HBO Promotes the Differentiation of Neural Stem Cells via Interactions Between the Wnt3/ß-Catenin and BMP2 Signaling Pathways.

Hyperbaric oxygen (HBO) therapy may promote neurological recovery from hypoxic-ischemic encephalopathy (HIE). However, the therapeutic effects of HBO and its associated mechanisms remain unknown. The canonical Wnt/ß-catenin signaling pathways and bone morphogenetic protein (BMP) play important roles in mammalian nervous system development. The present study examined whether HBO stimulates the differentiation of neural stem cells (NSCs) and its effect on Wnt3/ß-catenin and BMP2 signaling pathways. We showed HBO treatment (2 ATA, 60 min) promoted differentiation of NSCs into neurons and oligodendrocytes in vitro. In addition, rat hypoxic-ischemic brain damage (HIBD) tissue extracts also promoted the differentiation of NSCs into neurons and oligodendrocytes, with the advantage of reducing the number of astrocytes. These effects were most pronounced when these two were combined together. In addition, the expression of Wnt3a, BMP2, and ß-catenin nuclear proteins were increased after HBO treatment. However, blockade of Wnt/ß-catenin or BMP signaling inhibited NSC differentiation and reduced the expression of Wnt3a, BMP2, and ß-catenin nuclear proteins. In conclusion, HBO promotes differentiation of NSCs into neurons and oligodendrocytes and reduced the number of astrocytes in vitro possibly through regulation of Wnt3/ß-catenin and BMP2 signaling pathways. HBO may serve as a potential therapeutic strategy for treating HIE.

The role of ß-catenin signaling pathway on proliferation of rats neural stem cells after hyperbaric oxygen therapy in vitro.

ß-catenin, a protein that functions in both cell adhesion and Wnt signaling, plays a key role in mammalian neural development. To investigate the role of ß-catenin in hyperbaric oxygen therapy (HBO)-induced neurogenesis after hypoxic ischemic brain damage (HIBD), we transfected ß-catenin siRNA and negative control siRNA into neural stem cells (NSCs) after HIBD. We found that HBO promoted NSCs differentiate into neurons or oligodendrocytes, and inhibited NSCs differentiate into astrocytes; HIBD brain tissue extract conditioned cultures promoted NSCs differentiate into neurons; ß-Catenin siRNA decreased the NSE-positive neurons and increased GFAP-positive astrocytes in the NSCs in vitro. Furthermore, the expression of Ngn1 protein and mRNA in NSCs was increased when HBO promoted NSCs differentiate into neurons after HIBD, and the expression of BMP-4 protein and mRNA was decreased when HBO depressed NSCs differentiate into astrocytes after HIBD. These results showed that ß-catenin-mediated transcriptional activation functions in the decision of NSCs to proliferate neurogenesis during HBO-induced after HIBD, and suggested that HBO therapy promotes the proliferation of neural stem cells in vitro, an effect that may be correlated with ß-catenin protein and HBO therapy could promote neurogenesis by ß-catenin-induced activated Ngn1 gene and repress astrocytogenesis by ß-catenin-induced down-regulated BMP-4 gene.

[Effect of hyperbaric oxygen administered at different pressures and different exposure time on differentiation of neural stem cells in vitro].

OBJECTIVE: To study the effect of hyperbaric oxygen (HBO) administered at different pressures and different exposure time on the differentiation of neural stem cells (NSCs) in vitro. METHODS: The cerebral cortices from newborn rats (0-3 days old) were sterilely collected, digested, and centrifuged. After removal of the supernatant, the cells were re-suspended with DMEM/F12 medium containing B27, bFGF and EGF. The NSCs of 2-3 passages were randomly divided into seven groups: a control (untreated) and 6 HBO treatment groups that NSCs were subjected to HBO treatment of different pressures (1, 2 or 3 ATA) and different exposure time (30 or 60 minutes). The differentiated NSCs were examined by neuron-specific enolase (NSE) immunocytochemistry 24 hrs later. Percentage of NSE positive cells differentiated from NSCs was assessed by fluorescent microscopy. RESULTS: The percentage of NSE positive cells differentiated from NSCs was the highest in the HBO 2ATA-60 min group (9.17+/-0.50%) (P<0.01), followed by the HBO 3ATA-60 min (7.89+/-0.62%), HBO 2ATA-30 min (6.72+/-0.76%), HBO 3ATA-30 min (6.08+/-0.57%), HBO 1ATA-60 min (5.45+/-0.52%), HBO 1ATA 30 min (3.85+/-0.44%) and control groups (3.72+/-0.88%). In addition to the HBO 1ATA-30 min group, the other HBO treatment groups had increased significantly percentage of NSE positive cells compared with the control group (P<0.01). Under the same pressure, the 60 min treatment groups had increased significantly percentage of NSE positive cells compared with the 30 min treatment groups (P<0.01). CONCLUSIONS: HBO treatment (2 ATA, 60 minutes) produces a best effect in the differentiation of NSCs into neurons.

