代谢型谷氨酸受体Ⅰ组在成年大鼠脑的神经生发区SVZ的表达

成年哺乳动物侧脑室外侧壁的脑室下带(SVZ)具有持续性的神经生发功能。但其受哪些物质调节还不清楚。本研究采 用原位杂交和免疫组织化学方法,显示了代谢型谷氨酸受体I组的mGluR1和mGluR5在SVZ及其邻近室管膜细胞有明显表 达,mGluR5还在侧脑室内侧壁室管膜细胞表达。本研究结果提示,脑室下带细胞可能受谷氨酸的调节。     

The Notch pathway mediates expansion of a progenitor pool and neuronal differentiation in adult neural progenitor cells after stroke

Molecular mechanisms by which stroke increases neurogenesis have not been fully investigated. Using neural progenitor cells isolated from the subventricular zone (SVZ) of the adult rat subjected to focal cerebral ischemia, we investigated the Notch pathway in regulating proliferation and differentiation of adult neural progenitor cells after stroke. During proliferation of neural progenitor cells, ischemic neural progenitor cells exhibited substantially increased levels of Notch, Notch intracellular domain (NICD), and hairy enhancer of split (Hes) 1, which was associated with a significant increase of proliferating cells. Blockage of the Notch pathway by short interfering ribonucleic acid (siRNA) against Notch or a γ secretase inhibitor significantly reduced Notch, NICD and Hes1 expression and cell proliferation induced by stroke. During differentiation of neural progenitor cells, Notch and Hes1 expression was downregulated in ischemic neural progenitor cells, which was coincident with a significant increase of neuronal population. Inhibition of the Notch pathway with a γ secretase inhibitor further substantially increased neurons, but did not alter astrocyte population in ischemic neural progenitor cells. These data suggest that the Notch signaling pathway mediates adult SVZ neural progenitor cell proliferation and differentiation after stroke.     

Selective upregulation of 3-phosphoglycerate dehydrogenase (Phgdh) expression in adult subventricular zone neurogenic niche

In the adult rodent brain, constitutive neurogenesis occurs in two restricted regions, the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus, where multipotent neural stem/progenitor cells generate new neurons. Using Western blotting and immunohistochemistry for established markers, we demonstrated that the expression of 3-phosphoglycerate dehydrogenase (Phgdh), an enzyme involved in de novo synthesis of l-serine, was upregulated in the SVZ. The expression was selective to cells having morphological features and expressing markers of astrocyte-like primary neural stem cells (type B cells) and their progeny, actively proliferating progenitors (type C cells). By contrast, Phgdh protein expression was virtually absent in committed neuronal precursors (type A cells) derived from type C cells. High levels of Phgdh were also expressed by glial tube cells located in the rostral migratory stream (RMS). Interestingly, ensheathment of type A cells by these Phgdh-expressing cells was persistent in the SVZ and RMS, suggesting that l-serine mediates trophic support for type A cells via these glial cells. In vitro neurosphere assays confirmed that growth-factor-responsive, transient amplifying neural progenitors in the SVZ, but not differentiated neurons, expressed Phgdh. In the aged brain, a decline in Phgdh expression was evident in type B and C cells of the SVZ. These observations support the notion that availability of l-serine within neural stem/progenitor cells may be a critical factor for neurogenesis in developing and adult brain.     

