In situ detection of AP sites and DNA strand breaks bearing 3 '-phosphate termini in ischemic mouse brain

Our aims were to examine whether oxidative DNA damage was elevated in brain cells of male C57BL/6 mice after oxidative stress, and to determine whethe r neuronal nitric oxide synthase (nNOS) was involved in such damage. Oxidat ive stress was induced by occluding both common carotid arteries for 90 min , followed by reperfusion, Escherichia coli exonuclease LII (Exo III) remov es apyrimidinic or apurinic (AP) sites and 3'-phosphate termini in single-s trand breaks, and converts these lesions to 3'OH termini. These ExoIII-sens itive sites (EXOSS) can then be postlabeled using digoxigenin11-dUTP and Kl enow DNA polymerase-I, and detected using fluorescein isothiocyanate-IgG ag ainst digoxigenin, Compared with the non-ischemia controls, the density of EXOSS-positive cells was elevated at least 20-fold (P < 0.01) at 15 min of reperfusion, and remained elevated for another 30 min. EXOSS mainly occurre d in the cell nuclei of the astrocytes and neurons. Signs of cell death wer e detected at 24 h of reperfusion and occurred mostly in the neurons. Both DNA damage and cell death in the cerebral cortical neurons were abolished b y treatment with 3-bromo-7-nitroindazole (30 mg/kg, intraperitoneal), which specifically inhibited nNOS. Our results suggest that nNOS, its activator (calcium), and peroxynitrite exacerbate oxidative DNA damage after brain is chemia.