver preservation and perfusion conditions obtained in the first part were used to establish a rat heart death model and a normal temperature mechanical perfusion model.The livers from DCD rats were exposed to 30 min of warm ischemia and 8 h cold preservation followed by 2 h NMP with or without an ATF6 activator in the perfusate.The perfusion fluid and liver were collected to detect the expression of ATF6,the expression of ATF6 target genes PPARα and RCAN1,liver function,and the expression of inflammatory factors TNFα and IL6.Extract liver tissue mitochondrial protein and cytoplasmic protein to detect the protein expression of cytochrome C in mitochondria and cytoplasm.HE staining and Suzuki standard were used to assess liver pathological damage,immunohistochemical detection of neutrophil and macrophage activation indicators of myeloperoxidase(MPO)and CD68 expression,detection of liver tissue lipid peroxidation product malondialdehyde(MDA)content,superoxide dismutase(SOD)activity,GSH content,GSSG content and ATP content.ResultsIn experimental groups during NMP,the perfusion flows were set to increase when portal pressures in Group Ml and M2 were kept stable.The perfusion flows during NMP 60-120min were significantly higher than during NMP 0-20min.After NMP with full portal pressure,hepatic sinusoidal congestion,hepatocyte necrosis and steatosis and Suzuki criteria in Group M1 were evaluated lower than Group CS(P<0.05).Compared with Group M1,lower hepatic injury was characterized with a lower change of liver enzymes(P<0.05),a better histological evaluation(P<0.05),the lower MDA level and higher SOD activity(P<0.05),and the lower expression of CD68 and MPO(P<0.05).When using the more suitable liver preservation perfusion conditions obtained in the first part for mechanical perfusion.DCD livers with NMP were associated with ATF6 overexpression and activation based on IHC and WB(P<0.05).The ATF6 activator downregulated perfusate aminotransferases,decreased the Suzuki score,downregulated CD68 and MPO based on IHC,reduced TNFa and IL6 levels based on RT-qPCR and ELISA,downregulate the protein expression of cytochrome C in the cytoplasm and upregulate the protein expression of cytochrome C in the mitochondria,decreased MDA levels,GSSG levels,and increased SOD activity,GSH levels and ATP levels in the perfused livers(P<0.05).ConclusionThe ex vivo dual NMP with oxygenated perfusion via hepatic artery mimics the liver perfusion under the physiological conditions.NMP with a lower portal pressure can decrease hepatic ischemia-reperfusion injury,and may confer a better protection against liver damage from DCD.ATF6 is important for liver retrieval,and an exogenous ATF6 activator accelerates liver retrieval from DCD rats in an ex vivo NMP model.