Effect of nitric oxide synthase inhibition during post-hypoxic reoxygenation on Bax and Bcl-2 protein expression and DNA fragmentation in neuronal nuclei of newborn piglets

Previous studies have shown that cerebral tissue hypoxia results in increased generation of oxygen-free radicals including nitric oxide (NO), expression of the proapoptotic protein Bax and fragmentation of nuclear DNA. The present study tests the hypothesis that post-hypoxic reoxygenation for 6 h following hypoxia (FiO2 = 0.06 for 1 h) results in continued hypoxia-induced, NO-mediated expression of the Bax protein and nuclear DNA fragmentation in the cerebral cortex of newborn piglets. Piglets were divided into normoxic (Nx), hypoxic (Hx, FiO2 = 0.06 for 1 h), hypoxic with 6 h reoxygenation (Hx + reox) and hypoxic with 6 h reoxygenation injected with 7-nitroindazole sodium salt (7-NINA), a selective nNOS inhibitor, immediately after hypoxia (Hx + 7-NINA). Cerebral tissue hypoxia was documented by levels of ATP and phosphocreatine (PCr). Bax and Bcl-2 were analyzed by Western blot and DNA fragmentation was determined by agarose gel electrophoresis. ATP and PCr values in Hx, Hx + reox and Hx + 7-NINA were significantly different from Nx (P < 0.05 vs. Nx). Bax protein (OD × mm2) was 128.9 ± 38.7 in Nx; 223.6 ± 45.8 in Hx (P < 0.05 vs. Nx); 340.5 ± 73.2 in Hx + reox (P < 0.05 vs. Nx, Hx and Hx + 7-NINA); and 202.2 ± 34.8 in Hx + 7-NINA (P = NS vs. Hx). Bcl-2 protein (OD × mm2) was 14.9 ± 2.7 in Nx, 12.4 ± 2.1 in Hx, (P < 0.05 vs. Nx), 15.7 ± 3.8 in Hx + reox, (P < 0.05 vs. Hx) and 13.1 ± 2.2 in Hx + 7-NINA (P = NS among groups). Nuclear DNA fragmentation (OD × mm2) was 147 ± 15 in Nx; 797 ± 84 in Hx (P < 0.05 vs. Nx); 1134 ± 127 in Hx + reox (P < 0.05 vs. Nx, Hx and Hx + 7-NINA); and 778 ± 146 in Hx + 7-NINA (P = NS vs. Hx, P < 0.05 vs. Hx + reox). The results show that post-hypoxic reoxygenation results in increased expression of Bax protein without affecting Bcl-2 protein and increased fragmentation of nuclear DNA, which are prevented by 7-NINA. We conclude that during post-hypoxic reoxygenation the increase in Bax protein expression and fragmentation of nuclear DNA are mediated by NO derived from nNOS. We propose that in addition to NO-mediated nuclear DNA damage, the hypoxia-induced increased ratio of Bax/Bcl-2 protein will lead to caspase-activated cascade of hypoxic neuronal death during post-hypoxic reoxygenation.