Effects Of ATRA On The Differentiation Of Neural Stem Cells/Neural Precursors From Neonatal Rats

Objective:This experiment aimed at exploring the effects of ATRA (all trans-retinoic acid)aloneor combined with BDNF(brain-derived neurotrophic factor), GDNF(glial cell line-derivedneurotrophic factor) on the differentiation of NSCs/NPs (neural stem cells/neuralprecursors) from neonatal rats in vitro.Methods:The forebrain lateral ventricular SVZ (subventricular zone) region or hippocampuscontaining SGZ (subgranular zone) from neonatal SD rats (0～48h) was dispersed andcultivated. The EGF-responsive secondary neurospheres from SVZ region were used toobserve the effects of ATRA, BDNF, or GDNF alone and ATRA combined with BDNF orGDNF on differentiation. Immunocytochemistry was used to identify the expression of cellmarkers nestin, MAP-2 (microtubule-associated protein 2), GFAP (glial fibrillary acidicprotein) and MBP (myelin basic protein). Immunofluorescence was used to observe thecoexpression of nestin/GFAP and nestin/MAP-2.Results:1. EGF (epidermic growth factor), bFGF (basic fibroblast growth factor) andEGF+bFGF all could promote the proliferation of the primary cells from neonatal rats'SVZ region or SGZ region. The diameters of EGF-responsive neurospheres exceeded theones of bFGF-responsive neurospheres. The effect of EGF+bFGF exceeded that of EGFalone. Cells from SVZ region were more sensitive to EGF than the cells from SGZ region.2. Both primary and secondary EGF-responsive neurospheres from SVZ region werenestin&GFAP-positive. Double labeling immunofluorescence indicated that there wereneurosphere cells with coexpression of nestin and GFAP.3. Cells of secondary neurosphere cultivated for 6～7 days could differentiate into MAP-2-positive neurons (9.02±0.71%), GFAP-positive astrocytes(29.86±8.41%) andoccasionally into MBP-positive oligodendrocytes (trace), but the majority of cells werestill nestin-positive (84.45±5.36%). Coexpression of nestin/GFAP, but not nestin/MAP-2,could be observed in differentiatied cells.4. The number of MAP-2-positive cells was significantly increased in ATRA(1μM)group (29.14±5.00%) as compared with that of the control group(7.19±1.21%), while nodifference in the number of MAP-2-positive cells was observed between the ATRA groupand the groups combined with BDNF (10 ng/ml)or GDNF(10 ng/ml). GFAP and nestinexpressions were similar between each group.Conclusions:1. Cells from SVZ region and SGZ region of neonatal rats could response to EGFand/or bFGF, and EGF was more effective than bFGF.2. There were EGF-responsive NSCs/NPs in the SVZ region of neonatal rats and underthe natural conditions, the majority of cells turned into the cell phenotype of glia.3. ATRA could enhance the differentiation of neural stem cells/neural precursors intothe phenotype of neurons. No significant synergistic effect was observed when ATRA wascombined with BDNF or GDNF.