Early decrease in DNA repair proteins, Ku70 and Ku86, and subsequent DNA fragmentation after transient focal cerebral ischemia in mice

Background and Purpose-Ku70 and Ku86, multifunctional DNA repair proteins, bind to broken DNA ends, including double-strand breaks, and trigger a DNA repair pathway. To investigate the involvement of these proteins in DNA fra gmentation after ischemia/reperfusion, Ku protein expression was examined b efore and after transient focal cerebral ischemia (FCI) in mice. Methods-Adult male CD-I mice were subjected to 60 minutes of FCI by intralu minal suture blockade of the middle cerebral artery. Ku protein expression was studied by immunohistochemistry and Western blot analysis. DNA fragment ation was evaluated by gel electrophoresis and terminal deoxynucleotidyl tr ansferase-mediated dUTP nick end-labeling (TUNEL). The spatial relationship between Ku expression and DNA fragmentation was examined by double labelin g with Ku and TUNEL after reperfusion. Results-Immunohistochemistry showed constitutive expression of Ku proteins in control brains. The number of Ku-expressing cells was decreased in the e ntire middle cerebral artery territory as early as 4 hours after reperfusio n and remained reduced until 24 hours. Western blot analyses confirmed the significant reduction of these proteins (59.4% and 57.7% reduction in optic al density at 4 hours of reperfusion from the normal level of Ku70 and Ku86 bands, respectively; P <0.001). DNA gel electrophoresis demonstrated DNA l addering 24 hours after reperfusion, but not at 4 hours. Double staining wi th Ku and TUNEL showed a concomitant loss of Ku immunoreactivity and TUNEL- positive staining. Conclusions-These results suggest that the early reduction of Ku proteins a nd the loss of defense against DNA damage may underlie the mechanism of DNA fragmentation after FCI.