Early cell death in the brain of fetal preterm lambs after hypoxic–ischemic injury

The objective of the present study was to evaluate using premature fetal lambs the effect of cerebral hypoxia–ischemia induced by partial occlusion of the umbilical cord on the type of cell death which occurs in different brain regions and to ascertain some of the neural pathways which may underlie the associated pathologies. Lambs were sacrificed either immediately after a 1 h hypoxic–ischemic insult or 3 h later. Brains were fixed by perfusion and blocks of the different brain territories were processed for light microscopy (hematoxylin–eosin, Nissl staining), electron transmission microscopy and quantification of apoptosis by the TUNEL method. Other fixed brains were dissociated and labeled by nonyl acridine orange to determine mitochondrial integrity. Non-fixed brains were also used for membrane asymmetry studies, in which cell suspensions were analyzed by flow cytometry to quantify apoptosis. In both hypoxic–ischemic groups, necrotic-like neurons were observed mainly in the mesencephalon, pons, deep cerebellar nuclei and basal nuclei, whereas apoptotic cells were extensively found both in white and gray matter and were not limited to regions where necrotic neurons were present. The 3 h post-partial cord occlusion group, but not the 0 h group, showed a generalized alteration of cell membrane asymmetry and mitochondrial integrity as revealed by Annexin V/PI flow cytometry and nonyl acridine orange studies, respectively. Our results show that the apoptotic/necrotic patterns of cell death occurring early after hypoxic–ischemic injury are brain-region-specific and have distinct dynamics and suggest that therapeutic strategies aimed at rescuing cells from the effects of hypoxia/ischemia should be aimed at blocking the apoptotic components of brain damage.