Hypoxia-induced generation of nitric oxide free radicals in cerebral cortex of newborn guinea pigs

Previous studies have shown that brain tissue hypoxia results in increased N-methyl-D-aspartate (NMDA) receptor activation and receptor-mediated increase in intracellular calcium which may activate Ca++-dependent nitric oxide synthase (NOS). The present study tested the hypothesis that tissue hypoxia will induce generation of nitric oxide (NO) free radicals in cerebral cortex of newborn guinea pigs. Nitric oxide free radical generation was assayed by electron spin resonance (ESR) spectroscopy. Ten newborn guinea pigs were assigned to either normoxic (FiO(2) = 21%, n = 5) or hypoxic (FiO(2) = 7%, n = 5) groups. Prior to exposure, animals were injected subcutaneously with the spin trapping agents diethyldithiocarbamate (DETC, 400 mg/kg), FeSO(4.)7H(2)O (40 mg/kg) and sodium citrate (200mg/kg). Pretreated animals were exposed to either 21% or 7% oxygen for 60 min. Cortical tissue was obtained, homogenized and the spin adducts extracted. The difference of spectra between 2.047 and 2.027 gauss represents production of NO free radical. In hypoxic animals, there was a difference (16.75 +/- 1.70 mm/g dry brain tissue) between the spectra of NO spin adducts identifying a significant increase in NO free radical production. In the normoxic animals, however, there was no difference between the two spectra. We conclude that hypoxia results in Ca2+- dependent NOS mediated increase in NO free radical production in the cerebral cortex of newborn guinea pigs. Since NO free radicals produce peroxynitrite in presence of superoxide radicals that are abundant in the hypoxic tissue, we speculate that hypoxia-induced generation of NO free radical will lead to nitration of a number of cerebral proteins including the NMDA receptor, a potential mechanism of hypoxia-induced modification of the NMDA receptor resulting in neuronal injury.