In situ detection of neuronal DNA strand breaks using the Klenow fragment of DNA polymerase I reveals different mechanisms of neuron death after global cerebral ischemia

Ischemic cell injury in the brain may involve a cascade of programmed cell death. DNA damage may be either a catalyst or a consequence of this cascade . Therefore, the induction of DNA strand breaks in the rat brain following transient global ischemia was examined using (a) the Klenow labeling assay, identifying DNA single-strand breaks (SSBs) or double-strand breaks (DSBs) with protruding 5' termini, and (b) terminal deoxynucleotidyl transferase- mediated dUTP nick end-labeling (TUNEL), detecting DNA DSBs with protruding 3' termini or blunt ends. Klenow-positive staining occurred within 2 h of reperfusion and increased with increasing durations of reperfusion. DNA dam age detected with the Klenow labeling assay preceded that of TUNEL expressi on in the caudate putamen, reticular thalamus, thalamus, and cortex. Howeve r, in CA1, DNA SSBs were not detected until 72 h of reperfusion and occurre d simultaneously with DSBs. Thus, the time course and fragmentation charact eristics of DNA damage differ between the hippocampal CA1 and other selecti vely vulnerable brain regions. This distinct pattern suggests that the dela yed neuronal death in CAI following transient global ischemia may occur via an apoptotic mechanism different from that of other brain regions.