[Hyperbaric oxygen promotes the migration and differentiation of endogenous neural stem cells in neonatal rats with hypoxic-ischemic brain damage].

OBJECTIVE: To explore the effects of hyperbaric oxygen (HBO) treatment on the migration and differentiation of endogenous neural stem cells (NSCs) in neonatal rats with hypoxic-ischemic brain damage (HIBD). METHODS: Seven-day-old Sprague-Dawley rats were randomly divided into the normal control (CON), the HIBD model and the HBO groups (HBO treatment was administered at 2 ATA, once daily for 7 days within 3 hrs after HIBD). HIBD model was prepared according to the classic Rice-Vannucci method. BrdU/DCX, BrdU/beta-tubulin, BrdU/GFAP and BrdU/O4 immunofluorescence were examined by confocal microscopy in the subventricular zone (SVZ) and the cortex 7, 14 and 28 days after HBO treatment. RESULTS: The BrdU(+)DCX(+) cells in the SVZ (84 +/- 21 cells/mm2) in the HBO group were significantly higher than those in the CON group (39 +/- 14 cells/mm2) (p<0.05) and the HIBD model group (68 +/- 17 cells/mm2) (p<0.05) 7 days after HBO treatment. Fourteen days after HBO treatment, the BrdU(+) DCX(+) cells decreased in the SVZ and more cells were observed in the cortex in the HBO group as compared with the CON group (p<0.01). The BrdU(+) beta-tubulin(+), BrdU(+)GFAP(+) and BrdU(+) O4(+) cells were observed in the cortex, and more BrdU(+)beta-tubulin(+) and BrdU(+) O4(+) cells were observed in the HBO group as compared with the CON and the HIBD model groups (p<0.05) 28 days after HBO treatment. CONCLUSIONS: HBO treatment may promote endogenous NSCs to migrate to the cortex and differentiate into mature neurocytes in neonatal rats with HIBD.

[Protective effects of delayed multiple course hyperbaric oxygen treatment against hypoxic-ischemic brain damage in neonatal rats].

OBJECTIVE: To study the protective effects of multiple course hyperbaric oxygen (HBO) treatment against hypoxic-ischemic brain damage (HIBD) in neonatal rats when HBO treatment is delayed (96 hrs after the HIBD event). METHODS: Eighty-eight 7-day-old Sprague-Dawley rat pups were randomly assigned to control, HIBD and HBO groups. The HBO group was subdivided into cohorts receiving treatment 2 h, 48 h and 96 h, respectively, after HIBD was induced. The three subgroups comprising different therapeutic windows were further randomly assigned to receive 1, 2 or 3 courses of HBO treatment ("HBO-1, -2 and -3 sub-groups"). HBO was administered once daily (2 ATA), a course lasting for seven days. There was an interval of three days between the courses. All pups were sacrificed at the end of HBO treatment (31 days after HIBD). TUNEL staining was used for testing neuronal apoptosis in the cortex and the CA1 of the hippocampus, and NSE staining was used to ascertain cortical neuronal population. RESULTS: 1.There were significantly more TUNEL positive cells in the HIBD group than in the control group; NSE positive cells were significantly lower than in controls (P<0.01). 2. With the more delayed therapeutic window, the effects of apoptosis inhibition and neuronal protection of a single course of HBO were gradually reduced. 3. With increasing courses of HBO treatment, the effects of apoptosis inhibition and neuronal protection of HBO increased gradually in rats receiving treatment 48 and 96 hrs after HIBD. In the HBO group receiving treatment 2 hrs after HIBD, the number of apoptotic cells and NSE positive cells were close to that of the control group after one course of HBO treatment. CONCLUSIONS: One course of HBO administered within 2 hrs after HIBD can effectively inhibit neuron apoptosis and protect neurons. The effects of apoptosis inhibition and neuron protection of HBO can be increased through increasing the number of HBO treatment courses in neonatal rats with HIBD even if initiation of treatment is delayed after HIBD.