胰岛素样生长因子1在新生大鼠短暂脑缺血损伤修复中的关键作用

目的7日龄新生大鼠短暂脑缺血诱导侧脑室室管膜下区（SVZ）神经干细胞增殖,观察胰岛素样生长因子1（IGF-1）对神经干细胞增殖的影响。方法 7日龄新生SD大鼠64只,随机分为缺血组（n =24）、假手术组（n =24）、缺血阻断剂组（n =8）和缺血生理盐水组（n=8）。利用免疫组织化学方法观察缺血组和假手术组在缺血后1、4、7d SVZ新生细胞数量和IGF-1表达的变化,并观察缺血阻断剂组和缺血生理盐水组在IGF-1受体阻断剂（JB1）阻断7d后SVZ新生细胞数量和IGF-1表达的变化。结果 与同时间点假手术组比较,缺血后1、4、7d的SVZ新生细胞和IGF-1阳性细胞数量均显著增加,差异有统计学意义（P ＜0.05）;使用JB1后,缺血阻断剂组IGF-1的表达被阻断,IGF-1阳性细胞缺如,而缺血生理盐水组IGF-1表达正常;使用JB1后,缺血阻断剂组SVZ新生细胞数量显著减少,与缺血生理盐水组相比,差异有统计学意义（P ＜0.05）。结论 新生大鼠缺血再灌注损伤上调了IGF-1的表达,IGF-1表达增加促使SVZ神经干细胞增殖;使用JB1后,IGF-1的表达被阻断,神经干细胞增殖也显著减少,提示IGF-1在脑缺血损伤修复中起关键作用。     

Cilostazol attenuates ischemic brain injury and enhances neurogenesis in the subventricular zone of adult mice after transient focal cerebral ischemia

Evidence suggests that neurogenesis occurs in the adult mammalian brain, and that various stimuli, for example, ischemia/hypoxia, enhance the generation of neural progenitor cells in the subventricular zone (SVZ) and their migration into the olfactory bulb. In a mouse stroke model, focal ischemia results in activation of neural progenitor cells followed by their migration into the ischemic lesion. The present study assessed the in vivo effects of cilostazol, a type 3 phosphodiesterase inhibitor known to activate the cAMP-responsive element binding protein (CREB) signaling, on neurogenesis in the ipsilateral SVZ and peri-infarct area in a mouse model of transient middle cerebral artery occlusion. Mice were divided into sham operated (n=12), vehicle- (n=18) and cilostazol-treated (n=18) groups. Sections stained for 5-bromodeoxyuridine (BrdU) and several neuronal and a glial markers were analyzed at post-ischemia days 1, 3 and 7. Cilostazol reduced brain ischemic volume (P<0.05) and induced earlier recovery of neurologic deficit (P<0.05). Cilostazol significantly increased the density of BrdU-positive newly-formed cells in the SVZ compared with the vehicle group without ischemia. Increased density of doublecortin (DCX)-positive and BrdU/DCX-double positive neural progenitor cells was noted in the ipsilateral SVZ and peri-infarct area at 3 and 7 days after focal ischemia compared with the vehicle group (P<0.05). Cilostazol increased DCX-positive phosphorylated CREB (pCREB)-expressing neural progenitor cells, and increased brain derived neurotrophic factor (BDNF)-expressing astrocytes in the ipsilateral SVZ and peri-infarct area. The results indicated that cilostazol enhanced neural progenitor cell generation in both ipsilateral SVZ and peri-infarct area through CREB-mediated signaling pathway after focal ischemia.     

Characterization of novel monoclonal antibodies able to identify neurogenic niches and arrest neurosphere proliferation and differentiation

Two monoclonal antibodies (Nilo1 and Nilo2) were generated after immunization of hamsters with E13.5 olfactory bulb-derived mouse neurospheres. They are highly specific for neural stem and early progenitor cell surface antigens. Nilo positive cells present in the adult mouse subventricular zone (SVZ) were able to initiate primary neural stem cell cultures. Moreover, these antibodies added to neurosphere cultures induced proliferation arrest and interfered with their differentiation. In the lateral ventricles of adult mice, Nilo1 stained a cell subpopulation lining the ventricle and cells located in the SVZ, whereas Nilo2 stained a small population associated with the anterior horn of the SVZ at the beginning of the rostral migratory stream. Co-staining of Nilo1 or Nilo2 and neural markers demonstrated that Nilo1 identifies an early neural precursor subpopulation, whereas Nilo2 detects more differentiated neural progenitors. Thus, these antibodies identify distinct neurogenic populations within the SVZ of the lateral ventricle.     

