Figure 3. The effect of Salvianolic acid B on BrdU-incorporation in cultured NSPCs. Dissociated NSPCs were cultured on poly-L-lysine- coated 12-well chamber slides with or without Sal B for 12 h and were subsequently incubated with BrdU (10 mg/ml) for other 12 h. After treatment, cells were fixed, immunostained with anti BrdU antibody. (A) Visualization of BrdU-positive cells by the Immunofluorescence staining assay on NSPCs. Scale bar: 100 mm. (B) BrdU-positive cells were counted in 10 randomly selected fields from three different chambers. Data represent the mean 6 S.D. from three independent experiments. *Significant difference from the control group at P,0.05. **Significant difference from the control group at P,0.01. doi:10.1371/journal.pone.0035636.g003 Salvianolic acid B increased the number of BrdU-positive cells in vivo In the following study, the effect of Sal B on recovery of brain functions after transient cerebral ischemia was evaluated. Firstly, we investigated whether Sal B (50 mg/kg) exposure in vivo would affect NSPCs behavior. Since BrdU is incorporated into dividing and proliferating cells, BrdU labeling has been widely used in many investigations concerning neurogenesis. BrdU (50 mg/kg IP) was administered to the rats in sham, vehicle, and ischemia groups once every 2 hours over a period of 8 hours at the 21th day after injection to label the proliferating cells after ischemia. The next day the rats were perfused with 0.01 M PBS (pH 7.2), then with 4% paraformaldehyde (PFA) in PBS. The dividing cells labeled with BrdU were visualized using BrdU immunohistochemistry. BrdU labeled neurons were found in all groups in the dentate gyrus of the hippocampus (Fig. 7). A few BrdU-positive cells were observed in the SGZ of hippocampal dentate gyrus in the control, and BrdU immunoreactivity did not significantly differ between vehicle and sham-operated animals in 21 days. We observed a significant 2.5-fold increase in the numbers of BrdU positive cells in the SGZ of hippocampal dentate gyrus in Sal B treated group (50 mg/kg) (n = 4, p,0.01), which is consistent with our in vitro findings that Sal B enhanced NSPCs proliferation. Salvianolic Acid B Promote Neurogenesis Figure 4. Salvianolic acid B up-regulated the self-renew genes of NSPCs. (A) The mRNA levels of NSPCs self-renew markers were analyzed by RT-PCR. (B) The expression levels were semi-quantified by densitometric measurements, normalized with GAPDH internal control, compared with control, and expressed as means 6 S.D from three independent experiments. *Significant difference from the control group at P,0.05. **Significant difference from the control group at P,0.01. doi:10.1371/journal.pone.0035636.g004 Salvianolic acid B protected the learning and memory functions Since cerebral ischemia can cause prolonged spatial memory disturbance in rats [33], we performed Morris water-maze test to examine the hippocampus involvement and Sal B effect on in learning and memory. Each animal was tested twice a day for 5 consecutive days on their escape latency to the hidden platform. Fig. 8A showed the escape latencies of the three groups of animals during acquisition training (5 days). In all the groups, escape latencies became shorter across training sessions (F(2,30) = 31.54, P,0.01, Repeated measures ANCOVA). The decreased escape Figure 5. Salvianolic acid B promoted NSPCs proliferation in a PI3K/Akt -independent pathway. NSPCs were cultured in the proliferation medium containing Sal B (20 mM) in the presence and absence of the PI3K inhibitor Ly294002 (20 mM), MEK inhibitor U0126 (10 mM) or Notch inhibitor DAPT (10 mM) for 2 days. Cell survival was assessed by MTS assay. Data represent the mean 6 S.D. from three independent experiments. **P,0.01 as compared with con- trol,##P,0.01 as compared with Sal B-treated cells. doi:10.1371/journal.pone.0035636.g005 PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e35636