Study Of Ischemia-reperfusion Injury In Miniature Swine Pancreas Transplantation

Background and ObjectiveAlthough simultaneous pancreas-kidney transplantation (SPK) has now became the standard procedure in the treatment of the typeⅠdiabetic recipients with end-stage renal disease and achieved normoglycemia in the majority of them, graft pancreatitis remains a important problem which incidence rate is about 35%. It has also been confirmed that the recovery of pancreas function, the occurrence of rejection, infection and thrombosis were all related to ischemia-reperfuson injury. The pathophysiologic mechanisms of ischemia-reperfusion injury is complicated and involved lots of factors. Recent studies have demonstrated apoptosis could be induced by ischemia-reperfusion injury. Now, to investigate the endogenetic protection of grafts is a new hotpot in transplantation and heat shock proteins (HSP) are one of the most interested proteins which will be up-regulated by various stress events including ischemia-reperfusion injury and fulfill their function of molecular chaperones, even more, antiapoptosis. By establishing a model of miniature swine pancreas transplantation, we sought to characterize the pattern of ischemia-reperfusion injury on the pancreas and clarify the role of apoptosis associated with it, as well as investigate the expression of HSP70 during ischemia-reperfusion injury and determine whether up-expression of HSP70 could prevent apoptosis of pancreatic cells, thus to provide basic and necessary experience for preventing ischemia-reperfusion injury in the clinical pancreas transplantation.Materials and Methods Experiments of heterotopic pancreaticoduodenal allotransplantation with enteric drainage were performed in 20 miniature swine under general anaesthesia with ketamine and etomidate. The grafts were perfused in situ with 6% HAES-Sterial solution and stored for about 2 hours in this solution at 4°C. Biopsies were taken from the pancreatic tail before perfusion in situ, after pancreas removal, 2 hours after cold ischemia and 1 hour after reperfusion. Target parameters were measured, including apoptosis (detected by TUNEL and DNA Ladder), morphological damage (examined by light and transmission electronmicroscopy), immunohistochemistry (for HSP70, Bcl-2 and Bax protein), double staining of apoptosis and HSP70. Blood samples were taken exactly 0 min, 30 min, and 1 hour after reperfusion for measuring the serum amylase and lipase. The results of immunohistochemistry were assessed by optical density (OD) and apoptosis was quantified by apoptosis index (AI). The methods of statistics adopted were analysis of variance of repeated data and t test. In addition, the analysis of partial correlation between HSP70 expression and AI was also performed.Results ⑴Of the 20 miniature swine recipients studied, there were 18 cases survived exceeding 1 day and 9 cases survived exceeding 5 days. The successful operation rate was 90%; ⑵The levels of serum amylase and lipase significantly increased 30 min, 1 hour after reperfusion (P<0.01); ⑶DNA breaks of pancreatic cells were detected by TUNEL method and agarose gel electrophoresis. Strikingly increasing numbers of apoptotic cells quantified by AI can be seen, after pancreas removal, 2 hours after cold ischemia, 1 hour after reperfusion versus before perfusion in situ (P<0.01). Agarose gel electrophoresis showed typical pattern of DNA ladder, especially the DNA obtained from the pancreas 1 hour after reperfusion; ⑷Continuous morphological alterations were observed on all time points checked. The tissue damage scores were significantly higher after pancreas removal, 2 hours after cold ischemia and 1 hour after reperfusion than before perfusion in situ (P<0.01). The pancreatic tissue obtained 1 hour afer reperfusion manifested apparent ischemia-reperfusion injury characterized by interstitial or cell edema, sparse leukocyte infiltration and scattered hemorrhage. The alterations of ultrastructure were conformed with the histopathology and typical appearance of apoptosis can be seen; ⑸Immunohistochemistry for HSP70 showed ob...