Neuronal Type 1 Apoptosis After Unilateral Focal Ischemia With Reperfusion in the P7 Neonatal Rat Djomilo Aggoun-Zouaoui, PhD,Yezekiel Ben-Ari, PhD, and Christiane Charriaut-Marlangue, PhD Apoptotic cell death occurs frequently after cerebral ischemia, although prevailing necrosis could also be observed in older animals. The mechanisms that lead to delayed cell death after hypoxic-ischemic injury in the developing brain are not yet known. With silver staining, we showed increased dying neurons after ischemia and 4 to 24 hours of reperfusion in 7-day-old rats. Using electron microscopic studies, we investigated the presence of chromatin condensation. Injured neurons exhibited 2 different morphological types of apoptosis: Type 1 (aggregation of dense masses of chromatin beneath the intact nuclear membrane) predominately, and Type 2 (cytoplasmic vacuolization). Electron microscopy analysis also showed that the fragmented chromatin and neuronal debris were phagocytosed by surrounding glial cells, and associated with an inflammatory reaction leading to leukocyte infiltration. These results suggest that the immature brain may be more prone to apoptotic death with neuronal loss by neighboring cells. This experimental model may be useful for identifying the biochemical mechanisms that initiate and mediate Type 1 neuronal apoptosis after ischemia and reperfusion in neonate rodents. Key Words: Neuron--Glial cells--Cell death--Mitosis. Cerebral hypoxia-ischemia (HI) remains a major con- tributor to perinatal mortality and long-term neurological morbidity in survivors. 1 Only a few reports have de- scribed the chronological sequence of the morphological changes that occur after an ischemic-hypoxic insult in immature animals. 2,3These studies were performed in the traditional model of neonatal cerebral HI, which subjects 7-day-old rats to unilateral common carotid artery ligation followed by an hypoxic episode of several hours. 4 Results of these studies showed light micro- scopic evidence of irreversible brain injury. The main neuropathologic alterations consisted of spongy neuropil, condensation of nuclear chromatin, and various degrees of clearing of neuronal perikarya. Condensation and microvacuolation of neuronal perikarya were observed From the Universit6 Ren6 Descartes, INSERM U29, Paris, France. Received March 8,1999; accepted May 18,1999. Address reprint requests to Christiane Charriaut-Marlangue, PhD, Laboratoire de Pharmacologie, Facult6 de Pharmacie, 4 avenue de l'Observatoire, 75006Paris, France. Copyright 9 2000by National Stroke Association 1052-3057/00/0901-000253.00/0 only in occasional neurons. These findings are different from those found in adult models of HI, in which large numbers of apoptotic cells can be found 12 to 48 hours after the end of the ischemic injury, as shown by ultrastruc- tural analysis in models of global s and focal6,7 ischemia. The purpose of this study was to obtain ultrastructural microscopic evidence of in situ genomic DNA condensa- tion and fragmentation that is morphologically consistent with apoptosis and inflammatory responses in rat pups after transient occlusion. We recently developed a new model of unilateral focal ischemia with reperfusion in the P7 neonatal rat s because, in the filament model of revers- ible middle cerebral artery (MCA) occlusion, size limita- tions precluded performing this procedure before 14 to 18 days. 9 Up-regulation of the apoptosis-associated proteins, p53 and Bax, during the first day of reperfusion was shown in our model of transient ischemia in immature rats. TM Experimental Procedures Focal Ischemia Model Experiments were performed in strict accordance with the guidelines of the National Institutes of Health and the 8 Journal of Stroke and Cerebrovascular Diseases, Vol. 9, No. 1 (January-February), 2000: pp 8-15