快速老化模型小鼠学习记忆行为及有关脑区单胺递质水平的增龄性变化

目的 研究快速老化模型小鼠(senescence accelerated mice，SAM)学习记忆能力及其大脑皮层、海马和下丘脑单胺递质含量的增龄性变化及它们之间的关系。方法 分别采用跳台实验和穿梭箱实验测定SAM的被动和主动回避反应能力，采用高效液相色谱电化学检测法测定脑内单胺递质的含量。结果 2月龄快速老化亚系SAM-prone/8(SAMP8)的被动和主动回避反应能力已较同龄抗快速老化亚系SAM-resistance／1(SAMR1)明显降低，且其主动回避反应能力随增龄进一步降低。同时，SAMP8大脑皮层、海马及下丘脑内单胺递质水平多明显高于同龄SAMR1，且随增龄明显增高。结论 SAMP8学习记忆能力的衰退可能与其相关脑区单胺递质的变化密切相关。     

脑缺血后内源性激活神经干细胞的机制研究

1 脑缺血后内源性神经干细胞的自身激活 近年来研究表明,脑缺血后自身的神经干细胞有大量的增殖分化[1,2],说明神经干细胞可能参与脑缺血的病理生理过程.Zhang等通过建立沙鼠大脑中动脉缺血(MCAO)模型,观察缺血再灌注后不同时期的室管膜下层(SVZ)、海马颗粒细胞层(DG)、嗅球以及梗死灶周边皮质的神经干细胞增殖、分化情况,发现7 d后在梗死同侧脑SVZ出现神经干细胞增殖高峰,14 d达最高,而DG区则无增殖,且14 d后远离梗死区的嗅球有大量的神经干细胞增殖,但28 d后,标记Brdu阳性细胞大量减少,说明脑缺血只引起短暂的神经干细胞的增殖,Zhang认为是脑缺血损伤的应急保护反应[3].     

经鼻滴入海人藻酸引起C57BL/6小鼠嗅球和海马区神经元的退行性病变

目的 应用海人藻酸(KA)在C57BL／6小鼠建立神经退行性病变动物模型并观察其对嗅球神经元的影响。方法 经鼻滴人KA应用尼氏和嗜银染色观察海马及嗅球的病理变化，免疫组化检测Cyclooxygenase2(COX-2)的表达。结果 经鼻滴入KA成功地在C57BL／6小鼠建立了神经退行性病变动物模型，KA通过嗅神经引起双侧嗅球和海马损伤，其病变程度与小鼠体重和滴入KA剂量有关，同时KA引起了脑内明显的胶质细胞增生和炎症因子COX-2在嗅球部的表达。结论 经鼻滴入KA能够引起嗅球和海马的损伤。     

快速老化动物模型SAMP8小鼠及其相关研究进展

SAMP8鼠由日本京都大学竹田俊男教授开发成功，主要以学习记忆功能呈增龄性加速衰退，中枢神经系统如皮质、海马等部位发生病理改变为主，是一种比较理想的研究脑老化和痴呆的模型，亦是目前研究快速衰老的唯一哺乳类模式动物。行为学、形态学、神经生化和分子生物学为此提供了较充足的证据。SAMP8鼠可被广泛应用于阐明年龄相关的学习记忆功能改变及认知缺陷的基本机制，研究老化相关性疾病，评价治疗老化关联疾病和改善学习记忆机能的益智药物。但SAMP8所涉及的相关疾病主要是阿尔茨海默病，能否用于其他衰老相关性疾病如帕金森病仍需进一步研究。     

