切除嗅球对成年大鼠嘴侧迁移流的影响

我们以前的研究观察到嗅球切除后室管膜下层(SVZ)仍能产生新细胞,但新细胞迁移的通路尚不清楚。为此,本研究建立了成年SD雄性大鼠右侧嗅球切除模型,利用Nissl染色、PSA-NCAM和GFAP免疫组织化学染色的方法观察了成年SD大鼠嗅球切除后存活不同时间两侧嘴侧迁移流(RMS)的形态学特征及RMS细胞的密度和单个细胞的面积,并进行统计学分析;同时利用Westernblot方法检测PSA-NCAM在RMS的表达。结果观察到:(1)嗅球切除后不同时间点,嗅球切除侧RMS的细胞数和面积增加,PSA-NCAM免疫阳性细胞数增加,而GFAP免疫阳性细胞数在嗅球切除后2周和4周有明显增加;(2)嗅球切除后两侧RMS的细胞密度和单个细胞的面积没有明显改变;(3)从矢状切片可见嗅球切除后RMS的形态和路径没有明显改变,但在切除断端细胞堆积明显。这些结果提示嗅球切除后仍有年轻神经元沿RMS向嘴侧迁移,SVZ的神经生发活动与嗅球的存在与否可能没有必然的联系。     

嗅球切除对成年大鼠侧脑室外侧壁新生细胞增殖和分化的影响

目的 研究嗅球切除后成年大鼠侧脑室外侧壁(SVZ)新生细胞增殖和分化的情况,进一步探讨嗅球对SVZ神经生发活动的影响.方法 建立成年SD雄性大鼠右侧嗅球切除模型,并分别存活4周和12周,利用Nissl染色、多唾液酸神经细胞黏附分子(PSA-NCAM)和BrdU免疫组织化学染色的方法观察了成年SD大鼠嗅球切除后存活不同时间两侧吻侧迁移流(RMS)BrdU阳性细胞数占总细胞百分比的变化以及两侧RMS PSA-NCAM阳性细胞的形态学变化.结果 1.嗅球切除后不同时间点,嗅球切除侧RMS的细胞数增加,BrdU免疫阳性细胞数增加,但BrdU免疫阳性细胞数占总细胞数的百分比随嗅球切除后大鼠存活时间的延长而下降,且以RMS的吻侧部分下降更明显;2.嗅球切除后,在切除侧断端吻侧颗粒层和RMS均出现较对照侧更多的具有较长突起的PSA-NCAM阳性细胞.结论 嗅球切除后仍有新生神经元沿RMS向吻侧迁移,但其增殖率随时间延长下调;嗅球的切除似乎并没有影响成神经细胞的分化.     

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。     

GDNF、GFAP和NGFRp75在成年大鼠和金黄地鼠嗅球成鞘细胞的免疫组织化学研究

目的　研究GDNF在成年大鼠和金黄地鼠嗅球成鞘细胞的表达 ,探索成鞘细胞在中枢神经再生中的作用。　方法　用免疫组织化学ABC法 ,显示GDNF在成年大鼠和金黄地鼠嗅球成鞘细胞的表达和分布 ,同时用NGFRp75和GFAP染色作为阳性对照。　结果　在成年大鼠和金黄地鼠嗅球的纤维层和小球层内均可见深棕色的GDNF免疫组织化学反应的成鞘细胞。在小球层与纤维层分界处和小球层与分子层分界处及嗅小球之间密集分布 ,在嗅小球之内较稀疏。同时在同一嗅球组织的另两组切片的相同部位 ,分别出现GFAP和NGFRp75免疫反应性细胞 ,间接说明GDNF免疫反应的结构是嗅球成鞘细胞。　结论　嗅球成鞘细胞含有胶质细胞源性神经营养因子     

