is software. Data are expressed as percentage infarct volume. Percentage infarct volume was calculated as described: 6100%, V C is the volume of the control hemisphere, and V L is the volume of the non-infarcted tissue in the lesioned hemisphere. Immunohistochemistry At 24 h after MCAO reperfusion, rats were anesthetized with pelltobarbitalum natricum and then perfused transcardially with 200 mL of saline followed by 300 mL of 4% paraformaldehyde solution. Brains were removed and fixed in 4% paraformaldehyde solution at 4uC for three days and then dehydrated and embedded in paraffin blocks. Five-micrometer-thick sections were cut through the dorsal hippocampus. Immunohistochemistry was performed with antibodies for cleaved caspase-3. In brief, tissue sections were deparaffinized and wetted with graded alcohol. The tissue sections were incubated with 3% H2O2, 5% normal goat serum and 0.5% triton X-100 at room temperature for 30 min each. After anti-cleaved caspase-3 antibody was applied to the sections and incubated overnight at 4uC, the sections were incubated with anti-mouse IgG horseradish peroxidase for 1 h at 37uC. The sections were subsequently incubated with diaminobenzidine and counter-stained with hematoxylin. A cleaved 5 20-Hydroxyecdysone Inhibits Cerebral Injury caspase-3-positive cell was indicated by brown coloration. The total cell number and the number of cleaved caspase-3-positive cells were determined in the five areas of the cerebral cortex in every section using a square micrometer eyepiece at 2006 magnification. Five sections were selected from each 
experimental rat. Data are expressed as the ratio of cleaved caspase-3-positive cells to total cells. and Tukey’s test. Neurological behavior scores were analyzed by using a nonparametric KruskalWallis test and Dunn’s Multiple Comparison Test. p,0.05 was considered to be significant. Results 20E attenuated oxidative stress-induced injury in B35 neural cells The MTT assay revealed that H2O2 decreased cell viability in a concentration- dependent manner. As shown in Fig. 1B, the viability of the cells exposed to H2O2 at concentrations of 100 to Statistical analysis Data were expressed as the mean 6 S.E.M., and statistical significance was assessed by one-way analysis of variance 20-Hydroxyecdysone Inhibits Cerebral Injury 500 mM for 12 h decreased to 91.8162.45%, 49.87%64.39%, 38.8663.18%, 35.2862.22% and 28.6065.74% of the control value, respectively. Based on this result, a Salianic acid A biological activity treatment of 200 mM H2O2 for 12 h was used to induce B35 neural cell injury in subsequent experiments. To examine the neuroprotective effects of 20E on H2O2-induced cytotoxicity, B35 neural cells were pretreated with 20E for 24 h before the onset of H2O2 treatment. Application of 20E at 25 mM did not attenuate H2O2-induced cytotoxicity. We therefore increased the amount of 20-Hydroxyecdysone to 50, 100, 200, or 400 mM. The cell viability significantly increased to 60.6562.15%, 65.75%63.53%, 70.5664.18%, and 78.4065.42% of the control value at 50, 100, 200 and 400 mM, respectively. In another experiment, 48 h of treatment with various concentrations of 20E alone did not lead to any apparent increase in the viability of cells. These findings demonstrate that 20E protects B35 neural cells against H2O2-induced cytotoxicity in a dose-dependent manner. The neuroprotective effects of 20E were confirmed by the LDH assay, TUNEL analysis and immunocytochemistry stain. As shown in Fig. 1D, the cells treated with 2