Immunohistochemical analysis of DNA fragmentation in gerbil hippocampus after transient forebrain ischemia

Abstract

Pyramidal neurons in the hippocampal CAI sector are especially vulnerable to ischemic insult and reveal morphological cell death at several days after transient forebrain ischemia designated as delayed neuronal death (DND). The mechanism of DND remains unknown, although recent studies have indicated that apoptosis is involved in DND demostrations evidence of DNA ladder formation on agarose gel electrophoresis. We examined the time course of DNA fragmentation in the hippocampus after 5 min forebrain ischemia in the gerbil, using immunohistochemical labeling of DNA 3-OH ends generated by DNA fragmentation. In situ end-labeling by the TUNEL method (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling) showed that DNA fragmentation in the hippocampal CAl sector first appeared at 72 hours after transient ischemia and remained up to 7 days in our observations. Our results were similar to those of a previous report describing the time course of DNA fragmentation with agarose gel electrophoresis (Okamoto et al. BBRC, 1993). The present findings indicate that in situ end-labeling by the TUNEL method is appropriate for detecting DNA fragmentation and suggest that apoprosis is at least partially involved in DND.