Neuroprotection by Selective Nitric Oxide Synthase Inhibition at 24 Hours After Perinatal Hypoxia-Ischemia

Abstract

Background and Purpose-Perinatal hypoxia-ischemia is a major cause of neonatal morbidity and mortality. Until now no established neuroprotective intervention after perinatal hypoxia-ischemia has been available. The delay in cell death after perinatal hypoxia-ischemia creates possibilities for therapeutic intervention after the initial insult. Excessive nitric oxide and reactive oxygen species generated on hypoxia-ischemia and reperfusion play a key role in the neurotoxic cascade. The present study examines the neuroprotective properties of neuronal and inducible but not endothelial nitric oxide synthase inhibition by 2-iminobiotin in a piglet model of perinatal hypoxia-ischemia. Methods-Twenty-three newborn piglets were subjected to 60 minutes of hypoxia-ischemia, followed by 24 hours of reperfusion and reoxygenation. Five additional piglets served as sham-operated controls. On reperfusion, piglets were randomly treated with either vehicle (nϭ12) or 2-iminobiotin (nϭ11). At 24 hours after hypoxia-ischemia, the cerebral energy state, presence of vasogenic edema, amount of apparently normal neuronal cells, caspase-3 activity, amount of terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end labeling (TUNEL)-positive cells, and degree of tyrosine nitration were assessed. Results-A 90% improvement in cerebral energy state, 90% reduction in vasogenic edema, and 60% to 80% reduction in apoptosis-related neuronal cell death were demonstrated in 2-iminobiotin-treated piglets at 24 hours after hypoxiaischemia. A significant reduction in tyrosine nitration in the cerebral cortex was observed in 2-iminobiotin-treated piglets, indicating decreased formation of reactive nitrogen species. Conclusions-Simultaneous and selective inhibition of neuronal and inducible nitric oxide synthase by 2-iminobiotin is a promising strategy for neuroprotection after perinatal hypoxia-ischemia.

Pathological apoptosis in the developing brain

“…Elimination of nNOS neurons through injections of quisqualic acid into the cortex of P7 rats rendered these animals resistant to HI [195], and nNOS deficiency through genetic targeting was also neuroprotective [196]. Administration of the combined iNOS/nNOS inhibitor 2-iminobiotin reduced caspase-3 activation [197] and conferred tissue protection [198]. The iNOS inhibitor aminoguanidine [199] (IL-1) also reduced brain injury after HI, further indicating that NO production exerts cytotoxic effects in the developing brain such insults.…”

Pathophysiological role of inflammatory molecules in paediatric ischaemic brain injury

“…The development of novel animal models of childhood stroke and the optimization of cerebral perinatal HI and MCAO models appear as the most important approaches to pursue this issue. As the majority of basic research studies have been focused on inflammation in perinatal HI model, a stronger scientific effort should be N-acetylcysteine Oxygen radical scavenger Reduction in cerebral oxidative stress and cerebral injury; improvement in myelin expression and neurological outcome in neonatal rats (combined with hypothermia) (HI model) [83] Melatonin Oxygen radical scavenger Reduction in white matter damage and promotion of repair by induction of axonal regrowth or sprouting in newborn mice (ibotenate-mediated excitotoxic brain injury) [84] Decrease in oxidative damage mediators in 7-day-old rats (HI model) [85] 2-iminobiotin Selective nNOS and iNOS inhibitor Improvement in cerebral energy state, reduction in vasogenic oedema and neuronal death (HI model) [60] 7-nitroindazole Selective nNOS inhibitor Suppression of both two peaks of NO metabolites (during hypoxia and during the re-oxygenation period) (HI model) [82] Aminoguanidine Selective iNOS inhibitor Suppression only the NO metabolites peak in the re-oxygenation period. Reduction in cerebral injury in a neonatal rats (HI model) [61,62] DETANONOate (Z)-1-[N-(2-amino-ethyl)-N-(2-ammonio-ethyl)amino] diazen-1-ium-1,2-diolate NO donor Reduction in neuropathologic injury score in newborn rats (HI model) [79] Hypothermia Nonselective anti-inflammatory effects Improvement in survival and neurologic outcomes in human neonates [89][90][91] More benefit in combined pharmacological hypothermia treatment (HI model) [92,93] Simvastatin Nonselective antioxidant and anti-inflammatory effects Reduction in cerebral damage in neonatal rats (HI model) [94] *HI, Hypoxia-ischaemia.…”

“…In 7-day-oldrats, HIprotocol up regulated themRNAexpressionofseveralchemokines(suchasmacrophage [60]. Increase frequency of polymorphisms )308 G ⁄ A in children with sickle-cell anaemia and stroke [61] Up regulation in ischaemic brain in perinatal rats (HI model) [35].…”

“…Nitric oxide (NO) is a free radical gas, mainly mediating neuroprotection (when produced by endothelial nitric oxide synthase [eNOS]) or neurotoxic effects (when produced by neuronal [nNOS] and inducible NOS [iNOS]) [59][60][61][62]. In particular, both genetic and pharmacological inhibition of iNOS reduced cerebral injury in HI animal protocols [59][60][61][62].…”

“…In particular, both genetic and pharmacological inhibition of iNOS reduced cerebral injury in HI animal protocols [59][60][61][62]. Moreover, NO produced by iNOS has been shown to increase the activity of cyclooxygenase-2 (COX-2, an enzyme expressed in neurons at the border of ischaemic regions) in an adult rat model of transient MCAO [63].…”