Therapeutic window of hyperbaric oxygen therapy for hypoxic-ischemic brain damage in newborn rats.

Previous studies showed that hyperbaric oxygen (HBO) promoted cell proliferation in hypoxic-ischemic (HI) neonate rats. Neural stem cells (NSC) existed in the brain lifelong and can be activated. This study was undertaken to assess whether HBO treatment promoted the proliferation of NSC and repaired the brain damage regardless of when it is started, thus to explore the therapeutic window of HBO treatment. Seven-day-old Sprague-Dawley rats underwent left carotid ligation followed by 2 h of hypoxic stress (8% O(2) at 37 degrees C). Hyperbaric oxygen therapy was administered 3, 6, 12, 24, and 72 h after HI. 5-bromo-2'-deoxyurindine and 5-bromo-2'-deoxyuridine/nestin were detected by immunofluorescence and nestin was examined by western blot analysis 10 days after HI. T-maze forced alternation, the foot-fault test, and the radial arm maze were conducted at P 22 days (14 days after HI), P 30 days, and P 34 days. Thereafter, cerebral morphology was examined by Nissl-staining 28 days after HI. There were remarkable increases in the proliferation of neural stem cells in the HBO-treated group, 3, 6, 12, and 24 h after HI, as compared with the HIBD group. The HBO-treated group, 3, 6, and 12 h after HI, performed better in the behavioral test and had less neural loss in the hippocampal CA1 region as compared with the HIBD group. The therapeutic window for effective HBO treatment could be delayed up to 12 h after HIBD, while the effect decreased 24 h after HI.

Proliferation of neural stem cells correlates with Wnt-3 protein in hypoxic-ischemic neonate rats after hyperbaric oxygen therapy.

Hyperbaric oxygen therapy promoted brain cell proliferation. Wnt-3 is closely associated with the proliferation of neural stem cells. We examined whether hyperbaric oxygen promoted neural stem cells to proliferate and its correlation with Wnt-3 protein in hypoxic-ischemic neonate rats. Hyperbaric oxygen therapy was administered 3 h after hypoxia ischemia daily for 7 days. The proliferating stem cells and Wnt-3 protein were examined dynamically in the subventricular zone. Results showed that stem cells proliferated and peaked 7 days after hyperbaric oxygen therapy. Wnt-3 protein increased to the higher levels 3 days after therapy. Linear regression analysis showed that nestin protein correlated with Wnt-3 protein. We propose that hyperbaric oxygen treatment promote stem cells to proliferate, which is correlated with Wnt-3 protein.

[Effect of hyperbaric oxygen therapy administered at different time on white matter damage following hypoxic-ischemic brain damage in neonatal rats].

OBJECTIVE: A recent study has suggested that hyperbaric oxygen (HBO) therapy administered within 3 hrs following hypoxic-ischemic brain damage (HIBD) may alleviate brain white matter damage (WMD) in neonatal rats. However it is unclear whether a delayed HBO therapy (more than 3 hrs following HIBD) has neuroprotective effects in neonatal rats. This study aimed to explore the effect of HBO therapy administered at different time points following HIBD on WMD in neonatal rats. METHODS: The HIBD model was prepared according to the Rice-Vannucci procedure in 7-day-old Sprague-Dawley rats. HBO therapy was administered at 3, 6, 12, 24 or 72 hrs after HIBD, once daily for consecutive 7 days. T-maze test, the foot-fault test and the radial arm maze test were performed after 14 days of HIBD. Myelin basic protein (MBP) in the callositas and corpora striata was examined by immunohistochemical method 28 days after HIBD. RESULTS: The rats receiving HBO therapy at 3, 6 and 12 hrs after HIBD performed significantly better in the T-maze test, the radial arm maze test and the foot-fault test than the untreated HIBD rats. There were no significant differences in the behavioral test results between the HBO-treated groups administered HBO at 24 and 72 hrs after HIBD and the untreated HIBD group. The MBP expression in the HBO-treated groups treated within 12 hrs after HIBD was significantly higher than that in the untreated HIBD group (P < 0.05). When the HBO therapeutic window was delayed to 24 hrs after HIBD, there were no significant differences in the MBP expression between the HBO-treated and the untreated HIBD groups. CONCLUSIONS: HBO therapy administered within 12 hrs following HIBD can alleviate brain WMD in neonatal rats, but the efficacy of HBO therapy administered 24 hrs after HIBD does not appear to be satisfactory.