Leukemia inhibitory factor participates in the expansion of neural stem/progenitors after perinatal hypoxia/ischemia

Subsequent to perinatal hypoxia/ischemia there is an increase in the number of neural stem/progenitor cells (NSP) within the subventricular zone (SVZ). Gene expression analyses have implicated Notch signaling in the expansion of these tripotential cells but there are limited data as to which signals are stimulating Notch activation. There is evidence that the leukemia inhibitory factor receptor (LIFR)/gp130 receptor heterodimer induces Notch1 to maintain NSP populations during normal development. LIF and ciliary neurotrophic factor (CNTF) bind to these receptor components and they coordinate injury responses in the CNS. Therefore, the aim of these studies was to investigate whether CNTF and/or leukemia inhibitory factor (LIF) participate in NSP expansion in the rat SVZ after hypoxia/ischemia (H/I) as well as to characterize the downstream events that regulate NSP numbers. We report that LIF mRNA is induced 48 h post-insult by 13-fold but that it returns almost to baseline by 72 h. Commensurate with increased LIF expression there is a corresponding increase in phosphorylated Stat-3 within the SVZ. Modeling the changes that occur in vivo, we show that LIF induces Stat-3 phosphorylation in neurospheres to enhance Delta-like-1 and Notch1 expression as well as to increase Notch1 activation. LIF also expands neurosphere number and size in vitro. Whereas CNTF can mimic the effects of LIF in vitro, CNTF expression in the SVZ was unchanged during recovery from H/I. Cumulatively, these data implicate LIF and not CNTF in the expansion of NSPs in the rat SVZ after perinatal brain injury. As both LIF expression and the endogenous regenerative response after brain injury are time-delimited, these findings provide insights into strategies to expand the endogenous pool of NSPs to repopulate the damaged brain.     

兴奋性氨基酸抑制胎鼠神经干细胞的增殖

目的：研究兴奋性氨基酸对体外培养胎鼠神经干细胞（NSCs）增殖的影响。 方法:体外分离、培养与鉴定SD胚胎大鼠脑室下带(SVZ)神经干细胞，利用MTT比色法测定兴奋性氨基酸N-甲基-D-天冬氨酸（NMDA）和谷氨酸（Glu）对细胞增殖的作用。 结果:成功分离出具有胚胎源性的神经干细胞，NMDA和Glu均能导致神经干细胞的增殖活性下降。 结论:培养的SVZ区域细胞具有自我更新和多分化潜能，是中枢神经系统干细胞，兴奋性氨基酸能有效地抑制神经干细胞的增殖。     

成年和老年大鼠局灶性脑缺血后脑室下区细胞的增殖分化

目的：比较老年和成年大鼠局灶性脑缺血后脑室下区（SVZ）神经干细胞的增殖与分化。方法：制作大脑中动脉梗死模型,用免疫组化法检测SVZ的5-溴脱氧尿核苷（BrdU）、神经元核抗原（NeuN）及胶质纤维酸性蛋白（GFAP）阳性细胞数的变化。结果：SVZ的BrdU阳性细胞在正常组和假手术组成年大鼠明显多于老年大鼠。实验组成年和老年大鼠均在缺血后增加,28d时仍高于正常水平,但成年大鼠各时间点均明显高于老年大鼠。在新生细胞中部分细胞是Brdu／NeuN或BrdU／GFAP双标细胞,但老年大鼠Brdu／NeuN双标细胞明显少于成年大鼠。结论：大鼠脑缺血激活SVZ神经干细胞增殖能力,成年大鼠明显强于老年大鼠,且新生细胞分化为神经元的比例也明显高于老年大鼠。     

Beta-catenin signaling promotes proliferation of progenitor cells in the adult mouse subventricular zone

The subventricular zone (SVZ) is the largest germinal zone in the mature rodent brain, and it continuously produces young neurons that migrate to the olfactory bulb. Neural stem cells in this region generate migratory neuroblasts via highly proliferative transit-amplifying cells. The Wnt/beta-catenin signaling pathway partially regulates the proliferation and neuronal differentiation of neural progenitor cells in the embryonic brain. Here, we studied the role of beta-catenin signaling in the adult mouse SVZ. beta-Catenin-dependent expression of a destabilized form of green fluorescent protein was detected in progenitor cells in the adult SVZ of Axin2-d2EGFP reporter mice. Retrovirus-mediated expression of a stabilized beta-catenin promoted the proliferation of Mash1+ cells and inhibited their differentiation into neuroblasts. Conversely, the expression of Dkk1, an inhibitor of Wnt signaling, reduced the proliferation of Mash1+ cells. In addition, an inhibitor of GSK3 beta promoted the proliferation of Mash1+ cells and increased the number of new neurons in the olfactory bulb 14 days later. These results suggest that beta-catenin signaling plays a role in the proliferation of progenitor cells in the SVZ of the adult mouse brain.     

