Figure 8. Delayed post-ischemic treatment with salvianolic acid B improved cognitive impairment in Morris water maze task. (A) Place learning with multiple trials. There was a decrease in escape latencies with training in all three groups. (B) In the transfer task, the escape latencies (mean 6 S.D.) are compared among sham- operated, untreated ischemic, and Sal B treated groups (n = 8). And the animals from the Sal B-treated group spent more time in the quadrant that contained the escape platform during the place learning than untreated group. *P,0.05 as compared with sham group, #P,0.05 as compared with model group. doi:10.1371/journal.pone.0035636.g008 90 s without the escape platform and the time the animal spent to arrive the position and in the quadrant which had the escape platform during the training experiment was recorded. Time spent data were shown in Fig. 8. In relation to the transfer of this task, animals in untreated group remained for a similar total period of time in the four quadrants. Animals from sham and Sal B (50 mg/ kg) groups spent longer in the quadrant which had contained the escape platform during the initial place learning than in the remaining quadrants. These results showed that Sal B could improve the learning and memory ability of cerebral ischemic animals. Discussion NSPCs proliferation is important to produce of neurogenesis. Compounds that can promote neural stem cell proliferation are seen to support neurogenesis. In the present study, we selected a total of 45 natural compounds from medicinal materials which are extensively used in China to treat stroke clinically, and tested their proliferation-inducing activities on NSPCs using a MTS assay. The screening results showed that several natural compounds such as berberine and Sal B displayed marked effects on the proliferation of NSPCs. In the further research, we found that although berberine could increase the value of MTS assay, it reduced the BrdU incorporation. The action of berberine was proved to be an illusion since BrdU incorporation was considered as an important sign of cell proliferation. Additionally, morpho- Salvianolic Acid B Promote Neurogenesis logically berberine could cause cell swelling and decrease the cell number (see Figure S1 in the Supporting Information). Therefore berberine was cytotoxic to neural stem cell and could not promote proliferation. To explore whether Sal B could promote the NSPCs proliferation, we next systemic examined the promotive effects of Sal B on NSPCs proliferation. Cortical NSPCs were isolated at embryonic day 13.5, the cells were plated in non-adherent conditions, evaluation with MTS assays demonstrated that Sal B induced NSPCs proliferation in a dose- and time-dependent manner. On the molecular level, DNA synthesis of NSPCs in Sal B-treated cultures was determined by BrdU incorporation assay. Immunostaining results showed that the BrdU positive cells were up regulated by Sal B treatment. Consistent with the MTS assay, the BrdU incorporation studies established that metabolic activity of cells was enhanced with the presence of Sal B when compared with the control. These observations suggested that Sal B indeed promoted the proliferation of the NSPCs. Next we conducted a RT-PCR analysis with NSPCs self-renew marker, Nestin and Notch-1. The results showed that the nestin and Notch-1 mRNA expression was up-regulated significantly by Sal B treatment. The results were consisted with the promotion of NSPCs proliferation induced by Sal B. In summary, these observations suggest that Sal B could maintain the NSPCs self-renew and promote proliferation. Several intricate cell signaling cascades are crucial for determining whether NSPCs proliferation or differentiation. Notch, MEK/ERK and PI3K/Akt pathways are the most frequently ones that associated with regulation of cell growth, survival, and differentiation [34]. We sought to determine which signaling pathway was activated by which Sal B facilitated the NSPCs self-renew. If these signal pathways play important proliferation roles in NSPCs, inhibition of PI3K/Akt should reduce Sal B-induced proliferation. MTS results showed that the absorbance value could be increased by Sal B, and PI3K/Akt specific inhibitor Ly294002 but not Notch specific inhibitor DAPT and MEK/ERK specific inhibitor U0126 could block this effect. The results suggested that PI3K/Akt signal pathway was involved in the Sal B-induced proliferation of NSPCs. To gain further insight into the mechanisms by which Sal B modulates PI3K/Akt signalling and by which PI3K/Akt mediates Sal B induced self-renew, we evaluated the role of the PI3K/Akt pathway. Akt is a serine/threonine kinase, which can be activated by phosphorylation and subsequently enhance cell survival by activates multiple downstream targets [35,36]. We demonstrated that exposure to Sal B induced the activation of Akt. If PI3K/Akt signal pathway plays an important prolifeation role, inhibition of PI3K/Akt should reduce Sal B-induced phosphorylation of Akt. Consistent with our hypothesis, the results showed that Sal B- induced activation of Akt was completely inhibited by the PI3K/ Akt-inhibitor, Ly294002. In short, these findings supported the hypothesis that PI3K/Akt signal pathway was the signal mediator in Sal B-stimulated NSPCs proliferation. In order to examine the effects of Sal B on proliferation of NSPCs in vivo, transient cerebral ischemic rats and BrdU labeling were employed. New neurons can be generated in the hippocam- pal of adult rats after transient ischemia of the forebrain [37–39]. Many researchers had reported that cerebral ischemic could transient stimulated neurogenesis in the adult hippocampus. As far as we know, Liu et al. [40] first demonstrated increased neurogenesis in the gerbils hippocampus after transient global ischemia, the BrdU incorporation peaks 11 days after ischemia and that incorporation subsides to control levels by 3 weeks after ischemia. Similar results were described after transient global ischemia in mice [41] and rats [42]. But most of the cells were not PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e35636