DNA fragmentation in ischemic core and penumbra in focal cerebral ischemiain rats

Although apoptotic cell death has been suggested to be involved in ischemic injury of the brain, the precise mechanisms of ischemic neuronal cell deat h are unknown. Here, we examined the biochemical feature of apoptosis (i.e. DNA fragmentation) in male spontaneously hypertensive rats (5-7 months old ) subjected to photothrombotic distal middle cerebral artery (MCA) occlusio n. After MCA occlusion, the brain was cut in a cryostat to produce a standa rd coronal block and samples were dissected from the regions corresponding to the ischemic core, penumbra and contralateral control areas. Changes in cerebral blood Row (CBF) were monitored at I mm posterior and 2-4 mm latera l to the bregma by means of a laser-Doppler flowmetry. After MCA occlusion, CBF was decreased to 72 +/- 18 (+/-S.D.), 50 +/- 14, and 35 +/- 11% of the control values at 2, 3, and 4 mm from the midline, respectively. DNA fragm entation characteristics of apoptosis were examined in these samples by con ventional and pulse-field gel electrophoresis. On the conventional gel elec trophoresis, nucleosomal DNA fragmentation was detected in the penumbral zo ne at 6 h after MCA occlusion. Large DNA fragments of 50 and 20 kbp were de tected in the penumbral zone and also in the ischemic core region at 3 h af ter distal MCA occlusion. The large DNA fragments seen on the pulse-field g el elecrophoresis were further degraded to small DNA fragments at 6 h after MCA occlusion in the penumbral zone but not in the core regions. The evolv ing DNA fragmentation was observed between 3 and 6 h after the onset of bra in ischemia in the penumbra, suggesting that apoptosis may contribute to th e development of ischemic infarction. (C) 2001 Elsevier Science B.V. All ri ghts reserved.