Endogenous hepatocyte growth factor is a niche signal for subventricular zone neural stem cell amplification and self-renewal

Neural stem cells persist in the adult mammalian brain, within the subventricular zone (SVZ). The endogenous mechanisms underpinning SVZ neural stem cell proliferation, self-renewal, and differentiation are not fully elucidated. In the present report, we describe a growth-stimulatory activity of liver explant-conditioned media on SVZ cell cultures and identify hepatocyte growth factor (HGF) as a major player in this effect. HGF exhibited a mitogenic activity on SVZ cell cultures in a mitogen-activated protein kinase (MAPK) (ERK1/2)-dependent manner as U0126, a specific MAPK inhibitor, blocked it. Combining a functional neurosphere forming assay with immunostaining for c-Met, along with markers of SVZ cells subtypes, demonstrated that HGF promotes the expansion of neural stem-like cells that form neurospheres and self-renew. Immunostaining, HGF enzyme-linked immunosorbent assay and Madin-Darby canine kidney cell scattering assay indicated that SVZ cell cultures produce and release HGF. SVZ cell-conditioned media induced proliferation on SVZ cell cultures, which was blocked by HGF-neutralizing antibodies, hence implying that endogenously produced HGF accounts for a major part in SVZ mitogenic activity. Brain sections immunostaining revealed that HGF is produced by nestin-expressing cells and c-Met is expressed within the SVZ by immature cells. HGF intracerebroventricular injection promoted SVZ cell proliferation and increased the ability of these cells exposed in vivo to HGF to form neurospheres in vitro, whereas intracerebroventricular injection of HGF-neutralizing antibodies decreased SVZ cell proliferation. The present study unravels a major role, both in vitro and in vivo, for endogenous HGF in SVZ neural stem cell growth and self-renewal.     

Cell division and apoptosis in the adult neural stem cell niche are differentially affected in transthyretin null mice

Thyroid hormones (THs) are fundamental in regulation of growth and development, particularly of the brain. THs are required for full proliferative activity of neural stem cells in the subventricular zone (SVZ) of adult mouse brains, and also affect the normal fate of progenitor cells: apoptosis. Transthyretin (TTR) is a TH distributor protein in the blood and cerebrospinal fluid. TTR secretion by the choroid plexus is involved in transport of THs from blood into cerebrospinal fluid. We investigated the regulation of neural stem cell cycle in the SVZ of adult TTR null mice. Markers for neural stem cell mitosis that are reduced during hypothyroidism, did not differ between genotypes. However, in TTR null mice the level of apoptosis, the fate of most progenitor cells, was as low as that in brains of hypothyroid wildtype mice. Thus, lack of TTR results in reduced availability of TH to progenitor cells in the SVZ. We show that proliferation and apoptosis in the SVZ neural stem cell niche are differentially affected by the lack of TTR synthesis.     

Tumor necrosis factor-alpha modulates survival, proliferation, and neuronal differentiation in neonatal subventricular zone cell cultures

