Correlation between caspase-3 activation and three different markers of DNA damage in neonatal cerebral hypoxia-ischemia

Caspase-3 has been identified as a key protease that, by targeting a limite d number of proteins, can disrupt essential homeostatic processes and initi ate an orderly disassembly of cells, including degradation of genomic DNA. We demonstrate the usefulness of an antibody specific for activated caspase -3 in a model of neonatal rat hypoxia-ischemia (HI) and correlate the spati al and temporal activation of caspase-3 with three different markers of DNA damage and with the loss of a neuronal marker [microtubule-associated prot ein 2 (MAP 2)]. An oligonucleotide hairpin probe (HPP) with one base overha ng in the 3' end displayed a close colocalization with caspase-3 activation at 3 h post-HI, whereas terminal deoxynucleotidyl transferase-mediated dUT P nick end-labeling (TUNEL) appeared later (24 h post-HI). A monoclonal ant ibody against single-stranded DNA appeared to stain an entirely different p opulation of cells, not positive for active caspase-3, HPP, or TUNEL at thi s time point. After 24 h of reperfusion, however, when cellular injury is e xtensive, all markers stained a large number of cells with a high degree of colocalization, and all markers delineated regions with loss of MAP 2. We conclude that the HPP shows the best correlation with pathological caspase- 3 activation in this model.