Cerebral Hypoperfusion Increases Neuronal Nitric Oxide Synthase Abundance in the Ovine Fetal Brainstem

Nitric oxide (NO) is potent vasorelaxant in vascular smooth muscle and serves as a neurotransmitter in the brainstem, influencing the neural pathways that control cardiovascular function. Hypotension, and the resultant cerebral hypoperfusion, stimulates a series of cellular responses that modify the reflex responses to subsequent periods of hypotension. This study was performed to test the influence of brachiocephalic occlusion (BCO) on the abundance of nNOS in the fetal brainstem. Fifteen chronically catheterized, late gestation fetal sheep were subjected to a 10 minute period of cerebral hypoperfusion followed by reperfusion. Fetuses were euthanized 0, 0.5, 3, 8, and 18 hours after the onset of BCO (n=3 at each timepoint). Brain regions were rapidly dissected, snap‐frozen, and later processed for isolation of protein for Western Blot analysis. We tested for an upregulation in fetal brainstem nNOS abundance in response to brachiocephalic occlusion using anti‐nNOS antiserum (Transduction Labs) and anti‐β‐actin (Sigma) for normalization of protein loading. After normalization with β‐actin, nNOS protein abundance appeared to increase 3 hours after the onset of a 10‐min period of BCO. When analyzed by one‐way ANOVA, the apparent increase was not statistically significant (p=0.079). Analysis of a smaller data set, in which the 18 hour values were excluded yielded a statistically significant change (p=0.022). Analysis of the individual means by Bonferroni test revealed that the nNOS abundance was greater at 3 hours than at earlier or later times. We conclude that nNOS abundance is increased in fetal brainstem after a period of cerebral hypoperfusion, and we speculate that increased nNOS activity increases brainstem NO responses to subsequent periods of hypotension.