[Changes of Wnt-3 protein during the proliferation of endogenous neural stem cells in neonatal rats with hypoxic-ischemic brain damage after hyperbaric oxygen therapy].

OBJECTIVE: Previous studies suggest that hyperbaric oxygen (HBO) treatment promotes the proliferation of neurocytes in neonatal rats following hypoxic-ischemic brain damage (HIBD). The Wnt signaling pathway is associated with neurogenesis. This study examined whether HBO promoted neural stem cells (NSCs) proliferation after HIBD, and whether that the proliferation correlated with Wnt-3 protein expression. METHODS: Seven-day-old Sprague-Dawley rats were randomly divided into three groups: normal control, hypoxia-ischemia (HI), and HI-HBO. HI was induced by the ligation of left common carotid artery, followed by a 2-hr exposure to 8% O2 in the latter two groups. HBO was administered 3 hrs after HI in the HI-HBO group for continuous 7 days (2 atmospheres absolute, once daily). The proliferating NSCs in the subventricular zone (SVZ) was examined by BrdU/nestin immunofluorescence and the expression of Wnt-3 protein in NSCs was examined by nestin/Wnt-3 immunofluorescence at 6 and 24 hrs and at 3, 7 and 14 days of HI. The cellular expressions of nestin and Wnt-3 protein were analyzed by laser scanning confocal microscopy. The linear regression analysis was used to evaluate the correlation between cellular Wnt-3 and nestin protein. The expressions of nestin and Wnt-3 protein in the ischemic cerebral hemisphere were analyzed with Western blotting. RESULTS: The number of BrdU/nestin positive cells in the SVZ increased 3 hrs after HBO therapy, peaked at 7 days and remained at a higher level until 14 days after HBO therapy in the HI-HBO group compared with the normal control and the HIBD groups. The level of Wnt-3 protein in NSCs increased significantly 3 hrs after HBO therapy, peaked at 3 days and remained at high levels until 14 days after HBO therapy in the HI-HBO group compared with the normal control and the HIBD groups. The level of cellular nestin protein was closely correlated with the level of cellular Wnt-3 protein (r = 0.893, P < 0.05). The Western blotting analysis demonstrated increased Wnt-3 and nestin protein expressions in the ischemic cerebral hemispheres. CONCLUSIONS: HBO treatment promotes the proliferation of NSCs in HIBD neonatal rats, which is correlated with the activation of Wnt signaling.

[Protective effects of baicalin pretreatment on hypoxic-ischemic brain damage in neonatal rats].

OBJECTIVE: Previous research suggests that dexamethasone (Dex) pretreatment protects neonatal rats against hypoxic-ischemic brain damage (HIBD). Some of the pharmacological effects of baicalin (a traditional Chinese medicine extracted from Scutellaria baicalensis Georgi) are similar to Dex. This study was designed to explore the effect of baicalin on the neuronal apoptosis following HIBD in neonatal rats. METHODS: Six-day-old Sprague-Dawley rats were randomly assigned into Control (without HI), HIBD, Dex-pretreatment and post-treatment, Baicalin-pretreatment and -post-treatment groups. HIBD was induced by ligating the left common carotid artery, followed by exposure to hypoxia. In the pretreatment groups either baicalin (16 mg/kg) or Dex (0.1 mg/kg) was administered to the rats 24 hrs before HIBD; in the post-treatment groups baicalin or Dex was given 30 minutes after HIBD. The rat pups were sacrificed on postnatal day 10, and brain tissues were harvested. Brain water content was determined, morphological changes were observed under a light microscope, and neuronal apoptosis was measured by terminal deoxynucleotidyl transferase mediated dUTP biotin nick end labeling (TUNEL) staining. RESULTS: The brain water content and the number of apoptotic cells were significantly higher in the HIBD group than those of the Control group (P < 0.05). Both baicalin and Dex pretreatment decreased the brain water content from 88.9 +/- 1.7 % (HIBD group) to 87.4 +/- 0.7% (baicalin) or 87.3 +/- 0.6% (Dex) (P < 0.05) and the number of apoptotic cells were reduced from 251 +/- 28 (HIBD group) to 102 +/- 47 (baicalin) or 75 +/- 26 (Dex) (P < 0.05). Baicalin and Dex post-treatment had no effects on the brain water content and the number of apoptotic cells. Loss and degeneration of neurons could be observed in the HIBD group. Baicalin and Dex pretreatment significantly alleviated neuronal injury, but post-treatment did not. CONCLUSIONS: Pretreatment with baicalin, as with Dex, has a protective effect against HIBD in neonatal rats, but baicalin or Dex post-treatment do not reverse the neuronal injuries.

