HYPOXIC PRECONDITIONING AMELIORATES ISCHEMIA-INDUCED DAMAGE OF ISOLATED RAT ISLETS

Abstract

Aim: Islet isolation results in disruption of the native islet vascular network until posttransplant islet revascularisation is completed. Prolonged exposure to a hypoxic and nutrient-depleted environment has detrimental effects on morphological and functional integrity of isolated islets. In solid organ transplantation it was shown that short sublethal periods of anoxia can induce adaptive mechanisms in the affected tissue offering protection against subsequent potentially lethal hypoxic insults. This study was aimed to explore the potential of hypoxic preconditioning for isolated islets. Methods: Rat islets, isolated from 20 non-heartbeating donors, were subjected either to 2 x 2 min-periods of anoxia, intermitted by a 2 min-period of oxygen supply, or to sham-treatment. Hypoxic preconditioning and sham-treatment were followed by 4 hours of recovery in normoxia prior to 24 hr-culture at 1.5% oxygen. Islet characterisation included yield of islet equivalents (IEQ) or islet particle number (IN), viability (FDA-PI), DNA (pico-green) and ATP (luciferase assay) content, intraislet reactive oxygen species (ROS) production (DCFH-DA), gene expression (qRT-PCR), and glucose stimulation index (2 vs 20 vs 2 mM). Data were normalised to islet DNA content and are presented as mean ± SEM. Results: Hypoxic preconditioning had no effect on islet survival when compared with sham-treatment (64 ± 7 vs 61 ± 7%). Compared with preconditioned islets, sham-treated controls were characterised by increased fragmentation when determined as IN/IEQ ratio (1.51 ± 0.18 vs 1.24 ± 0.13, p<0.05). Fragmentation corrrelated inversely with viability of sham-treated and preconditioned islets (61 ± 3 vs 66 ± 3 %, p<0.01). ATP (0.66 ± 0.14 vs 0.73 ± 0.20 pM/ng DNA) and insulin content (51.1 ± 12.5 vs 46.3 ± 11.7 ng/ng) were not altered by hypoxic preconditioning compared with sham-treatment. ROS production was reduced after hypoxic preconditioning compared with sham-treatment (645 ± 73 vs 858 ± 88 AU/ng, p<0.01). Islets of both experimental groups responded with insulin release after glucose challenge, but the stimulation index of preconditioned islets was significantly larger compared with sham-treated islets (2.39 ± 0.37 vs 2.09 ± 0.33, p<0.05). Whilst pro-apoptotic BAX and CHOP gene expression was reduced by 54 ± 15% (p<0.05) and 49 ± 8% (p<0.05), hypoxic preconditioning increased HIF-1 alpha and VEGF-A gene expression by 90 ± 41% (p<0.01) and 184 ± 65% (p<0.05), respectively. Conclusion: Although hypoxic preconditioning could not ameliorate ischaemia-induced loss of islets, islet morphology and function was significantly improved presumably as result of reduced intraislet ROS production. qRT-PCR-determined gene expression suggests that hypoxic preconditioning induces rescue mechanisms islets and mediates a significant anti-apoptotic effect. This study describes a new option for pretransplant treatment of isolated human islets. European Union’s Cooperation Programme FP7 (HEALTH-F2-2012-305746). Oxford NIHR Biomedical Research Centre.