大鼠受损视神经中神经前体细胞的变化及预变性腓总神经的调节

为了研究大鼠受损视神经内神经前体细胞的变化及调节,本研究建立了成年雄性大鼠视神经损伤及自体腓总神经移植模型,分正常组、损伤组和移植组,体外培养视神经的神经前体细胞,在相差显微镜下观测各组神经前体细胞神经球的形态和数目,以免疫荧光细胞化学方法对神经球细胞进行鉴定。结果显示:正常组神经球较小、较少,多数细胞表达nestin、GFAP或半乳糖脑苷脂(GC);视神经损伤后神经球的总数有所增加,但大神经球数明显减少,nestin、GFAP或GC阳性细胞也明显减少;神经移植后增加了各类神经球数,nestin、GFAP或GC阳性细胞也明显增加。本研究提示成年大鼠视神经内神经前体细胞较少,增殖能力较弱;视神经损伤也伤及其神经前体细胞并抑制其增殖;自体神经移植能保护神经前体细胞并促进其增殖。     

帕金森病MPTP食蟹猴模型嗅球细胞凋亡

目的 建立1-甲基-4-苯基-1, 2, 3, 6-四氢吡啶（MPTP）帕金森病（PD）模型,探讨模型组嗅球细胞凋亡和胶质细胞增生情况。方法 3只成年健康食蟹猴,静脉注射MPTP建立PD模型,另3只静脉注射生理盐水作为对照。取出嗅球,免疫组织化学染色检测Caspase-3、Bcl-2、离子钙接头蛋白分子1（Iba-1）和胶质纤维酸性蛋白（GFAP）在食蟹猴嗅球中的表达情况,采用Image J v1.8.0软件分析模型组和对照组之间的差异。 结果 MPTP损伤后,与对照组相比,模型组嗅球突触小球层Caspase-3阳性细胞数明显增加,而Bcl-2表达减少;与对照组相比,模型组嗅球突触小球层和外网状层的GFAP和Iba-1阳性细胞数增加。 结论 MPTP可诱导食蟹猴嗅球突触小球层细胞凋亡,并伴有星形胶质细胞增生和小胶质细胞激活,这可能与帕金森病的功能障碍有关。     

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.     

成年大鼠脑室下区吻侧迁移流的细胞形态学研究

为了探讨成年大鼠脑室下区吻侧迁移流(RMS)神经干细胞的分布及特点,我们利用Nissl染色,BrdU、GFAP、PSA-NCAM和nestin免疫组织化学染色,nestin与GFAP免疫荧光双标记染色等方法观察了正常成年SD大鼠RMS的组织化学和免疫组织化学特征;同时观察了正常RMS的超微结构特征。结果观察到:(1)在光镜下,Nissl染色切片的RMS由深染和浅染细胞组成,其中深染细胞为PSA-NCAM、BrdU及nestin免疫反应阳性,浅染细胞为GFAP及nestin免疫反应阳性;(2)电镜下,RMS存在数量不等、由同一种类型的细胞聚集形成的细胞团,以及星型胶质细胞和少量少突胶质细胞,细胞团中可看到正在分裂的细胞。结果提示:(1)除少突胶质细胞外,RMS主要由两种类型细胞组成,即成神经细胞和星型胶质细胞;(2)成神经细胞存在于RMS全长并保持分裂特性,属神经元前体细胞;(3)目前还没有充分的证据证实RMS中存在干细胞。     

高压氧对新生大鼠缺氧缺血性脑损伤后在体神经干细胞的影响

目的： 新生鼠的神经生发区-室管膜下区（SVZ） 和海马齿状回（DG）聚集了多种不同发育阶段神经干细胞（NSCs），它们可分化产生新的神经元和胶质细胞。本研究探讨缺氧缺血性脑损伤（HIBD）对脑生发区NSCs的损伤及高压氧（HBO）对此损伤的影响。从在体NSCs 探讨HBO对新生大鼠HIBD的保护作用机制。 方法： 新生7 d龄SD大鼠随机分为4组：① 正常对照组（CON，n=16）；② HIBD 模型组（HIBD，n=16）；③ 高压空气组（HBA，n=16）；④HBO治疗组（n=16）。Rice法复制HIBD模型，并予HBA或HBO治疗，每天1次共7 d。BrdU免疫组化显示在体NSCs。并取损伤侧脑SVZ区组织，体外NSCs培养并进行神经干细胞球计数。 结果： HIBD组生发区在体BrdU 阳性细胞和体外培养的神经干细胞球数目明显少于对照组。HBO组SVZ区的BrdU阳性细胞增多；体外培养的神经干细胞球增多。HBA组增加不明显。 结论： 新生大鼠HIBD后生发区NSCs减少，HBO治疗可以减轻HIBD后NSCs的死亡。HBA治疗无明显作用。     