Tumor necrosis factor (TNF)-alpha has been reported to modulate brain injury, but remarkably, little is known about its effects on neurogenesis. We report that TNF-alpha strongly influences survival, proliferation, and neuronal differentiation in cultured subventricular zone (SVZ) neural stem/progenitor cells derived from the neonatal P1-3 C57BL/6 mice. By using single-cell calcium imaging, we developed a method, based on cellular response to KCl and/or histamine, that allows the functional evaluation of neuronal differentiation. Exposure of SVZ cultures to 1 and 10 ng/ml mouse or 1 ng/ml human recombinant TNF-alpha resulted in increased differentiation of cells displaying a neuronal-like profile of [Ca2+](i) responses, compared with the predominant profile of immature cells observed in control, nontreated cultures. Moreover, by using neutralizing antibodies for each TNF-alpha receptor, we found that the proneurogenic effect of 1 ng/ml TNF-alpha is mediated via tumor necrosis factor receptor 1 activation. Accordingly, the percentage of neuronal nuclear protein-positive neurons was increased following exposure to mouse TNF-alpha. Interestingly, exposure of SVZ cultures to 1 ng/ml TNF-alpha induced cell proliferation, whereas 10 and 100 ng/ml TNF-alpha induced apoptotic cell death. Moreover, we found that exposure of SVZ cells to TNF-alpha for 15 minutes or 6 hours caused an increase in the phospho-stress-activated protein kinase/c-Jun N-terminal kinase immunoreactivity initially in the nucleus and then in growing axons, colocalizing with tau, consistent with axonogenesis. Taken together, these results show that TNF-alpha induces neurogenesis in neonatal SVZ cell cultures of mice. TNF-alpha, a proinflammatory cytokine and a proneurogenic factor, may play a central role in promoting neurogenesis and brain repair in response to brain injury and infection.     

局灶性脑缺血后老年大鼠室管膜下区和颗粒下层细胞增殖与分化的实验研究

目的观察老年大鼠局灶性脑缺血后室管膜下区（SVZ）和颗粒下层（SGZ）神经干细胞的增殖与分化.方法取老年大鼠制作大脑中动脉梗塞模型.用5-溴脱氧尿核苷（BrdU）脉冲标记结合免疫组织化学单标记技术,观察正常组、假手术组、脑缺血后3、7、14、21、28 d组SVZ和SGZ区BrdU阳性细胞的变化;用BrdU累积标记结合免疫组织化学双标技术,观察脑缺血14 d后SVZ和SGZ区BrdU/NeuN和BrdU/GFAP双标阳性细胞的数量.结果在正常组、假手术组及各脑缺血组大鼠的双侧SVZ和SGZ均可观察到BrdU阳性细胞.与正常组和假手术组相比,脑缺血后SVZ和SGZ区BrdU阳性细胞明显增加.缺血组SVZ区BrdU阳性细胞在脑缺血后7 d时达到高峰,28 d时仍高于正常水平;SGZ区BrdU阳性细胞在脑缺血后14 d时达到高峰,28 d时仍高于正常水平.通过BrdU累积标记和免疫组织化学双标发现：脑缺血14 d后,老年大鼠SVZ区有部分细胞显示BrdU/NeuN（0.98%）或BrdU/GFAP（12.56%）双标阳性,而SGZ区未见双标细胞.结论局灶性脑缺血可激活老年大鼠室管膜下区和颗粒下层的神经干细胞明显增殖,并且室管膜下区有部分增殖细胞可分化为神经元或神经胶质.     

EphB2 induces proliferation and promotes a neuronal fate in adult subventricular neural precursor cells

The subventricular germinal zone (SVZ) retains an active population of stem cells and neural precursor cells throughout adulthood. EphrinB signaling mediates angiogenesis and vasculogenesis in the developing and adult brain. Recent studies indicate that molecules involved in angiogenesis often influence neurogenesis as well. However, little work has been done considering a role for EphB2/EphrinB in adult neural precursor cells. We therefore examined whether the EphB2 receptor tyrosine kinase could directly effect proliferation of SVZ neural precursors and/or direct the cell fate of SVZ cells in vitro. Here, we found that clustered EphB2 increased bromodeoxyuridine (BrdU) incorporation and proliferation of SVZ neurosphere cultures. Immunostaining and RT-PCR analysis for beta-tubulin III (Tuj1) and GFAP indicated 4-day treatment with EphB2 promoted a neuronal phenotype, suggesting that the EphB2 receptor might also direct SVZ cell fate. EphB2 transiently down-regulated SVZ cell mRNA of Notch1 and Zic1, genes that regulate neurogenesis and neuronal differentiation. Notch1 has been implicated in apoptosis of neural precursors, however, a cell viability assay revealed no statistical difference between EphB2-treated and control cultures. When SVZ neurospheres were cultured upon Matrigel, EphB2 attenuated radial migration of SVZ cells in vitro. These results demonstrate that EphB2/EphrinB signaling directly induces SVZ proliferation, decreases migration, and promotes a neuronal fate of SVZ neural precursors independent of cell survival.     

