fully mature during the 2- to 5-week period after ischemia and could not form a functional link [42]. Therefore, in our view, the behavior performance could not be better at that time. In accordance with previous reports, the NSPCs responded to the injury by proliferation in this study. Cerebral ischemic made the NSPCs proliferation after 7 days (See Figure S2 in the Supporting Information), but the proliferation ability of the rats in the vehicle group declined quickly 28 days after cerebral ischemic. However, Sal B (25 mg/kg) treatment significantly increased the number of BrdU-positive cells in the dentate gyrus 21 days after injection, suggested that Sal B exposure could maintain the ability of the NSPCs proliferation in vivo. Since Sal B promote NSPCs proliferation in vitro and in vivo, we next sought to correlate the regeneration of new neurons and recovery of brain functions. The present study demonstrated that delayed post-ischemic treatment (7 days after ischemic stroke) with Sal B (25 mg/kg) improved cognitive impairment after stroke in rats. Since NSPCs proliferation in hippocampus was important site for spatial learning and memory [43,44], we speculated that the enhancement of functional recovery by Sal B might be dependent on its action on NSPCs proliferation. But more extensive experiments were still necessary to demonstrate that Sal B could cross over the blood–brain barrier and act on NSPCs. Further study will be carried out to clarify this point. Although our present study showed that Sal B promoted the adult hippocampus neurogenesis and improved the cognitive functions in cerebral ischemia rats, there is no direct evidence to show that Sal B could pass through the blood-brain barrier [45], and the exact mechanism(s) by which Sal B acts on adult neurogenesis remain unclear. More advanced research is needed in the future to further clarify the pathways and mechanisms of Sal B in promoting adult neurogenesis including using appropriate tool drugs to block certain pathways to confirm NSPCs proliferation by Sal B on contributing to the cognitive improvement. In conclusion, the results of this work clearly demonstrated that Sal B was capable of promoting proliferation of NSPCs and improving the learning and memory ability of cerebral ischemic rats. Additionally, we confirmed that Sal B promoteed NSPCs self- renew and neurogensis were at least in part mediated by the PI3K/Akt signaling pathway. These findings suggested that Sal B may act as a potential drug in treatment brain injury or neurodegenerative disease. Materials and Methods Reagents Salvianolic acid B (Sal B, purity .99%) was purchased from the Chinese National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), When used, it was freshly prepared in phosphate buffer solution (PBS); B27 (without retinoic acid) was purchased from Invitrogen (Carlsbad, CA, USA); Recombinant human fibroblast growth factor 2 (FGF-2) was from Millipore (Temecula, CA); Antibodies for ERK1/2, phospho- ERK1/2, Akt, phosphor-Akt (Ser-473) were obtained from Cell Signaling Technology (Beverly, MA). Antibody for b-actin was purchased from senta; 1,4-diamino-2,3-dicyano-1,4-bis(2-amino- phenylthio)-butadiene (U0126) and 2-(4-morpholinyl)-8-phenyl- 4H-1-benzopyran-4-one (Ly294002) was from CalBiochem (San Diego, CA). U0126 and Ly294002 were solubilized in dimethyl- sulphoxide. Cell culture Primary neurospheres were isolated from the cerebral cortex of 13.5-day-embryonic Wistar rats, according to the method Salvianolic Acid B Promote Neurogenesis described by Davis et al [46]. Briefly, the cerebral cortex was carefully isolated from adjacent tissues, and collected in cold serum-free medium consisting of DMEM/F-12 (1:1; Invitrogen, Carlsbad, CA, USA). The tissue was digested at 37uC for 10 min by acctuase (Millipore, Temecula, CA), and mechanically disrupted into single cells by filtering through a nylon mesh of 70 mm. The dissociated cells were then plated at a concentration of 26105 cells/ml in T75 culture flasks for 20 ml, and cultured in neurosphere proliferation media consisting of Dulbecco’s Modified Eagle’s Medium (DMEM): F12 supplemented with 2% (v/v) B27 supplement (Invitrogen, Carlsbad, CA, USA), 20 ng/ml epidermal growth factor (EGF; PEPROTECH, Rocky Hill, NJ, USA), and 20 ng/ml fibroblast growth factor 2 (FGF2). After 6 days in culture, the proliferating cells formed the neurospheres, which were suspended in the medium. Subsequently, the neurospheres were passaged by treatment with accutase about 5 min at 37uC until they were gently dissociated, and then subcultured as single cells in a new T75 culture flask at a density of 20,000 cells/ml in the fresh culture medium. The procedure of subculture was repeated again to achieve the purified cortical NSPCs and proliferating neurospheres. The 3–5 passages of NSPCs were used for the following experiments. MTS assay For in vitro cell proliferation assay, A Cell Titer 96 AQueous One Solution Cell Proliferation Assay (Promega, Charbonnie`res-les- Bains, France) was used. NSPCs were plated at 30, 000 cells/well in a 96-well plate in the presence and absence of Sal B (0.5, 1, 5, 10, 20 mM), U0126 (10 mg/ml), DAPT (10 mg/ml) and Ly294002 (20 mg/ml) in a 96-well plate. Cell proliferation was assessed at day 2 of cell culture. According to the manufacturer’s recommenda- tions, 40 ml MTS solution was then added into each of the wells. Then cells were incubated for 2 h at 37uC in the humidified 5% CO2 atmosphere incubator, and results were obtained at a wavelength of 490 nm using a microplate reader (FlexStation 3, Molecular Devices, USA). The same volume of medium without cells was used as blank. Results were expressed in Optical Density (OD). In vitro BrdU-incorporation assay For in vitro BrdU-incorporation assay, cultured NSPCs were incubated with or without Sal B for 24 h, the cells were labeled with BrdU (10 mg/ml) during the last 12 h of incubation, then the cells were plated onto poly-L-lysine coated slides for 2 h, fixed in 4% paraformaldehyde, washed with phosphate-buffered saline (PBS) and incubated in 2 N HCl at 37uC for 10 min. After washing with PBS, the cells were incubated with mouse anti-BrdU antibody (1:100, senta) at 4uC for 24 h in PBS. After washing in PBS, they were then incubated at room temperature for 1 h in PBS containing FITC conjugated anti-mouse IgG secondary antibody (1:200, senta). BrdU-positive cells were evaluated using a fluorescent microscope (Leica, German). BrdU positive cells were counted in 10 randomly selected fields from three different chambers. RT-PCR analysis Cells were harvested and total RNA was isolated from treated or untreated NSPCs with TRIzol (Roche), the first strand cDNA was synthesized from 0.4 mg of total RNA using Reverse Transcriptase (Takala) and Random primer as described in the manufacture’s instructions. After synthesis, 5 ml of cDNA was used in PCR reaction with gene-specific primers, The sequences of the PCR primer pairs (59 to 39) that were used for each gene are as follows: rat nestin, 59- TTCCCTTCCCCCTTGCCTAATACC-39 (for- PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e35636