TEMPORAL PROFILE OF IN-SITU DNA FRAGMENTATION AFTER TRANSIENT MIDDLE CEREBRAL-ARTERY OCCLUSION IN THE RAT

We measured the temporal profile and anatomic distribution of cells ex hibiting DNA fragmentation at various durations of reperfusion after m iddle cerebral artery (MCA) occlusion in the rat. Focal cerebral ische mia was induced in male Wistar rats (n = 62) using an intraluminal mon ofilament blockade of the MCA. After 2 h of MCA occlusion, the animals were killed at different durations of reperfusion (0.5, 3, 6, 9, and 12 h and 1, 2, 4, 7, 14, 21, and 28 days, n = 4 per time point). Sham- operated rats (n = 4) and normal rats not subjected to any surgical pr ocedure (n = 4) were used as controls. Coronal brain sections (5 mu m) were analyzed, using an in situ Ap-opTag kit, hematoxylin and eosin, and immunohistochemical double-staining methods. Six rats subjected to 2 h of MCA occlusion were killed at 24 h for measurement of DNA fragm entation by gel electrophoresis. Our data indicate that within a coron al section, DNA fragmentation was present in zero to three cells in ea ch hemisphere of normal and sham-operated rats as well as in the contr alateral hemisphere of ischemic rats. The number of cells exhibiting D NA fragmentation increased as early as 0.5 h (8 +/- 6), peaked at 24-4 8 h (213 +/- 59), and persisted for 4 weeks (10 +/- 2) after onset of reperfusion (p < 0.01). Groups of cells exhibiting DNA fragmentation ( >95% neurons) were located primarily in the inner boundary zone of the infarct. With use of gel electrophoresis, purified DNA obtained from the ischemic tissue exhibited the characteristic nucleosome ladder pat tern associated with apoptosis. The presence and anatomical location o f cells exhibiting DNA fragmentation after transient MCA occlusion sug gest that apoptosis contributes to the development of ischemic infarct . In addition, the prolonged presence of DNA fragmentation after the o nset of ischemia suggests that apoptotic ischemic brain damage is a dy namic ongoing process.