Expression and localization of Fas-associated proteins following focal cerebral ischemia in rats

The aim of this study was to investigate the changes of expression of Fas-associated proteins and its cellular localization in the peri-infarct region following transient focal cerebral ischemia. Adult male Sprague–Dawley rats underwent right middle cerebral artery occlusion (MCAo) for 2 h and reperfusion for 1, 3, 6, 12 and 24 h. The expression of Fas-associated death domain protein (FADD), Fas-associated phosphatase-1 (FAP-1) caspase-8 and death-associated protein (Daxx), the pro-apoptotic genes, were examined by methods of RT-PCR, immunohistochemistry and Western blot. The results showed that the expression levels of mRNA and protein for FADD and caspase-8 increased significantly at 1–3 h after reperfusion, peaked at 12 h, then declined markedly at 24 h. The time course change of FAP-1 was consistent with that of FADD. The expression level of mRNA and protein for death-associated protein (Daxx) increased significantly at 3 h after reperfusion and persisted for 24 h at a high level. Immunofluorescence double-staining laser scanning showed that the immunoreactivity of FADD was localized in cytoplasm, and Daxx immunoreactivity was translocated from nucleus to cytoplasm at 3 h after reperfusion. The TUNEL-positive cells could be found in peri-infarct region at 3 h and increased with time after reperfusion. Our findings suggest a possible association between expression of FADD, caspase-8, Daxx and FAP-1 genes and apoptosis following ischemia.