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INHIBITION OF APOPTOSIS IN HUMAN ISLETS ISOLATED FROM PANCREAS STORED IN PERFLUOROCARBONS.
S. Ramachandran, Ph.D, N. Benshoff, B. Olack, N. Desai, M.D, M. Jendrisak, M.D and T. Mohanakumar, Ph.D. St. Louis MO, USA, Wash. Univ School of Med, 63110, Surgery.

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Introduction: Enzymatic digestion and cold storage of pancreas prior to islet isolation results in cell death by apoptosis. Use of two layer UW/PFC (perfluorocarbon) immediately after procurement or after cold storage of pancreas has been shown to increase the quantity and quality of islets. The objective was to study islet apoptosis following isolation and methods to inhibit.
Methods: Islets were isolated from 3 groups of pancreata with varying preserving conditions(1. immediately on UW/PFC, 2. stored 12 h. in UW followed by storage in UW/PFC or 3.in UW alone). Donors were administered N-acetyl cysteine (NAC), an antioxidant, prior to pancreas harvest to study its effect on apoptosis. Pro- and anti-apoptotic gene expression profile was analyzed byApoptotic gene array. Caspase (CS) activation was profiled using Caspase profiling kit. Expression of Bcl2, BAD, pSurvivin, cytochrome (CY) B/C, Apaf1 and AIF1 were analyzed by western blot using specific antibodies.
Results: A 3 fold decrease in bad, bax, CS and the TRAF and significant reduction in activated CS-2 (50%), CS-3 (7%) and CS-8 (100%) levels were observed in islets from pancreas immediately preserved in PFC. Preservation in PFC even after 12 h. of CIT down regulated pro-apoptotic genes and inhibited CS. Significant reduction in CY-C release (65%) was observed in islets stored in PFC. Administration of NAC, further decreased levels of pro-apoptotic genes in islets.
Conclusion: Isolated islets undergo apoptosis through mitochondrial pathway. The reported improvement in islet function following PFC preservation or by administration of NAC could be due to improvements in redox potential and significant up-regulation of anti-apoptotic and inhibition of pro-apoptotic genes.