DNA Damage and Fragmentation

Reactive oxygen species can damage DNA. DNA strand breaks are detected at as early as 3 hr of reperfusion and increase over a 24-hr period in selectively vulnerable regions of striatum, cerebral cortex, and thalamus after global ischemia (125,139). In contrast, DNA strand breaks in CA1 hippocampus can be delayed by 72 hr, which is close to the time that the pyramidal neurons undergo cell death. These findings support the hypothesis for a different molecular mechanism of cell death in the CA1 region. Expression of apurinic/apyrimidinic endonuclease (APE/ref-1) was found to decrease 1 day prior to cell death (140). Overexpression of Cu,Zn-SOD attenuated the decrease in APE and protected CA1 neurons (141). The delayed decreased activity of this DNA repair enzyme was postulated to contribute to delayed cell death in the CA1 region.

Classic programmed cell death is associated with internucleosomal DNA fragmentation that results in a laddered pattern during electrophoresis, whereas necrosis results in DNA fragments of random size and a smeared electrophoretic pattern (142,143). After global cerebral ischemia, the laddered pattern of DNA fragmentation is not prominent, although the pattern is not completely random (136). Initially, 10-kilo-base pairs (kbp) fragments with a high proportion of 5'-OH ends and 50-kbp fragments with a high proportion of 3'-OH ends are found (143). The 50-kbp fragments are degraded into a laddered oligonucleosomal pattern. Use of ligation-mediated polymerase chain reaction (PCR) indicated that most of the DNA fragments after global cerebral ischemia have staggered ends with a 3' recess of 8 to 10 nucleotides, whereas apoptosis results in blunt-ended double-strand breaks (144 ). These findings imply that different sequences of endonucleases are activated in global ischemia versus classic apoptosis.

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