Interleukin-1 (IL-1) converting enzyme (ICE) is a cysteine protease that cleaves inactive pro-IL-1beta to active IL-1beta. The pro-inflammatory cytokine IL-1beta is implicated as a mediator of hypoxic-ischemic (HI) brain injury, both in experimental models and in humans. ICE is a member of a family of ICE-like proteases (caspases) that mediate apoptotic cell death in diverse tissues. The authors hypothesized that in neonatal mice with a homozygous deletion of ICE (ICE-KO) the severity of brain injury elicited by a focal cerebral HI insult would be reduced, relative to wild-type mice. Paired litters of 9- to 10-day-old ICE-KO and wild-type mice underwent right carotid ligation, followed by 70 or 120 minutes of exposure to 10% O2. In this neonatal model of transient focal cerebral ischemia followed by reperfusion, the duration of hypoxia exposure determines the duration of cerebral ischemia and the severity of tissue damage. Outcome was evaluated 5 or 21 days after lesioning; severity of injury was quantified by morphometric estimation of bilateral cortical, striatal, and dorsal hippocampal volumes. In animals that underwent the moderate HI insult (70-minute hypoxia), damage was attenuated in ICE-KO mice, when evaluated at 5 or 21 days post-lesioning. In contrast, in mice that underwent the more severe HI insult (120-minute hypoxia), injury severity was the same in both groups. Reductions in intra-HI CBF, measured by laser Doppler flow-metry, and intra- and post-HI temperatures did not differ between groups. These results show that ICE activity contributes to the progression of neonatal HI brain injury in this model. Whether these deleterious effects are mediated by pro-inflammatory actions of IL-1beta and/or by pro-apoptotic mechanisms is an important question for future studies.

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Platelet-activating factor (PAF) is overproduced in ischemic brain. Although postischemic PAF antagonist administration protects the mature brain in some models, little is known about the effects of PAF antagonists in the immature brain. We hypothesized that the PAF antagonist BN 52021 would attenuate perinatal cerebral hypoxic-ischemic injury. To elicit focal hypoxic-ischemic brain injury, 7-d-old (P7) rats (n = 111) underwent right carotid ligation, followed by 2.5-3.25 h of hypoxia (fractional concentration of inspired O2 = 0.08). BN 52021 neuroprotection was evaluated in three groups of experiments: 1) 25 mg/kg/dose, 0 and 2 h posthypoxia; 2), 25 mg/kg/dose immediately before and 1 h after hypoxia; and 3) posthypoxia-ischemia treatment with BN 52021 12.5, 25, or 50 mg/kg/dose in 2 doses 0 and 2 h after hypoxia. All experiments included concurrent vehicle-injected controls. To quantitate severity of injury, bilateral regional cross-sectional areas (groups 1 and 2) or hemisphere weights (group 3) were evaluated on P12. Both pre- and posthypoxic treatment with BN 52021 (25 mg/kg/dose, two serial doses) decreased the incidence of cerebral infarction from 90% to about 30% (p < 0.02, Fisher's exact test). Measurement of cross-sectional areas confirmed neuroprotection and indicated some benefit of pre- over posthypoxic-ischemic treatment in hippocampus and cortex. Over the dose range tested, the neuroprotective effect of BN 52021 administration was not dose-dependent. In contrast, BN 52021 did not attenuate N-methyl-D-aspartate-induced hippocampal excitotoxic injury in P7 rats. Either prophylactic or "rescue" administration of PAF antagonists decreases the incidence and severity of brain injury associated with an episode of perinatal cerebral hypoxia-ischemia.

