brain, another subgroup of mice subjected to IPC was randomly selected to receive intraventricular injection with 3-MA, 2 L wortmannin, or an equivalent amount saline, at Debio 1347 chemical information bregma 0 mm, mediolateral 2 mm, and dorsoventral 3 mm, 2 h before the first surgery. After the second BCCAO procedure, all mice were sacrificed either 6 h later for immunohistochemical staining or 24 h later for Nissl staining. Western Blotting After extraction of total protein, the protein concentration was determined using Bradford assays, and 10 g of total protein was loaded for sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunoblotting was performed with antibodies directed against LC3, phospho-Akt, total Akt, and -actin. Immunohistochemical Staining Mice were deeply anesthetized and transcardially perfused with 4% paraformaldehyde at 6 h after lethal BCCAO. The brains were incubated sequentially in 10%, 20%, and 30% sucrose solutions overnight, embedded in paraffin, and sectioned PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19731986 to 5 m thickness through the dorsal and ventral hippocampus using a vibratome. After deparaffinization/rehydration, antigen was retrieved using 10 mM sodium citrate buffer. Next, sections were blocked with 10% goat serum 3 / 13 Effects of Ischemic Preconditioning in Mice for 1 h at room temperature, followed by incubation overnight at 4C with primary antibodies against LC3. Subsequently, antibodies were detected with the EnVision Detection System. Three areas of interest in the hippocampal CA1 and CA3 layers were chosen. Each observation was repeated five times. Images were captured using a Nikon ECLIPSE 80i microscope with Nikon Plan Fluor lenses. Nissl Staining Analysis For Nissl staining, 5-m-thick paraffin-embedded sections were incubated with 1% cresylviolet at 50C for 20 min. After rinsing with double distilled water, sections were dehydrated and mounted with Permount. The hippocampal CA1 and CA3 layers from each animal were analyzed. Six visual fields of CA1 and CA3 layers were photographed in each section. The number of stained cells in each field was counted at higher magnification. The data were represented as the number of cells per mm measured using the scale-bars under PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19729663 a high-power field. Statistical Analysis Data are presented as the mean standard error of the mean. Unless stated otherwise, we carried out statistical analysis with SPSS 13.0 statistical software using two-tailed Student’s t-tests if two groups were compared or one-way analysis of variance with Bonferroni’s multiple comparisons post-hoc test for comparisons of more than two groups. Differences were with p values of less than 0.05 were considered significant. Results Protective Effects of IPC on Hippocampal Neuronal Death during Ischemic Insult Hippocampal neurons were exposed to 055 min of OGD, followed by determination of cell survival by MTT assays 24 h later. Our results showed that exposure to 5, 10, 15, or 30 min of OGD stimulus 
and reperfusion for 24 h did not cause significant neuronal death. In contrast, exposure to 55 min of OGD decreased neuronal survival to 52% 12.9%, significantly lower relative to survival percentages after 030 min of OGD; therefore, the lethal OGD time was set as 55 min. To confirm whether different OGD times altered neuronal vulnerability to lethal OGD, we exposed hippocampal neurons to sublethal OGD for different times, followed by lethal OGD 24 h later. We found that 15 min of OGD conferred protection against the deleterious effects of lethal OGD by increasing n