Functional uncoupling of perivascular nitric oxide production following neonatal hypoxia/ischemia
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Background and Aims: Compromise of cerebral vascular function due to a loss of vascular reactivity may be a cause of cerebral injury after hypoxia-ischemia. A phenomenon that has gained increasing attention as a possible determinant of blood flow and a cause of vascular injury under pathological conditions is the uncoupling of endothelial cell nitric oxide synthase (eNOS, NOS3), which reduces NO production and results in the production of superoxide anion (O2-) by the enzyme. There have been few direct assessments of this phenomenon in the CNS, although eNOS coupling appears to be developmentally regulated in the neonate in other vascular beds. Using a histofluorescence method, we characterized acute changes in cerebral blood flow, vascular O2- anion, and nitric oxide (NO) in neonatal rat cerebral hypoxic/ischemic injury and their response to pharmacological manipulation and the eNOS substrate, tetrahydrobiopterin (BH4). Methods: Neonatal P7 rats were subjected to cerebral hypoxia ischemia by unilateral carotid ligation followed by hypoxia with hyperoxic resuscitation. Laser Doppler flowmetry was used to monitor CBF, and cerebral vascular wall NO and O2- were determined using i.p.-injected fluorescent dyes (DAF-2AC and DHE respectively) and stimulation with acetylcholine, assessed with fluorescence microscopy. The effect of the NADPH inhibitor apocyanin and the NOS inhibitor, L-NAME, injected i.p. at the onset of resuscitation on fluorescence patterns, and the functional effect of tetrahydrobiopterin supplementation were determined. Results: We found a progressive decline in perivascular NO production and an increase in perivascular O2- in the hours following hyperoxic resuscitation (Figure: ratio between ischemic/contralateral hemisphere for DAF-2AC and DHE fluorescence; p=0.01 at 24 hours). Reperfusion CBF declined by up to 30% in the first 3 hours relative to baseline. The ratio between perivascular O2- and NO fluorescence was restored by both apocyanin and L-NAME. Administration of tetrahydrobiopterin increased vascular wall NO, reduced vascular wall superoxide anion levels (Figure), and normalized CBF. Conclusions: These results demonstrate vascular dysfunction follows hyperoxic resuscitation after neonatal hypoxic/ischemic injury manifested by reduced CBF, a reduction in perivascular production of NO and an increase in O2-. Inhibitor studies implicate both NADPH oxidase and NOS and indicate these enzymes functionally disrupt vascular function. That tetrahydrobiopterin normalized these abnormalities suggests a possible role of substrate oxidation leading to uncoupling of eNOS due to tetrahydobiopterin deficiency with resultant implications for propagation of injury.