Abstracts

Rationale: Studies have shown that nitric oxide (NO) plays an important role in neuronal injury following hypoxia-ischemia in the immature brain. Neuronal NO synthase (nNOS) is the most abundant NOS isoform and plays a central role in NO production during a hypoxic-ischemic insult. Pharmacological inhibition of nNOS has been shown decrease hypoxia-ischemia induced brain injury however; the role of nNOS-mediated NO production in hypoxia-induced seizures is unclear. Therefore the goal of this study is to test the hypothesis that inhibition of nNOS during hypoxia-reoxygenation alters seizure generation in the neonatal mouse. Methods: To test this hypothesis, neonatal mice were exposed to acute hypoxia (4% FiO₂ for 4 min and 30 sec) and hypoxic and post-hypoxic seizures were observed using video-EEG. Results in wild-type mice were compared to those obtained in nNOS-/- mice. In addition, 7-nitroindazole (7-NI) was used to pharmacologically inhibit nNOS and was given by IP injection 30 min prior to hypoxia onset. Finally, vehicle (DMSO)-treated animals were also used as controls. For all groups, EEG data were obtained by stereotaxically implanting 4 monopolar insulated stainless steel electrodes (2 in the ventral hippocampus and 2 in the cortex) in postnatal day 7-8 anesthetized C57BL/6 neonatal mice. Pups were monitored via video-EEG throughout the procedure including baseline, hypoxia and reoxygenation. Electrographic seizures were defined as the appearance of high frequency (>2Hz) rhythmic sharp wave discharges with amplitude at least 3 times that of baseline and lasting greater than 10 sec with clear evolution Results: Hypoxic seizures were observed in 70% of control mice (n=17) and 71% of vehicle (DMSO)-treated mice (n=7). Animals pre-treated with 7-NI (n=7) and nNOS -/- mice (n=12) had a similar rate of hypoxic seizures: 67% and 92%, respectively (P = NS, chi-square). Further, there was no difference between the 4 groups in the incidence of seizures observed during the post-hypoxic period. Following reoxygenation, seizures were observed in 59% of control mice; 57% of vehicle-treated mice, 43% of 7-NI treated mice and 58% of nNOS -/- mice (P = NS, chi-square). Conclusions: Pharmacologic inhibition of nNOS does not protect against acute hypoxic and post-hypoxic seizures in the neonatal mouse. Further nNOS-/- status does not reduce acute hypoxic seizures in fact, hypoxic seizures appear to be increased in this group. Based on these observations, we conclude that the reported nNOS-mediated decrease in hypoxia-induced brain injury in immature animals is not mediated via an effect on hypoxia-induced seizures.