[Effect of hyperbaric oxygenation on neural stem cells and myelin in neonatal rats with hypoxic-ischemic brain damage].

OBJECTIVE: This study investigated the effect of hyperbaric oxygenation (HBO) on neural stem cells (NSCs) and myelin in neonatal rats following hypoxic-ischemic brain damage (HIBD) and aimed to explore the possible mechanism of the protective effect of HBO on HIBD. METHODS: Seven-day-old Sprague-Dawley rat pups were randomly assigned into 4 groups: Normal control, HIBD, hyperbaric air (HBA), and HBO groups (n=30 each). The HIBD model was produced by permanent occlusion of the left common carotid artery and 2 hrs hypoxemia exposure (8% O2 at 37 degrees C). HBA and HBO treatment was administered (2 ATA, once daily for 7 days) in the HBA and HBO groups respectively 1 hr after HIBD. BrdU immunohistochemistry was used to detect the NSCs in the sub-ventricle zone (SVZ) of the lateral ventricle and the dentate gyrus (DG) of the hippocampus. The myelin damage was assessed by myelin basic protein (MBP) immunostaining. RESULTS: The BrdU-positive cells in the SVZ and the DG of the ischemic hemisphere in the HIBD group were dramatically decreased compared with those of the Normal control group at 3 weeks post-HIBD (P < 0.01). The HBO treatment resulted in an increase of BrdU-positive cells in the DG from 153.7 +/- 37.0 to 193.7 +/- 38.8 (P < 0.05). The nestin expression in the HIBD and HBA groups was reduced compared with that in the Normal control group. There was no difference in the nestin expression between the HBO and the Normal control groups. Hypoxia-ischemia (HI) led to marked myelin damage at 1 week post-HIBD. HBO or HBA treatment alleviated the damage. CONCLUSIONS: The HBO treatment can result in the proliferation of BrdU-positive cells and alleviate the myelin damage following HIBD in neonatal rats, thereby offering neuroprotectivity against HI insults.

[Antagonistic effect of baicalin on oxidative stress injury in neurons and astrocytes of rats].

OBJECTIVE: To explore the experimental conditions for H2O2 to injure astrocytes and the effect of baicalin in protecting neurons and astrocytes from oxidative stress injury. METHODS: Neurons and astrocytes from forebrain of rats were cultured in vitro and treated with H2O2, baicalin and combination of the two, respectively. The cell viability or survival rate was determined using MTT. RESULTS: Effects of H2O2 in different concentrations on survival rate of astrocytes showed significant difference (F = 28.569, P < 0.01) in a dose-dependent manner. Degrees of H2O2 injury, with the same concentration of H2O2, on cells with different seeding density were also significantly different (F = 5.439, P < 0.01), and dose-dependently. Baicalin didn't influence the survival rate of neurons and astrocytes when the concentration was within 2.5-40 mumol/L (for neurons, F = 0.49, P > 0.05; for astrocytes, F = 1.001, P > 0.05), but baicalin showed significant antagonism to the injury of oxidative stress (for neurons, F = 24.384, P < 0.01; for astrocytes, F = 5.000, P < 0.01). The higher the concentration of bainalin, the higher the cell survival rate. CONCLUSION: A model of astrocytes oxidative injury induced by H2O2 is established. Baicalin shows no toxicity on neurons and astrocytes when the concentration is within 2.5-40 mumol/L, but could antagonize the H2O2 caused oxidative injury on cells in a dose-dependent manner.

[Protective effect and mechanism of pretreatment with curcumin on infectious brain edema in rats].

