The Impact Of Ischemic Preconditioning On Acute Rejection After Ischemia Reperfusion Injury Of Allogene Liver Transplantation In Rats

Part 1 The Model Construction of allogene Rat's Orthotopic Liver Transplantation(OLT)to study acute rejectionObjeetive:To study the operation technique of rat's orthotopic liver transplantation(OLT)and to provide a reliable model to study acute rejection after ischemia reperfusion injury of allogene OLT.Methods:120 cases of rats' OLT operations were performed by modified Kamada's cuff technique with hepatic arterial reconstruction and modified suture for suprahepatic inferior vena cava and Hepatic perfusing method to study acute rejection after ischemia reperfusion injury of allogene OLT.Results:80 cases formal operations were practiced.The anhepatic time was 16～21min and the successful rate was 85%(68/80)after the liver grafts undergoing 0～2 min of warm ischemia and 120 min of cold ischemia.Acute rejection was emerged in 3 days post-operation.The appearance was most typical in 7 days post-operation.Conclusion:The allogene rat's OLT model performed by Kamada's cuff technique with hepatic arterial reconstruction and modified suture for superior vena and Hepatic perfusing method was reliable to study acute rejection after ischemia reperfusion injury. Part 2 The relationship between degree of ischemia reperfusion injury and acute rejection after allogene Rat's Orthotopic Liver Transplantation.Objective:To investigate the relationship between degree of ischemia reperfusion injury and acute rejection after allogene rat's Orthotopic liver transplantation.Methods:Sprague-Dawley rats were used as donors and Lewis were used as recipients,rats' OLT operations were performed by modified Kamada's cuff technique with hepatic arterial reconstruction and modified suture for superior vena and Hepatic perfusing method.There were divided into Sham group(groupⅠ)and similargene experiment group(groupⅡ)and allogene experiment(groupⅢ-Ⅵ)according to warm and cold ischemia time.The animals were killed at 1,3,5,7d after graft reperfusion or sham operation respectively.Plasma samples were collected for ALT,AST,AKP and r-GT test and assessed the degree of ischemia reperfusion injury;Part of the liver tissue was made in paraffin-embedded specimens to detect rat liver histological change (HE)and classify rejection;apoptosis(TUNEL)and the protein expression of Fas,Perforin and granzyme B(immunohistochemical); Part of fresh liver tissue was collected to detect the expression of Fas,Perforin and granzyme B mRNA by RT-PCR.Results:The serum level of ALT,AST,AKP and r-GT were higher in group ofⅡ,Ⅲ,Ⅳ,ⅤandⅥthan group ofⅠ(P＜0.05)at 1,3,5, 7d after graft reperfusion.The serum level of ALT,AST,AKP and r-GT has significant difference(P＜0.05)at 1,3,5,7d after graft reperfusion group comparison among group ofⅡ,Ⅲ,Ⅳ,Ⅴ.Hepatic tissue was demonstrated ischemia reperfusion injury by histopathologic examination. The extent of disease and degree of Hepatic tissue was Paralleled with the degree of ischemia reperfusion injury;The enhancement of classify rejection was Paralleled with degree of ischemia reperfusion injury in group ofⅢ,ⅣandⅤ,The appearance was most typical in 7 days post-operation.No rejection occurred in group ofⅠ,ⅡandⅥ.The apoptosis index(AI)of liver tissue in group ofⅡ,Ⅲ,Ⅳ,ⅤandⅥwere higher than group ofⅠ(P＜0.05)at 1,3,5,7d after graft reperfusion.The apoptosis index(AI)of liver tissue in group ofⅢ,ⅣandⅤwere higher than group ofⅡ(P＜0.05)at 5,7d after graft reperfusion.The hepatic tissue mRNA and protein expression of Fas,Perforin and granzyme B in group ofⅢ,ⅣandⅤwere higher than group ofⅠ,ⅡandⅥ(P＜0.05)at 1,3,5,7d after graft reperfusion.The hepatic tissue mRNA and protein expression of Fas,Perforin and granzyme B has significant difference (P＜0.05)at 1,3,5,7d after graft reperfusion group comparison among group ofⅢ,Ⅳ,Ⅴ.Conclusion:(1)The incidence of acute rejection after allogene Rat's OLT was Paralleled with the degree of ischemia reperfusion injury.The incidence rate of acute rejection would decrease if The hepatic tissue emerges major injury because of ischemia reperfusion injury.(2)Ischemia reperfusion injury impacted acute rejection after allogene Rat's OLT.It was correlated with apoptosis and activation of Fas/FasL and Perforin/granzyme B.(3)The incidence rate of acute rejection would decrease if The hepatic tissue emerges major injury because of ischemia reperfusion injury.It was correlated with restraining apoptosis and restraining mRNA and protein expression of Fas,Perforin and granzyme B. Part 3 The Impact of ischemic preconditioning on acute rejection after ailogene Rat's Orthotopic Liver Transplantation.Objective:To investigate the impact of ischemic preconditioning on acute rejection after allogene Rat's Orthotopic Liver Transplantation.Methods:Sprague-Dawley rats were used as donors and Lewis were used as recipients,rats' OLT operations were performed by modified Kamada's cuff technique with hepatic arterial reconstruction and modified suture for superior vena and Hepatic perfusing method.There were divided into Sham group(groupⅠ)and similargene experiment group(groupⅡ)and allogene ischemia reperfusion group(groupⅢa,Ⅲb)and allogene ischemic preconditioning group(groupⅣa,Ⅳb) according to ischemic preconditioning or not.The animals were killed at 1,3,5,7d after graft reperfusion or sham operation respectively.Plasma samples were collected for ALT,AST,AKP and r-GT test and assessed the degree of ischemia reperfusion injury;Part of the liver tissue was made in paraffin-embedded specimens to detect rat liver histological change(HE)and classify rejection;apoptosis(TUNEL)and the protein expression of Fas,Perforin and granzyme B(immunohistochemistry); Part of fresh liver tissue was collected to detect the expression of FasmRNA,PerforinmRNA and granzyme B mRNA by RT-PCR.Results:The serum level of ALT,AST,AKP and r-GT were Lower in group ofⅣa than group ofⅢa,group ofⅣb than group ofⅢb(P＜0.05) at 1,3,5,7d after graft reperfusion.The extent of disease and degree of ischemia reperfusion injury and degree of acute rejection were Lower in group ofⅣa than group ofⅢia,group ofⅣb than group ofⅢb (P＜0.05)by histopathologic examination,at 1,3,5,7d after graft reperfusion.The hepatic tissue mRNA and protein expression of Fas,Perforin and granzyme B in group ofⅢa,Ⅲb,Ⅳa andⅣb were higher than group ofⅠ,Ⅱ(P＜0.05)at 1,3,5,7d after graft reperfusion.The hepatic tissue mRNA and protein expression of Fas,Perforin and granzyme B in group ofⅢa were higher than group ofⅣa(P＜0.05), group ofⅢ_b were higher than group ofⅣ_b(P＜0.05)at 1,3,5,7d after graft reperfusion. Conclusion:(1)Ischemic preconditioning can relieve the degree of ischemia reperfusion injury and degree of acute rejection because of the same cold and warm ischemia time.(2)Ischemic preconditioning can relieve the degree of acute rejection.It was correlated with restraining apoptosis and restraining mRNA and protein expression of Fas,Perforin and granzyme B.