[D-ala2,D-leU5]enkephalin (DADLE) reduces necrotic and apoptotic (panel B) cell death associated with ischemia. (A) Triphenyltetrazolium chloride (TTC) staining at 72 h post occlusion and reperfusion surgery of the middle cerebral artery (MCAor) revealed that the striata from ischemic animals exposed to ischemia/reperfusion surgery and pretreated with saline showed dehydrogenase-deficient tissue (negative TTC stains). In contrast, the striata from ischemic animals pretreated with DADLE, DADLE + naloxone methiodide, or DADLE + naltrexone did not reveal any dehydrogenase deficiency. (B) Ischemic animals exposed to ischemia/reperfusion injury and treated with saline showed a significant increase in mRNA expression of p-53 in their ischemic striata (65% increment compared with intact striatum of normal, control animals) at 24 h after stroke surgery. In contrast, ischemic animals treated with DADLE exhibited only a small increment in p-53 mRNA expression in their ischemic striata at the same time period (28% increment compared with intact striatum of normal, control animals; not significantly different from control values). Comparisons of ischemic striata between these two groups showed a marked reduction in p-53 mRNA expression in DADLE-treated ischemic animals compared with saline-treated ischemic animals. (C) Immunohistochemical analyses of phenotypic markers of apoptosis revealed that DADLE significantly reduced caspase-3- (panels a, b, c, d) and Fas-positive cells (panels e, f, g, h) in DADLE-treated ischemic animals compared to saline-treated ischemic animals. Quantitative data shown in bar graphs and represent means ± S.E.M. Asterisks correspond to statistical significance at P <0.05. To better capture the necrotic and apoptotic cells in saline-treated ischemic animals (panel d), higher magnification images are generated from propidium iodide (a) and caspase-3 (b) immunofluorescently labeled striatal cells, respectively. Scale bar = 50 μm (a), 100 μm (b).