BACKGROUND:It has been previously shown that hyperbaric oxygen may promote proliferation of neural stem cells and reduce death of endogenous neural stem cells(NSCs). OBJECTIVE:To explore the effects of hyperbaric oxygen on the differentiation of hypoxic/ischemic brain-derived NSCs into neuron-like cells and compare with high-concentration oxygen and high pressure. DESIGN,TIME AND SETrlNG:An in vitro contrast study,performed at Laboratory of Neurology, Central South University between January and May 2006. MATERIALS:A hyperbaric oxygen chamber(YLC 0.5/1A) was provided by Wuhan Shipping Design Research Institute;mouse anti-rat microtubule-associated protein 2 monoclonal antibody by Jingmei Company,Beijing;mouse anti-rat glial fibrillary acidic protein monoclonal antibody by Neo Markers, USA;mouse anti-rat galactocerebroside monoclonal antibody by Santa Cruz Biotechnology Inc., USA;and goat anti-mouse fluorescein isothiocyanate-labeled secondary antibody by Wuhan Boster Bioengineering Co.,Ltd.,China. METHODS:Brain-derived NSCs isolated from brain tissues of neonatal Sprague Dawley rats were cloned and passaged,and assigned into five groups:normal control,model,high-concentration oxygen,high pressure,and hyperbaric oxygen groups.Cells in the four groups,excluding the normal control group,were incubated in serum-containing DMEM/F_(12) culture medium.Hypoxic/ischemic models of NSCs were established in an incubator comprising 93%N_2,5%CO_2,and 2%O_2. Thereafter,cells were continuously cultured as follows:compressed air(0.2 MPa,1 hour,once a day) in the high pressure group,compressed air+a minimum of 80%O_2 in the hyperbaric oxygen group, and a minimum of 80%O_2 in the high-concentration oxygen group.Cells in the normal control and model groups were cultured as normal. MAIN OUTCOME MEASURES:At day 7 after culture,glial fibrillary acidic protein, microtubule-associated protein 2,and galactocerebroside immunofluorescence staining were examined to observe differentiation and calculate the percentage of NSCs differentiating into neuron-like cells or neuroglia-like cells. RESULTS:Neuron-like cells or neuroglia-like cells were visualized in all five groups.There were no significant differences in the percentage of differentiating cells between the hyperbaric oxygen group and the normal control group(P>0.05).The percentage of NSCs differentiating into neuron-like cells in the hyperbaric oxygen group was significantly greater than model,high-concentration oxygen,and high pressure groups;however,the percentage differentiating into neuroglia-like cells was significantly lower(P<0.01). CONCLUSION:Hyperbaric oxygen promotes the differentiation of brain-derived neural stem cells into neuron-like cells but inhibits differentiation into neuroglia-like cells.Furthermore,the efficacy of hyperbaric oxygen is superior to high-concentration oxygen and high pressure. BACKGROUND: It has been previously shown that hyperbaric oxygen may promote proliferation of neural stem cells and reduce death of endogenous neural stem cells (NSCs). OBJECTIVE: To explore the effects of hyperbaric oxygen on the differentiation of hypoxic / ischemic brain-derived NSCs into DESIGN, TIME AND SETrlNG: An in vitro contrast study, performed at Laboratory of Neurology, Central South University between January and May 2006. MATERIALS: A hyperbaric oxygen chamber (YLC Mouse / anti-rat microtubule-associated protein 2 monoclonal antibody by Jingmei Company, Beijing; mouse anti-rat glial fibrillary acidic protein monoclonal antibody by Neo Markers, USA; mouse anti-rat galactocerebroside monoclonal antibody by Santa Cruz Biotechnology Inc., USA; and goat anti-mouse fluorescein isothiocyanate-labeled secondary antibody by Wuhan Boster Bioengineering Co. , Ltd., China. METHODS: Brain-derived NSCs isolated from brain tissues of neonatal Sprague Dawley rats were cloned and passaged, and assigned into five groups: normal control, model, high-concentration oxygen, high pressure, and hyperbaric oxygen groups. Cells in the four groups, excluding the normal control group, were incubated in serum-containing culture medium. Hypoxic / ischemic models of NSCs were established in an incubator consisting of 93% N 2, 5% CO 2, and 2% O_2. After, cells were continuously cultured as follows: compressed air (0.2 MPa, 1 hour, once a day) in the high pressure group, compressed air + a minimum of 80% O_2 in the hyperbaric oxygen group, and a minimum of 80 % O_2 in the high-concentration oxygen group. Cells in the normal control and model groups were cultured as normal. MAIN OUTCOME MEASURES: At day 7 after culture, glial fibrillary acidic protein, microtubule-associated protein 2, and galactocerebroside immunofluorescence to observe differentiation and calculate the percentage of NSCs differentiating into neuron-like cells or neuroglia-like cells. RESULTS: Neuron-like cells or neuroglia-like cells were visualized in all five groups. There were no significant differences in the percentage of differentiating cells between the hyperbaric oxygen The percentage of NSCs differentiating into neuron-like cells in the hyperbaric oxygen group was significantly greater than model, high-concentration oxygen, and high pressure groups; however, the percentage differentiating into neuroglia -like cells was significantly lower (P <0.01). CONCLUSION: Hyperbaric oxygen promotes the differentiation of brain-derived neural stem cells into neuron-like cells but inhibits differentiation into neuroglia-like cells. Future Advanced, the efficacy of hyperbaric oxygen is superior to high-concentration oxygen and high pressure.