A model of transient unilateral focal ischemia with reperfusion in the P7 neonatal rat: morphological changes indicative of apoptosis.

Abstract

The mechanisms leading to delayed cell death after hypoxic-ischemic injury in the developing brain remain to be elucidated. The aim of this study was to develop a model of transient focal ischemia in the neonatal rat in an attempt to create a reperfusion phase since in the filament model of reversible middle cerebral artery occlusion, size limitations precluded performing this procedure before 14 to 18 days. We then analyze whether apoptosis or necrosis occurs in this model. Seven-day-old Wistar rat pups (n = 96) underwent permanent left middle cerebral artery occlusion in association with 1-hour occlusion of the left common carotid artery. Evolution of the brain infarction was studied from 24 hours to 3 months on cresyl violet-stained coronal sections. Infarct volume was determined with the use of the mitochondrial stain 2,3,5-triphenyltetrazolium chloride. Neuronal death was demonstrated by the silver staining method of Gallyas et al (1980). Chromatin condensation was shown by DNA fragmentation assessed with the use of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay in cryostat sections and electron microscopic analysis. Almost all of the animals who survived had reproducible cortical infarcts. The mean infarct volume was 31+/-7 mm3 (mean+/-SD). The ipsilateral hemisphere showed a well-delineated lesion in the frontoparietal cortex at 3-month recovery. Argyrophilic (dying) neurons were observed a few hours after reperfusion and increased with time. Cells exhibiting DNA fragmentation were shown as early as 6 hours, increased up to and peaked at 24 to 96 hours, then progressively decreased and persisted for several days, suggesting an ongoing process. Electron microscopy analysis demonstrated high condensation and clumping of chromatin beneath nuclear membrane in shrunken neurons. Our study demonstrates the feasibility of performing ischemia-reperfusion in 7-day-old rats that develop progressive neuronal death with features characteristic of apoptosis. The reperfusion phase mimics events that occur during neonatal human hypoxic-ischemic encephalopathy at birth, since perinatal intensive care most often permits recirculation.