Sustained Hypoxia-Ischemia Results in Production of Reactive Nitrogen Species in the Premature Fetal Rabbit Brain † 1154

Since nitric oxide is an important neurotransmitter in the brain, we hypothesized that reactive nitrogen species derived from nitric oxide may be formed in premature fetal rabbit brain following acute hypoxia-ischemia. The reaction of nitric oxide-derived species, such as peroxynitrite, with proteins results in the nitration of tyrosine residues to form 3-nitrotyrosine. An animal model with acute placental insufficiency from uterine ischemia was utilized for the present study. Pregnant rabbits, 29 day gestation (88-94% term), were randomized to either sustained uterine ischemia for 50 min(Hypoxia) or no ischemia (Controls). Immediately following uterine ischemia, a hysterotomy was performed and fetal brains obtained. Brain homogenate was processed and the processed protein solution was immunoprecipitated with Sepharose beads and polyclonal anti-nitrotyrosine antibody. Nitrotyrosine-containing proteins were then detected by Western Blot analysis with a mouse monoclonal anti-nitrotyrosine antibody. Nitration of tyrosine was detected only in hypoxic brains compared to the absence of nitration in control brains (n=3 in each group). As a control for nitrotyrosine specificity, the monoclonal antibody was pre-incubated with nitrotyrosine, which completely blocked immunoblot staining. Brain homogenate treated with peroxynitrite had extensive nitration of protein and served as positive controls. These positive control-stains were also almost completely blocked by pre-incubation with nitrotyrosine. These findings and the previously reported increase in nitrogen dioxides in hypoxic brains strongly suggest the formation of nitric oxide derived species. Thus, sustained hypoxia-ischemia results in the increased formation of reactive nitrogen species in premature fetal brains, which may include increased formation of the potent radical, peroxynitrite. It is speculated that reactive nitrogen species may contribute to free radical-mediated injury following acute hypoxia-ischemia in clinical situations of acute placental insufficiency.