| Objectives:Neural stem cell niche is a key factor to regulate the self-renewal, proliferation and differentiation of neural stem cells.However,little is known about how ischemia affects the neural stem cell niche in the premature brain.In order to clarify the mechanisms how ischemia effect on the neurogensis in the premature brain, we observed the proliferation and differentiation of neural stem cells and the changes of neural stem cell niche in a 3-day-old rat model following brain ischemia in this study.Furthermore,we elucidated the pathways which related to the neural stem cell niche through observing the effects of soluble factors secreted by vascular endothelial cells on proliferation and differentiation of neural stem cells.Besides,the effect of basic fibroblast growth factor(bFGF) on the proliferation and differentiation of neural stem cells after brain ischemia were investigated.The study will provide novel evidence for intervention of ischemic premature brain injury.Methods:Part 1.The bilateral common carotid artery of rats was occluded to prepare the premature brain ischemia models.Proliferating cells were labeled by bromodeoxyuridine(BrdU) through intraperitoneal injection.By immunfluorescence staining,we observed the proliferation and differentiation of neural stem cells in the subventricular zone(SVZ) at 24h,4d,7d,and 14d after ischemic injury.And by used cDNA microarry chip,we monitored the changes of genes expression profile after brain ischemia.The significant change genes were testified by real-time quantitative polymerase chain reaction(PCR) and Western blot analysis.Part 2.SVZ brain slices of 3-day-old SD rats were cultured in vitro,they were divided into 3 groups randomly, named co-culture group(co-culture with vascular endothelial cells),co-culture and siRNA group(co-culture with vascular endothelial cells and siRNA) and control group(only culture brain slices),respectively.A mouse vascular endothelial cell line (bEnd.3,No.CRL-2299TM) was used in the co-culture.RNA interference for knock-down VEGF gene expression was synthesized and transfection by liposome methods.At 4d,7d,and 10d of cultured,the proliferation and differentiation of neural stem cells in the brain slices were detected by immunfluorescence stain. Synchronously,the changes of genes involved neurogenesis and angiogenesis were analyzed by GeneChip and the relative signal pathways were further confirmed by real-time PCR.Part 3.One hundred and eight 3-day-old SD rats with brain ischemia were used in this experiment and half of them were received bFGF 10ng/g through lateral ventricles injection.Besides,fifty four 3-day-old SD rats without brain ischemia were used as normal control.Proliferating cells in brain were labeled by BrdU through intraperitoneal injection in a pulsed or a cumulative protocol.Rats were killed at 4d,7d,and 14d after operation,and the proliferation and differentiation of neural stem cells in the SVZ were observed by immunfluorescence assays,real-time PCR and Western blot analysis to evaluate the impact of bFGF on bain injury repair.Results:Part 1:(1) There were cerebral edema widely;neuronal cells necrosis and focal cerebromalacia formation in the ischemic rats.These damages were the most serious at the 4th day after the operation.(2) In brain ischemic rats, BrdU+/Nestin+ cells in the SVZ were significantly increased at all four time points and more neural stem cells differentiated into neurons,astrocytes,and oligodendrocytes,compared with the rats without brain ischemia(P＜0.01).(3) GeneChip analysis showed a 3- to 10-fold increase in the mRNA expression of vascular endothelial growth factor(VEGF),transforming growth factor-beta (TGF-beta),and their receptors in the brain SVZ of ischemic rats,compared with that of without ischemic rats.PCR and Western blot analyses also confirmed these results. The data indicated that vascular endothelial growth factor and transforming growth factor-beta might be two important factors that involved post-ischemic neural stem cell proliferation and differentiation.Part 2:(1) The positive stain cells of Nestin, Tuj1,GFAP and O4 in brain slices were significantly increased in the co-culture group,compared with that in the control group(P＜0.01).But,when VEGF siRNA was added into the co-culture all these four type cells were decreased markedly (P＜0.01 vs the control group and co-culture group).(2) GeneChip analysis showed that the expressions of 112 genes were up-regulated more than 2-fold,however, among them 81 genes were down-regulated more than 2-fold after administration with VEGF siRNA.(3) Analyzed by "significance analysis of microarray" software,we found these noticeable altered genes were classified into notch and pten signal pathways on the ground of KEGG database,and these changes were further confirmed by real-time PCR assayes.Part 3:(1) The brain pathological damages in ischemic rats described in part 1 were alleviative in bFGF group.(2) Treatment with bFGF significantly increased the number of new cells(BrdU positive stain) in the SVZ at all three time points(P＜0.01).The new neural stem cells(BrdU/Nestin double positive cells) were peaked at 7d after operation,and amount in the bFGF group was more than that in the control group(P＜0.01).While,the new neurons(BrdU/NeuN double positive cells),astrocytes(BrdU/GFAP double positive cells),and oligodendrocytes(BrdU/NG2 double positive cells) were peaked at 14d.Treatment with bFGF significantly enhanced the number of these three group cells at all three time points(P＜0.01).Moreover,Real-time PCR and Western blot assay confirmed these results.Conclusion:1.Ischemia induced the proliferation and differentiation of neural stem cells in the SVZ,which involved vascular endothelial growth factor and transforming growth factor-beta pathways.2.The soluble factors secreted by vascular endothelial cells enhance the proliferation and differentiation of neural stem cells in the SVZ.Moreover,inhibition of the expression of genes for angiogenesis attenuated the efficacy of vascular endothelial cells on neural stem cells.The effects of vascular endothelial cells on the neurogenesis were involved into many pathways,among them notch and pten signal pathways were important.3.bFGF stimulated the proliferation and differentiation of neural stem cells in neonatal rats after ischemic brain injury, which maybe is helpful for the neural repair after ischemia.
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