Exogenous low dose hydrogen peroxide increases hypoxia-inducible factor-1alpha protein expression and induces preconditioning protection against ischemia in primary cortical neurons

HIF-1 is believed to play a critical role in hypoxia/ischemia (H/I) preconditioning protection in neonatal brain. Recently, it has been shown that hydrogen peroxide (H2O2) may contribute to H/I preconditioning in rat primary neurons. We hypothesize that H2O2 produced during H/I preconditioning may increase HIF-1α protein expression and contribute to H/I preconditioning protection in the immature brain. To test this hypothesis, we used 6-8 days in vitro (DIV) primary cortical neurons from embryonic day 16 CD1 mouse brains and preconditioned them with 10 min of oxygen and glucose deprivation (OGD) or exogenous H2O2 at doses from 5 to 50 μM. Both OGD and low dose H2O2 (15 μM) preconditioning provided neuronal protection 24 h later against a 2 h OGD insult. Cell survival was 34.9 ± 1.8% and 35.8 ± 3.8% with OGD and H2O2 preconditioning respectively vs. 20.0 ± 0.4% without preconditioning (P < 0.01). After OGD preconditioning, HIF-1α protein increased at 4 h and peaked at 8 h, then declined at 18 h and increased again to reach another peak at 32 h. HIF-1α protein following H2O2 preconditioning increased at 8 h and peaked at 32 h. For both preconditioning paradigms, HIF-1α expression level declined to baseline at 72 h. Our results suggest that low levels of H2O2 may up-regulate HIF-1α protein and thereby mediate H/I preconditioning protection.