Inflammatory mediators are implicated in the pathogenesis of ischemic injury in immature brain. The phosphodiesterase inhibitor pentoxifylline inhibits production of tumor necrosis factor-alpha and platelet-activating factor. We hypothesized that pentoxifylline treatment would attenuate hypoxic-ischemic brain injury in immature rats. Seven-day-old rats (n = 79) underwent right carotid ligation, followed by hypoxia (FiO2 = 0.08). Rats received pentoxifylline immediately before and again after hypoxia (two doses, 25-150 mg/kg/dose, n = 34), or vehicle (n = 27). In separate experiments, rats received pentoxifylline treatment (40 mg/kg/dose, n = 8), or vehicle (n = 10) immediately and again 3 h after hypoxia-ischemia. Severity of injury was assessed 5 d later by visual evaluation of ipsilateral hemisphere infarction and by measurement of bilateral hemispheric cross-sectional areas. Pentoxifylline pretreatment reduced the incidence of liquefactive cerebral infarction, from 75% in controls to 10% with pentoxifylline, 40 mg/kg/dose (p<0.001, chi2 trend test). Quantification of hemispheric areas confirmed these findings. In contrast, posthypoxic-ischemic treatment with pentoxifylline resulted in only a modest reduction in cortical damage, without an overall reduction in incidence of infarction. Phosphodiesterase inhibition may be an effective strategy to use to decrease the severity of neonatal hypoxic-ischemic brain injury. Pretreatment regimens could be clinically relevant in settings in which an increased risk of cerebral ischemia can be anticipated, such as in infants undergoing surgery to correct congenital heart disease.

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Platelet-activating factor (PAF) is a lipid derived from breakdown of cell membranes that is postulated to be a mediator of cerebral ischemic injury. PAF regulates CNS gene transcription via intracellular binding sites. To test the hypothesis that PAF mediates CNS injury in part by modulating gene transcription, we evaluated the neuroprotective efficacy of the drug BN 50730, an antagonist of the intracellular (microsomal) CNS PAF binding site, in the neonatal rat model of unilateral cerebral hypoxiaischemia. Seven-day-old rats underwent right carotid ligation followed by a 2.5-h exposure to 8% O 2 , and were then treated with BN 50730 (2.5 or 25 mg/kg per dose) or vehicle, at 0 and 2 h after the end of hypoxia. Ipsilateral cortical, striatal, and hippocampal damage was quantitated either 5 d later, or at 5 wk after the insult. Treatment with BN 50730 resulted in approximately 60 -80% reduction in ipsilateral tissue loss at both times. Learning and memory were evaluated 5 wk after insult using the Morris Watermaze place navigation task. Severity of cortical and striatal damage correlated significantly with learning and memory deficits. These results support the hypothesis that PAF is a pathogenetic mediator of hypoxic-ischemic damage in the immature brain. Accumulating evidence suggests that PAF mediates its deleterious effects in the immature CNS via multiple mechanisms. PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent phospholipid mediator with distinct systemic and CNS actions. In the CNS, PAF is synthesized in neurons and microglia (1, 2), and PAF receptors are expressed predominantly in neurons and microglia and, to a lesser extent, on astrocytes and CNS endothelium (3-5). PAF has distinct neuronal actions, as a synaptic messenger, a transcriptional activator, and a critical determinant of normal brain development (6, 7). In addition to these CNS effects, PAF has potent proinflammatory systemic effects (8). PAF accumulates in ischemic CNS tissue in mature and immature animals (9 -12). In mature animals, most reports indicate that PAF receptor antagonist treatment attenuates cerebral ischemic damage (13-16). Several recent studies implicate PAF as a mediator of ischemic injury in the neonatal brain (12, 17).Brain PAF concentrations rise acutely after neonatal cerebral hypoxia-ischemia (12), most likely as a result of breakdown of membrane phospholipids. Treatment with the PAF receptor antagonist BN 52021 (ginkgolide B), beginning either before or immediately after hypoxia exposure, reduced hypoxicischemic brain injury in postnatal d 7 rats (17). Pretreatment with a Ginkgo biloba extract, which includes BN 52021, attenuated posthypoxic-ischemic PAF accumulation (12). These results suggest that PAF contributes to the pathogenesis of neonatal cerebral hypoxic-ischemic injury.PAF could mediate cerebral hypoxic-ischemic injury via either of two distinct neuronal PAF binding sites. Studies in adult rat cerebral cortex subcellular membrane fractions delineate distinct populations of neuronal PAF bind...