成年大鼠脑室下层细胞表达GFR-α_1的免疫组织化学研究

观察胶质细胞源性神经营养因子受体-α1 (GFR -α1 )在成年大鼠脑室下层(SVZ)细胞的表达,探讨胶质细胞源性神经营养因子(GDNF)对SVZ细胞的作用。成年大鼠SVZ组织冰冻切片,采用GFR -α1 结合5- 溴脱氧尿苷(BrdU)的免疫组织化学单标记与双标记方法,对成年大鼠SVZ进行观察。在成年大鼠SVZ可见GFR- α1 阳性细胞、BrdU阳性细胞和GFR- α1 /BrdU双标记细胞,且三种阳性细胞的分布趋势相似。结果提示GDNF可能参与调节成年哺乳动物脑内SVZ细胞的增殖、分化和迁移。     

切除嗅球对成年大鼠脑室下区神经生发功能的影响

为了探讨嗅球对成年哺乳动物侧脑室外侧壁脑室下区神经生发活动的影响 ,本研究在机械性切除成年大鼠一侧嗅球后采用连续半薄切片、克紫染色和细胞核表面三维重建技术 ,观察了侧脑室外侧壁脑室下区的细胞核表面特征及其空间位置 ,并测量了细胞核的形态学指标。结果发现 ,在切除嗅球 3个月后 ,同侧侧脑室外侧壁脑室下区的区域变小 ,特有的中、小型、形状不规则的胶质细胞核的数量比例增大 ,而具有成神经细胞核特征的细胞核 (体积较大、呈规则的椭圆体形 )数量明显减少 ,且其同型细胞核聚集现象消失。说明同侧此区的神经生发活动减弱而胶质生发 (gliogenesis)相对增强。本研究结果提示 ,哺乳动物嗅球对成年侧脑室外侧壁脑室下区的神经生发机能可能具有促进作用     

Short term treatment with estradiol decreases the rate of newly generated cells in the subventricular zone and main olfactory bulb of adult female mice

Adult neurogenesis occurs most notably in the subgranular zone (SGZ) of the hippocampal dentate gyrus and in the olfactory bulb (OB) where new neurons are generated from neural progenitors cells produced in the subventricular zone (SVZ) of the forebrain. As it is well known that gonadal steroid hormones, primarily estradiol, modulate neurogenesis in the hippocampus of adult female rodents, we wanted to determine whether estradiol would also affect the proliferation of progenitor cells in the SVZ and by consequence the rate of newly generated cells in the main OB. Thus a first group of adult female C57Bl6/J mice was ovariectomized and received a short term treatment with estradiol (single injection of 1 or 10 μg 17β-estradiol or Silastic capsule of estradiol during 2 days) before receiving a single injection with BrdU to determine whether estradiol would modulate the cell proliferation in the SVZ. A second group of adult ovariectomized female mice was submitted to the same estradiol treatment before receiving four BrdU injections, and was sacrificed 21 days later to determine whether a modulation in cell proliferation actually leads to a modulation in the number of newborn cells in the main OB. We observed a decrease in cell proliferation in the SVZ following either dose of estradiol compared to the controls. Furthermore, 21 days after their generation in the SVZ, the number of BrdU labeled cells was also lower in the main OB, both in the granular and periglomerular cell layers of estradiol-treated animals. These results show that a short term treatment with estradiol actually downregulates cell proliferation leading to a decreased number of newborn cells in the OB.     

