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.

The hypothesis was tested that treatment with allopurinol, a xanthine oxidase inhibitor, or deferoxamine, a chelator of nonprotein-bound iron, preserved cerebral energy metabolism, attenuated development of edema, and improved histologic outcome in the newborn piglet at 24 h after hypoxia-ischemia. Thirty-two newborn piglets were subjected to 1 h of hypoxia-ischemia by occluding both carotid arteries and reducing the fraction of inspired oxygen; five newborn piglets served as sham-operated controls. The depth of hypoxia-ischemia was controlled by phosphorous magnetic resonance spectroscopy. Upon reperfusion and reoxygenation, piglets received vehicle (n ϭ 12), allopurinol (30 mg/kg/d, n ϭ 10), or deferoxamine (12.5 mg/kg/d, n ϭ 10). The cerebral energy status was determined with phosphorous magnetic resonance spectroscopy. The presence of vasogenic edema was assessed by T2-weighted magnetic resonance imaging. Brain cell injury was assessed with caspase-3 activity, histology, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick end (TUNEL)-labeling. At 24 h after hypoxia-ischemia, the phosphocreatine/inorganic phosphate ratios were significantly decreased in vehicle-treated, but not in allopurinol-or deferoxamine-treated piglets. Water T2 values were significantly increased at 24 h after hypoxia-ischemia in cerebral cortex, thalamus, and striatum of vehicle-treated piglets, but not in allopurinol-and deferoxamine-treated piglets. No differences in caspase-3 activity, histologic outcome, or TUNEL-labeling were demonstrated between the three treatment groups. We suggest that allopurinol and deferoxamine may have an additional value in the treatment of perinatal hypoxia-ischemia with other neuroprotective agents or in combination with hypothermia. The neonatal brain appears to be vulnerable to oxidative stress after perinatal hypoxia-ischemia with reperfusion and reoxygenation due to excessive free radical production, relatively large amounts of NPBI production, and inadequate scavenging mechanisms to counteract these potentially neurotoxic events (1). During perinatal hypoxia-ischemia and upon reperfusion, a biochemical cascade occurs, including modification of the NMDA receptor-ion channel complex, leading to increased intracellular Ca 2ϩ and resulting in the conversion of xanthine dehydrogenase to xanthine oxidase (2). During the primary hypoxic-ischemic insult, ADP is degraded to hypoxanthine and then oxidated to xanthine and on to uric acid by xanthine oxidase upon reperfusion [for review, see Fellman and Raivio (3)]. During these reactions, superoxide and hydrogen peroxide are formed, which can be converted to the highly reactive hydroxyl radical through the Haber-Weiss reaction, catalyzed by ferrous iron (4). The reactive oxygen species Received October 28, 2002; accepted April 11, 2003

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