UNLABELLED: Curcumin is a natural compound extracted from the spice tumeric, possessing both anti-inflammatory antioxidant, and anti-carcinogenic effect, is a potent stimulator of the stress-induced expression of heat shock protein 70 kd (HSP70). OBJECTIVES: To study the protective effect of pretreatment with curcumin against infectious brain edema in rats, and investigate its mechanism by assessing the free radical, cytokine and HSP70 expression of the brain. METHODS: An animal model of infectious brain edema induced by injecting pertussis bacilli (PB) through carotid artery was used. SD rats were randomly divided into five groups: (1) Normal control group (NS group, n = 9); (2) Infectious brain edema group (PB group, n = 12); (3) DMSO control group (DMSO group, n = 9); (4) HS pretreatment group (HS group, n = 9); (5) Curcumin pretreatment group (CUR group, n = 13). The water content (WC), Na(+) and K(+) content in brain tissue were measured. The content of malondialdehyde (MDA) and super oxide dismitase (SOD) were assessed by chemical colorimetry. The levels of tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta) were detected by ELISA. The HSP70 expression was examined by Western blot analysis. RESULTS: (1) The WC and Na(+), MDA, TNF-alpha and IL-1beta were increased in PB group compared with NS group (P < 0.01); they were decreased in HS and CUR groups compared with PB group (P < 0.01 or P < 0.05). (2) The content of SOD was decreased in PB group than in NS group (P < 0.05), and was increased in HS and CUR group Compared with PB group, (P < 0.05). (3) Western blot analysis showed that the band density areas of HS, CUR and PB groups were higher than those in NS and DMSO groups, especially in CUR group (P < 0.01). CONCLUSION: Pretreatment with curcumin showed a protective effect against infectious brain edema in rats. The effect might be associated with antioxidant, inhibition of the activity of cytokines and inducing expression of HSP70 by curcumin.

[Study of protective effect and mechanism for baicalin on bacillus pertussis infected brain tissue and its dose-effect relationship].

OBJECTIVE: To study the possible mechanism of protective effect for Baicalin on Bacillus pertussis (BP) infected brain tissue and the dose-effect relationship. METHODS: Brain tissues slices were divided into 7 groups: (1) the normal group; (2) the model group: infected by 10% BP; (3) the baicalin group, which was pretreated with baicalin, infected by BP and subdivided into 5 sub-groups according to different doses of baicalin used; (4) the glutamic acid group: cultured with glutamic acid; (5) the baicalin plus glutamic acid group; (6) the peroxide group: cultured with hydrogen peroxide; and (7) the baicalin plus peroxide group. The lactate dehydrogenase (LDH) content in the supernatant of culture was determined and quantitative protein determination was conducted. RESULTS: The LDH releasing was higher in the model group, glutamic acid group and peroxide group as compared with that in the normal group, 15.10 +/- 4.89 u/g. protein (the same unit below), 15.49 +/- 5.66 and 16.54 +/- 5.47 vs 6.10 +/- 2.87 respectively (P < 0.01). After being pretreated with 0.25 mmol/L baicalin, LDH level decreased significantly to 8.65 +/- 2.43, which was significantly different from that in the model group (P < 0.01), LDH was also decreased in the baicalin plus glutamic acid group (9.93 +/- 2.89) and baicalin plus peroxide group (9.54 +/- 2.82), which was significantly lower than that in the glutamic acid group and the peroxide group respectively (P < 0.01). CONCLUSION: Pretreatment of baicalin has protective effect on BP caused nerve cell injury in rat brain slices, the protection is possibly related with the reduction of glutamic acid and hydrogen peroxide induced damage on nerve cells in vitro.

[Differentiation of rat bone marrow stromal cells into neuron induced by baicalin].

OBJECTIVE: To study the possibility that induced marrow stromal cells (MSCs) to neuron in vitro by baicalin, a kind of flavonoid isolated from an important medicinal plant Scutellariae Radix. METHODS: MSCs from adult SD rats were induced by baicalin in serum-free medium for 6 days. The expression of neuronal or glia specific markers and gene was evaluated by indirect immunofluorescence cytochemistry staining, Western blot, and reverse transcriptase-polymerase chain reaction (RT-PCR). The percentage of apoptotic cells at 6 days after baicalin treatment was measured by terminal deoxynucleotidyl transferase- mediated dUTP-biotin nick end-labeling (TUNEL) assay. Moreover, differentiating cells that labeled by green fluorescent protein vector were transplanted to the striatum of adult SD rats after 6d of baicalin treatment. RESULTS: After induction for 6 d, MSCs displayed neuronal morphologies, such as pyramidal cell bodies and processes formed extensive networks. The undifferentiated cells did not exhibit a neuronal or glial phenotype. The differentiating cells expressed nestin, a marker of neural stem cells, at 6 h, and disappeared at 6 d. In contrast, expression of neuron-specific markers and gene was detectable at 6 d. And glia-specific markers and gene was not expressed. The percentage of apoptotic cells was 12.2% +/- 2.8%. In addition, fluorescently labeled cells were readily detected in the brain section after 14d of transplantation. CONCLUSION: Baicalin may induce adult rat MSCs to neuron in vitro. It is a useful vehicles for autotransplantation in both cell and gene therapy for a variety of diseases of the central nervous system.