Birth asphyxia can cause moderate to severe brain injury. It is unclear to what degree apoptotic or necrotic mechanisms of cell death account for damage after neonatal hypoxia-ischemia (HI). In a 7-d-old rat HI model, we determined the contributions of apoptosis and necrosis to neuronal injury in adjacent Nisslstained, hematoxylin and eosin-stained, and terminal deoxynucleotidyl transferase-mediated UTP nick end-labeled sections. We found an apoptotic-necrotic continuum in the morphology of injured neurons in all regions examined. Eosinophilic necrotic neurons, typical in adult models, were rarely observed in neonatal HI. Electron microscopic analysis showed "classic" apoptotic and necrotic neurons and "hybrid" cells with intermediate characteristics. The time course of apoptotic injury varied regionally. In CA3, dentate gyrus, medial habenula, and laterodorsal thalamus, the density of apoptotic cells was highest at 24-72 hr after HI and then declined. In contrast, densities remained elevated from 12 hr to 7 d after HI in most cortical areas and in the basal ganglia. Temporal and regional patterns of neuronal death were compared with expression of caspase-3, a cysteine protease involved in the execution phase of apoptosis. Immunocytochemical and Western blot analyses showed increased caspase-3 expression in damaged hemispheres 24 hr to 7 d after HI. A p17 peptide fragment, which results from the proteolytic activation of the caspase-3 precursor, was detected in hippocampus, thalamus, and striatum but not in cerebral cortex. The continued expression of activated caspase-3 and the persistence of cells with an apoptotic morphology for days after HI suggests a prolonged role for apoptosis in neonatal hypoxic ischemic brain injury. Key words: apoptosis; necrosis; hypoxia-ischemia; cysteine proteases; caspase-3 cleavage; cell death continuum; cerebral palsy; newborn brain injury; developmental brainBirth asphyxia can cause cerebral hypoxic ischemic injury, resulting in severe neurological sequelae and death. Survivors of perinatal asphyxia frequently have moderate to severe brain injury for which there currently is no promising therapy (Johnston, 1997). The results of morphological, histochemical, and molecular studies indicate that apoptotic and necrotic mechanisms account for neuronal death after cerebral hypoxia-ischemia (HI) in different neonatal animal models (Mehmet et al., 1994; Charriaut-Marlangue et al., 1996a,b;Chopp and Li, 1996;Macaya, 1996;Yue et al., 1997;Banasiak and Haddad, 1998;Pulera et al., 1998;Renolleau et al., 1998). For example, a neonatal ischemia-reperfusion model showed terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL)-positive nuclei from 4 hr to 30 d after reperfusion (Renolleau et al., 1998). A human study demonstrated apoptotic and necrotic forms of cell death after hypoxic injury, whereas in some brains from stillbirths, only apoptotic figures were observed (Scott and Hegyi, 1997). The form of cell death also may depend on the severity of ischemic injury (Kerr...

Poly(ADP-ribose) polymerase-1 is over-activated in the adult brain in response to ischemia and contributes to neuronal death, but its role in perinatal brain injury remains uncertain. To address this issue, 7-day-old wild-type (wt ) and PARP-1 gene deficient (parp+/-and parp-/-) Sv129/CD-1 hybrid mice were subjected to unilateral hypoxia-ischemia and histologic damage was assessed 10 days later by two evaluators. Poly(ADP-ribose) polymerase-1 knockout produced moderate but significant (p < 0.05) protection in the total group of animals, but analysis by sex revealed that males were strongly protected (p < 0.05) in contrast to females in which there was no significant effect. Separate experiments demonstrated that PARP-1 was activated over 1-24 h in both females and males after the insult in neonatal wt mice and rats using immnocytochemistry and western blotting for poly(ADP-ribose). Brain levels of NAD + were also significantly reduced, but the decrease of NAD + during the early post-hypoxia-ischemia (HI) phase was only seen in males. The results indicate that hypoxia-ischemia activates Poly(ADP-ribose) polymerase-1 in the neonatal brain and that the sex of the animal strongly influences its role in the pathogenesis of brain injury.

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The effect of systemic erythropoietin pretreatment on hypoxic ischemic injury was examined in neonatal mice. Injury was significantly less in cortex, hippocampus, striatum and thalamus of erythropoietin-treated animals (5 U/g vs vehicle) 24 h after hypoxic ischemia and in all of these regions except hippocampus at 7 days. Activated caspase-3- and activated NFkappaB-immunoreactive neurons were observed in the injured areas; these areas were smaller in the erythropoietin group. To our knowledge, this is the first report demonstrating persistent neuroprotective effects of erythropoietin in neonatal mice.

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