Alteration in the differentiation-related molecular expression in the subventricular zone in a mouse model of Parkinson's disease

Enhancement of neurogenesis could be a suitable treatment approach to up-regulate dopaminergic neurons in Parkinson's disease (PD). In the present study, we focused on the kinetics of the subventricular zone (SVZ) in a mouse model of PD induced by MPTP injection. We showed recently the proliferation potential of neuronal stem cells (NSCs) prepared from the olfactory bulb of an animal model of PD [Hayakawa, H., Hayashita-Kinoh, H., Nihira, T., Seki, T., Mizuno, Y., Mochizuki, H., 2007. The isolation of neural stem cells from the OB of Parkinson's disease model. Neurosci. Res.]. In this study, we examined the relationship between proliferation and differentiation of NSCs in SVZ of both acute and chronic PD models. Only acute MPTP treatment significantly increased the areas of glial fibrillary acidic protein (GFAP)-expressing cells and decreased the areas of polysialylated neural cell adhesion molecule (PSA-NCAM)-expressing cells in the SVZ. In the case of caspase-11 knockout mice, MPTP did not induce alteration in the areas of GFAP-expressing cells and PSA-NCAM-expressing cells. Our results suggest that neuroinflammation related to the caspase-11 cascade in the striatum regulates differentiation of neural stem cells in the SVZ of our mouse model of PD.     

Deprivation of sensory inputs to the olfactory bulb up-regulates cell death and proliferation in the subventricular zone of adult mice

The main olfactory bulb (MOB) is the first relay on the olfactory sensory pathway and the target of the neural progenitor cells generated in the subventricular zone (SVZ) lining the lateral ventricles and which migrate along the rostral extension of the SVZ, also called the rostral migratory stream (RMS). Within the MOB, the neuroblasts differentiate into granular and periglomerular interneurons. A reduction in the number of granule cells during sensory deprivation suggests that neurogenesis may be influenced by afferent activity. Here, we show that unilateral sensory deafferentation of the MOB by axotomy of the olfactory receptor neurons increases apoptotic cell death in the SVZ and along the rostro-caudal extent of the RMS. The vast majority of dying cells in the RMS are migrating neuroblasts as indicated by double Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labeling/PSA-NCAM labeling. Counting bromodeoxyuridine-labeled cells in animals killed immediately or 4 days after tracer administration showed a bilateral increase in proliferation in the SVZ and RMS which was balanced by cell death on the operated side. These data suggest that olfactory inputs are required for the survival of newborn neural progenitors. The greatest enhancement in proliferation occurred in the extension of the RMS located in the MOB, revealing a population of local precursors mitotically stimulated following axotomy. Together, these findings indicate that olfactory inputs may strongly modulate the balance between neurogenesis and apoptosis in the SVZ and RMS and provide a model for further investigation of the underlying molecular mechanisms of this activity-dependent neuronal plasticity.     

The cellular composition and morphological organization of the rostral migratory stream in the adult human brain

The rostral migratory stream (RMS) is the major pathway by which progenitor cells migrate from the subventricular zone (SVZ) to the olfactory bulb (OB) in rodents, rabbits and primates. However, the existence of an RMS within the adult human brain has been elusive. Immunohistochemical studies utilising cell-type specific markers for early progenitor cells (CD133), proliferating cells (PCNA), astrocytes and type B cells (GFAP) and migrating neuroblasts (PSA-NCAM), reveal that the adult human RMS is organized into layers containing glial cells, proliferating cells and neuroblasts. In addition, the RMS is arranged around a remnant of the ventricular cavity that extends from the SVZ to the OB as seen by immunohistological staining analysis and electron microscopy, showing the presence of basal bodies and a typical 9 + 2 arrangement of tubulin in tufts of cilia from all levels of the RMS. Overall, these findings suggest that a pathway of migratory progenitor cells similar to that seen in other mammals is present within the adult human brain and that this pathway could provide for neurogenesis in the human forebrain. These findings contribute to the scientific understanding of adult neurogenesis and establish the detailed cytoarchitecture of this novel neurogenic niche in the human brain.     