The nuclear receptor tailless is required for neurogenesis in the adult subventricular zone

The tailless (Tlx) gene encodes an orphan nuclear receptor that is expressed by neural stem/progenitor cells in the adult brain of the subventricular zone (SVZ) and the dentate gyrus (DG). The function of Tlx in neural stem cells of the adult SVZ remains largely unknown. We show here that in the SVZ of the adult brain Tlx is exclusively expressed in astrocyte-like B cells. An inducible mutation of the Tlx gene in the adult brain leads to complete loss of SVZ neurogenesis. Furthermore, analysis indicates that Tlx is required for the transition from radial glial cells to astrocyte-like neural stem cells. These findings demonstrate the crucial role of Tlx in the generation and maintenance of NSCs in the adult SVZ in vivo.     

Disruption of Eph/ephrin signaling affects migration and proliferation in the adult subventricular zone

The subventricular zone (SVZ) of the lateral ventricles, the largest remaining germinal zone of the adult mammalian brain, contains an extensive network of neuroblasts migrating rostrally to the olfactory bulb. Little is known about the endogenous proliferation signals for SVZ neural stem cells or guidance cues along the migration pathway. Here we show that the receptor tyrosine kinases EphB1-3 and EphA4 and their transmembrane ligands, ephrins-B2/3, are expressed by cells of the SVZ. Electron microscopy revealed ephrin-B ligands associated with SVZ astrocytes, which function as stem cells in this germinal zone. A three-day infusion of the ectodomain of either EphB2 or ephrin-B2 into the lateral ventricle disrupted migration of neuroblasts and increased cell proliferation. These results suggest that Eph/ephrin signaling is involved in the migration of neuroblasts in the adult SVZ and in either direct or indirect regulation of cell proliferation.     

用组织块法培养成年和老年鼠神经干细胞

目的　探索体外培养扩增成年及老年动物室管膜下区 (SVZ)神经干细胞的实用方法。方法　取 8、14及 2 4三个月龄SD大鼠SVZ组织块置含bFGF的DMEM/F12 +B2 7培养液培养 ,Nestin免疫组化法鉴定细胞表型。结果　各月龄的SVZ组织块均能长出Nestin阳性的神经小球及神经干细胞 ,培养 7～ 10天产生的神经球最多 ,神经干细胞状态最佳。结论　用含bFGF的培养基培养成年和老年大鼠SVZ组织块能产生较多的神经干细胞 ;此法是一种具有实用价值的培养、扩增神经干细胞的手段。     

新生大鼠脑缺血后脑室下区基因表达谱的生物信息学分析

目的建立3日龄大鼠脑缺血模型,采用基因芯片分析新生大鼠未成熟脑缺血损伤后脑室下区(SVZ)基因表达谱的变化。方法同窝3日龄大鼠随机分为实验组和对照组,采用双侧颈总动脉结扎法制备缺血性脑损伤模型,于不同时点取SVZ组织,采用Affymetrix Rat230 2.0基因表达谱芯片观察SVZ基因表达变化,芯片数据分别用3种不同方法分析,并用实时PCR方法验证芯片结果。结果①通过差异基因筛选,发现3日龄大鼠脑缺血损伤后SVZ有17个基因发生表达变化,其中上调基因10个,下调基因7个,这些基因参与多种功能的调节。②基于基因功能的表达趋势分析显示,在所有参与增殖、凋亡功能的基因中,转化生长因子-β(TGF-β)在3日龄大鼠脑缺血损伤后SVZ微环境基因表达变化中起枢纽作用。实时PCR验证结果显示,TGF-β1及Smad2于缺血后1、4和7 d表达均上升,7 d达高峰。③在参与Wnt、TGF、BMP和血管内皮生长因子(VEGF)通路所有基因组成的基因功能相似性网络中,有13个基因在网络中起核心调控作用,构成信号通路串话节点。结论新生大鼠在脑缺血损伤后SVZ微环境中,参与神经新生的BMP、TGF、VEGF和Wnt通路间的串话...