ward) and 59- TGGGCTGAGCTGTTTTCTACTTTT-39 (re- verse); Notch-1, 59- ATGGCCTCCAACGATACTCCT-39 (for- ward) and 59- ACATGTACCCCCATAGTGGCA-39 (reverse). Products were analyzed on 1.5% agarose gel and visualized by ethidium bromide staining. The relative amount of each transcript was normalized to the level of b-actin. Western blot analysis Protein extracts were prepared and subjected to Western blot analysis. Cells were harvested and lysed with RIPA lysis buffer. The protein concentrations of the lysates were determined with a Bradford protein assay kit (Bio-Rad) according to the manufac- turer’s instructions. An equal amount of protein was fractionated by SDS-polyacrylamide gel electrophoresis (PAGE) and trans- ferred onto polyvinylidine difluoride membranes. After blocking with 5% skim milk in TBS-T, the membrane was probed with primary antibodies (goat anti-actin 1:1000, Santa Cruz; rabbit anti-ERK1/2 1:500, rabbit anti-phospho-ERK1/2 1:300, rabbit anti-Akt 1:500 or rabbit anti-phospho-Akt (Ser-473) 1:300, Cell Signaling Technology) in a blocking solution of non-fat milk (5%). Secondary horseradish peroxidase-conjugated rabbit anti-goat 1:2000, goat anti-rabbit 1:2000 (Santa Cruz) antibody in non-fat milk blocking solution (5%) was then applied. The immuno- reactivity was visualized with ECL Western blotting detection reagents (Millipore). Induction of the transient global ischemia Adult (8–10 week-old) male Wistar rats weighing 200–250 g were subjected to transient forebrain ischemia by a method combining those described previously [47,48]. The rats were housed singly in temperature-controlled conditions with a 12 hr light/dark cycle (lights on: 8:00 AM) following surgery. They had access to food and water ad libitum. Experimental procedures were conducted in accordance with recommendations of the Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine (TCM-2009-034-E01). In brief, twelve hours before the induction of ischemia, rats were anesthetized with sodium pentobarbital, the bilateral carotid arteries were exposed to facilitate the occluding on the following day, and then the vertebral arteries were irreversibly occluded by electrocoagulation. The next day, ischemia was induced by bilaterally occluding carotid arteries with aneurysm clips, and carotid arteries were clamped for 6 min exactly. Rats lost their righting reflex during ischemic, the clips were removed to restore cerebral blood flow. The rats that remain their righting reflex in 1 min after occluding of the both carotid arteries were considered to be failure of ischemia and eliminated. Sham-operated rats were anesthetized, the carotid arteries were isolated, but they were not clamped. Drug adminstration The study was carried out on rats divided into three groups (n = 13). (i) Sham group+vehicle (Sham), (ii) Ischemic group+- vehicle (Model) and (iii) Ischemic group+Sal B (Sal B). Seven days after cerebral ischemia, Sal B (50 mg/kg) was dissolved in distilled water, and injected i.p. once daily for 4 weeks, and three mice in each group were used for histological analysis at day 21 after innjection. All controls received an amount of vehicle equivalent to drug treatment conditions. In vivo proliferation assay For in vivo proliferation analysis of Sal B, that had not been behaviorally tested were evaluated for BrdU labeling. BrdU (50 mg/kg; Sigma) was injected i.p. once every 2 hours over a Salvianolic Acid B Promote Neurogenesis period of 8 hours at the 21th day after injection to label the proliferating cells. Twenty-four hours after the last BrdU injection, rars were anesthetized with ether and perfused with PBS, followed by a cold 4% paraformaldehyde solution. Brains were collected and post-fixed overnight in a 4% paraformaldehyde solution at 4uC. Coronal sections (20 mm thickness) were obtained throughout the hippocampus. For analysis concerning BrdU immunohisto- chemistry, sections were incubated in 50% formamide/26saline sodium citrate (SSC) for 2 h at 65uC, followed by a rinse with PBS. Sections were then incubated in 2 N HCl for 30 min at 37uC to denature double-stranded DNA, and rinsed in 0.1 M borate buffer (pH 8.5). After blocking for 2 h with 1% BSA in PBS, sections were incubated overnight at 4uC with mouse anti-BrdU monoclonal antibody (1:100; senta). Followed by rinsing in PBS, sections were incubated for 2 h at RT with biotinylated goat anti- mouse IgG (1:200; senta), and incubated for 2 h at RT with the ABC kit. BrdU positive cells were visualized by incubating sections with Vector DAB. Morris water maze task The learning and memory ability was examined using the Morris water-maze [49]. A cylindrical tank 1.5 m in diameter was filled with water (2261uC), and a transparent platform 10 cm in diameter was placed at a constant position in the center of one of the four quadrants within the tank. The platform was set 2 cm below the water level where the rats could not see it directly. Rats were allowed to swim freely for 1 min to become habituated to the apparatus. From the next day, in the hidden platform trials, acquisition trials were carried out 2 times per day for 5 days. In each trial, rats were placed into the water at a fixed starting position, and the time taken to escape onto the hidden platform and the swimming path length were measured. Rats were given 90 s to find the hidden platform during each acquisition trial. If it failed to locate the platform within 90 s, it was guided there. The rat was allowed to stay on the platform for 20 s. Performance was tested 24 h after the final training day in a probe trial during which the platform was removed, the rat was placed in the start and its behavior was monitored for 90 s. Statistical analysis SPSS11.5 for Windows (SPSS Inc.) was used to analyze the data. Data are expressed as the mean 6 S.D. and analyzed by ANOVA followed by the post-hoc Least Significant Difference (LSD) test. Differences were considered statistically significant if P values were less than 0.05. To analyze water-maze place- navigation performance, the average escape latency of 5 trials per day per animal was calculated and evaluated by repeated- measures ANCOVA. Supporting Information Figure S1 Berberine failed to promote the NSPCs proliferation. (A) Berberine increased the value of MTT assay. (B) Berberine reduced the BrdU incorporation. **Significant difference from the control group at P,0.01. (C) Morphologically berberine caused cell swelling and decreased the cell number. Scale bar: 200 mm. (TIF) Figure S2 Regeneration of Hippocampal NSPCs Follow- ing Ischemia. BrdU positive cells in the granule cell layer in the intact (A) and ischemic (B, DAI7) animals. The inset shows an enlarged display of a typical BrdU positive cell. Scale bar: 200 mm. (TIF) PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e35636