The Effect Of Nitric Oxide On The Differentiation Of Neural Stem Cells/Neural Precursors

ObjectiveTo observe the effect of inhibiton of endogenous nitric oxide (NO) production andexogenous nitric oxide on the differentiation of neural stem cells/neural precursors(NSCs/NPs) from newborn rats in vitro.Methods1. Conventional culture method was used to isolate subventricular zone (SVZ)region of newborn rats for NSCs/NPs culture. 5-bromo-2-deoxyuridine (BrdU) wasincorporated to mark proliferating cells.2. N-nitro-L-arginine methylester (L-NAME), inhibitor of neuronal nitric oxidesynthase (nNOS), was used to observe the effect of inhibihition of endogenous NOproduction on the differentiation of NSCs/NPs.3. Diethylenetriamine/NO (DETA/NO), NO donor, was used to observe the effect ofexogenous nitric oxide on the differentiation of NSCs/NPs.4. The immunocytochemistry was used to identify the expression of nestin (a markerof NSCs/NPs),β-Ⅲ-tubulin (Tuj-1, a marker of neurons), glial fibrillary acidic protein(GFAP, a marker of astrocytes), BrdU and nNOS.5. The release of NO was measured by Greiss assay in medium.Results1. Cells isolated from SVZ could form neurospheres of proliferating cells after 5-7days in vitro. Primary or subcultured neurospheres were nestin-positive and most cells ofneurospheres were BrdU-positive. Meanwhile, neurospheres could differentiate intoneurons which were Tuj-1 positive and astrocytes which were GFAP positive.2. After treatment with 100μM,150μM and 200μM L-NAME for 5 days, theconcentration of NO released was 28.4±3.0μM,27.9±2.7μM and 25.2±4.4μM,respectively, which was decreased significantly as compared with that of the control group(34.1±2.7μM) (P＜0.05). The percentage of differentiated neurons was 34.5±5.2 %,33.8±5.7% and 32.6±7.2% at 100μM,150μM and 200μM L-NAME, respectively,which was decreased significantly (P＜0.05) as compared with that of the control group(40.8±6.7%). The percentage of differentiated astrocytes was 27.5±4.1% and 27±7.5% at 150μM and 200μM L-NAME, respectively, which was also significantly (P＜0.05) as compared with that of the control group (36.2±4.3%).The expression of nNOSin the differentiated cells was 19.0±5.3%,18.3±3.0% and 17.8±4.9% at 100μM,150μM and 200μM L-NAME, respectively, which was decreased significantly (P＜0.05) ascompared with that of the control group (25.9±6.4%).3. After treatment with 40μM,50μM and 60μM DETA/NO for 5 days, theconcentration of NO released was 62.0±6.4μM,64.8±15.7μM and 68.5±11.6μM,respectively, which was increased significantly (P＜0.01) as compared with that of thecontrol group (25.6±16.4μM). The percentage of differentiated neurons attained 53.1±4.7%,54.1±6.9% and 63.8±9.5% at 40μM,50μM and 60μM DETA/NO, whilethe percentage of differentiated astrocytes was only 36.6±6.3%,38.2±6.7% and 41±10.5% at 40μM,50μM and 60μM DETA/NO, respectively.ConclusionsNO could promote the differentiation of NSCs/NPs in vitro, particularly into neurons.