EARLY ASSESSMENT OF APOPTOSIS IN ISOLATED ISLETS OF LANGERHANS1

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Abstract
There is substantial evidence to link early graft loss after islet transplantation to isolation-induced islet cell apoptosis. Measurement of caspase 3 activity and detection of the lost cell membrane asymmetry, revealed by annexin V binding, are newly available assays that allow the analysis of early events of apoptosis. In this study, we compared these tests with the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay and analysis of DNA fragmentation after gel electrophoresis in freshly isolated islets obtained from rats, before and after treatment with interleukin-1 β, interferon γ, and tumor necrosis factor α, cytokines known to induce islet cell damage. A measurable level of apoptosis was observed the day after isolation when caspase 3 activity and annexin V binding were used as assays, although no substantial DNA fragmentation was detected with TUNEL assay and DNA gel electrophoresis. Baseline caspase 3 activity was 0.8±0.3 U/100 islet equivalents and it increased to 1.4±0.45 U/100 islet equivalents 3 hr after cytokine stimulation (P <0.05 vs. unstimulated islets). The baseline level of apoptosis, as detected by annexin V binding, was 21.1%±5.8%, and it increased to 27.5%±8.1% 6 hr after addition of the cytokine cocktail (P <0.01 vs. unstimulated islets). An increase in the number of TUNEL-positive nuclei was detected 24 hr after stimulation and peaked at 48 hr. DNA laddering was also evident 24 hr after cytokine treatment. These data suggest that measurement of caspase 3 activity and annexin V binding analysis might represent reliable markers of early events of islet cell apoptosis.