Pancreas preservation by the 2-layer cold storage method before islet isolation protects isolated islets against apoptosis through the mitochondrial pathway

Abstract

Background Apoptosis in isolated islets has been implicated in primary nonfunction or early graft failure after islet transplantation. Recently, pancreas preservation by the 2-layer method (TLM) before islet isolation has been proved to improve the islet yield, quality, and transplant results not only in experimental models, but also in clinical settings. We examined the influence of TLM on apoptosis of isolated islets. Method Rat islets freshly isolated and after pancreas preservation by TLM or conventional cold storage in University of Wisconsin solution (UW) were examined and compared. Islet apoptosis was assessed by TUNEL and annexin V assays. The apoptosis pathways involved were investigated by measurement of caspase 3, 8, and 9 activities and by immunoblotting for total and phosphorylated c-Jun NH2-terminal kinase (JNK) and p38. Results Islet apoptosis in the UW group was significantly increased compared with the fresh and TLM groups. Both caspase 3 and 9 activities in the UW group were higher than in the fresh and TLM groups with an approximate increase of 2- to 3-fold. On the other hand, there was no significant difference in caspase 8 activity among these 3 groups. JNKs were strongly activated both in the TLM and UW groups; although they were not activated in the fresh group, p38 was activated to almost the same levels in these 3 groups. Conclusions Pancreas preservation by TLM before islet isolation protects isolated islets against apoptosis mainly through the mitochondrial pathway. Pancreas storage before islet isolation even with TLM triggers activation of JNKs in isolated islets.