Evidence of phosphorylation of Akt and neuronal survival after transient focal cerebral ischemia in mice

The serine-threonine kinase, Akt, prevents apoptosis by phosphorylation at serine-473 in several cell systems. After phosphorylation, activated Akt in activates other apoptogenic factors, such as Bad or caspase-9, thereby inhi biting cell death. The present study examined phosphorylation of Akt at ser ine-473 and DNA fragmentation after transient focal cerebral ischemia in mi ce subjected to 60 minutes of focal cerebral ischemia by intraluminal block ade of the middle cerebral artery. Phospho-Akt was analyzed by immunohistoc hemistry and Western blot analysis. The DNA fragmentation was evaluated by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biot in nick end-labeling (TUNEL). Immunohistochemistry showed the expression of phospho-Akt was markedly increased in the middle cerebral artery territory cortex at 4 hours of reperfusion compared with the control, Whereas it was decreased by 24 hours. Western blot analysis showed a significant increase of phospho-Akt 4 hours after focal cerebral ischemia in the cortex, wherea s phospho-Akt was decreased in the ischemic core. Double staining With phos pho-Akt and TUNEL showed different cellular distributions of phospho-Akt an d TUNEL-positive staining. Phosphorylation of Akt was prevented after focal cerebral ischemia by LY294002, a phosphatidylinositol 3-kinase inhibitor, which facilitated subsequent DNA fragmentation. These results suggest that phosphorylation of Akt may be involved in determining cell survival or cell death after transient focal cerebral ischemia.