营养不良可增加幼鼠齿状回颗粒下层的细胞增殖和神经生发(英文)

为了观察营养不良对幼鼠海马齿状回 (DG)和脑室下层 (SVZ)的细胞增殖和神经发生的影响 ,采用 5 -溴 -2 -脱氧尿苷(Brd U)标记结合免疫组织化学方法对脑切片分别进行 Brd U、Tu J1(β tubulin,β微管蛋白 )及 GFAP(胶质纤维酸性蛋白 )反应或双重反应。结果表明 ,营养不良幼鼠齿状回的细胞增殖和神经生发明显高于营养良好的幼鼠而脑室下层的细胞增殖数量在两者却无明显差异。在齿状回 ,新生的细胞中大约有 5 0 %为新生的神经元 ,10～ 2 0 %为神经胶质细胞。本文结果提示 ,幼鼠海马齿状回的细胞增殖和神经生发可能因营养不良而增加 ,这些新生的细胞可能对日后某些海马依赖性行为产生一定的影响     

Expression of amyloid precursor protein-like molecule in astroglial cells of the subventricular zone and rostral migratory stream of the adult rat forebrain

In adult mammals, new neurons in the subventricular zone (SVZ) of the lateral ventricle (LV) migrate tangentially through the rostral migratory stream (RMS) to the olfactory bulb (OB), where they mature into local interneurons. Using a monoclonal antibody for the beta-amyloid precursor protein (APP) (mAb 22C11), which is specific for the amino-terminal region of the secreted form of APP and recognizes all APP isoforms and APP-related proteins, immunoreactivity was detected in specific subpopulations of cells in the SVZ and RMS of the adult rat forebrain. In the SVZ, APP-like immunoreactivity was detected in the ependymal cells lining the LV and some of the subependymal cells. The latter were regarded as astrocytes, because they were positive for the glial markers, S-100 protein (S-100) and glial fibrillary acidic protein (GFAP). APP-like immunoreactive astrocytes exhibited strong labelling of the perinuclear cytoplasm and often possessed a long, fine process similar to that found with radial glia. The process extended to an APP-like immunoreactive meshwork in the RMS that consisted of cytoplasmic processes of astrocytes forming 'glial tubes'. Double-immunofluorescent labelling with a highly polysialylated neural cell adhesion molecule (PSA-NCAM) confirmed that the APP-like immunoreactive astrocytes in the SVZ and meshwork in the RMS made close contact with PSA-NCAM-immunopositive neuroblasts, suggesting an interaction between APP-containing cells and neuroblasts. This region of the adult brain is a useful in vivo model to investigate the role of APP in neurogenesis.     

Conditional ablation of Tbr2 results in abnormal development of the olfactory bulbs and subventricular zone‐rostral migratory stream

Development of the olfactory bulb (OB) is a complex process that requires contributions from several progenitor cell niches to generate neuronal diversity. Previous studies showed that Tbr2 is expressed during the generation of glutamatergic OB neurons in rodents. However, relatively little is known about the role of Tbr2 in the developing OB or in the subventricular zone‐rostral migratory stream (SVZ‐RMS) germinal niche that gives rise to many OB neurons. Results: Here, we use conditional gene ablation strategies to knockout Tbr2 during embryonic mouse olfactory bulb morphogenesis, as well as during perinatal and adult neurogenesis from the SVZ‐RMS niche, and describe the resulting phenotypes. We find that Tbr2 is important for the generation of mitral cells in the OB, and that the olfactory bulbs themselves are hypoplastic and disorganized in Tbr2 mutant mice. Furthermore, we show that the SVZ‐RMS niche is expanded and disordered following loss of Tbr2, which leads to ectopic accumulation of neuroblasts in the RMS. Lastly, we show that adult glutamatergic neurogenesis from the SVZ is impaired by loss of Tbr2. Conclusions: Tbr2 is essential for proper morphogenesis of the OB and SVZ‐RMS, and is important for the generation of multiple lineages of glutamatergic olfactory bulb neurons. Developmental Dynamics 243:440–450, 2014. © 2013 Wiley Periodicals, Inc.