低氧预适应刺激脑海马区内源性神经干细胞的增殖**

背景：缺血/缺氧/低氧等刺激均能导致内源性神经干细胞的增殖和分化,起到脑组织修复作用,但低氧预适应能否影响内源性神经干细胞的增殖尚不清楚。 目的：探讨低氧预适应对小鼠脑海马区内源性神经干细胞增殖的影响。 方法：清洁级Balb/c近交系小鼠随机分为3组,低氧对照组小鼠放入广口瓶内,立即用橡皮塞封紧,以动物出现第1次喘呼吸为低氧耐受极限的标志,完成低氧暴露1次;低氧预适应组小鼠按此法重复操作4次;正常对照组小鼠不进行低氧暴露。通过免疫荧光和激光共聚焦显微镜等技术,测定海马BrdU阳性细胞数和荧光强度。 结果与结论：与低氧对照组比较,低氧预适应组小鼠耐受时间显著延长(P < 0.01)。正常对照组海马区BrdU标记的内源性神经干细胞荧光强度微弱,海马区可见少量BrdU阳性细胞;低氧对照组、低氧预适应组荧光强度及BrdU阳性细胞数均明显增加(P < 0.01),且低氧预适应组增加幅度大于低氧对照组(P < 0.01)。证实在低氧预适应过程中,海马区内源性神经干细胞明显增殖,可能参与低氧预适应脑保护机制。     

内嗅皮质-海马神经投射通路的形成——体内及体外示踪研究

本研究对内嗅皮质一海马通路的各个亚支的发生进行了调查。方法：对不同龄大鼠脑用DiI、DiO、快兰示踪法及calretinin免疫细胞化学法处理。结果：槽通路、海马交通通路于胚胎16天（E16）开始发生,而穿通通路分别始见于胚胎17天海马的腔隙分子层和生后第2天齿状回外分子层。DiI的逆行标记显示内嗅皮质．海马通路主要来自内嗅皮质中Ⅱ到Ⅳ层神经元。另外,calretinin免疫细胞化学法显示Cajal—Retzius（CR）细胞早在胚胎16天存在于海马的腔隙分子层,DiI和calretinin免疫细胞化学法双重标记显示CR细胞和内嗅皮质转入纤维之间可能存在密切的接触关系。结论：嗅皮质-海马通路的各个亚支是按照上述各自的时间表进行发生,CR细胞和穿通纤维的发育时空关系提示该细胞对内嗅皮质传入纤维寻径具有引导作用。     

缺血缺氧可促进新生鼠神经干细胞的增殖

目的观察缺血缺氧对新生鼠皮层及海马神经干细胞的影响。方法制作新生鼠缺血缺氧性脑病(hypoxla-ischemic encephalopathy,HIE)模型,实验动物每天两次腹腔注射Brdu,每次剂量50mg/kg,用于标记各组动物的神经干细胞增殖情况。分别于 模型建立后3d、7d、14d和28d取脑组织行抗Brdu的免疫组化染色,分别观察HIE模型组和对照组动物之间海马及皮层Brdu阳性细 胞数目及细胞形态、分布情况;并比较他们之间的差异。结果正常新生SD(Sprague-Dsawley)大鼠脑内Brdu阳性细胞弥散分布于 各脑区,在海马的齿状回、脑室下区等部位有干细胞密集分布。缺血缺氧后新生动物脑内细胞坏死明显的区域见Brdu阳性细胞呈灶 状增生。各个阶段缺血缺氧组动物皮层及海马的Brdu阳性细胞数目均比假手术组明显增多,差别有统计学意义。假手术组动物皮 层及海马的Brdu阳性细胞数目和正常组差别无统计学意义。结论缺血缺氧可促进新生SD大鼠皮层及海马的神经干细胞增殖。     

去卵巢及穹窿海马伞切断复合模型大鼠不同脑区雌激素α受体mRNA的表达及倍美力的干预作用

目的观察倍美力对去卵巢及穹窿海马伞切断后大鼠脑皮质、基底前脑及海马CA_1区雌激素α受体(estrogen receptor alpha,ERa)mRNA表达的干预作用,探讨其脑保护作用的机制。方法健康雌性Wister大鼠30只,随机分成对照组、模型组、倍美力组;制备去卵巢及穹窿海马伞切断复合模型大鼠,采用Morris水迷宫观察其学习、记忆能力,利用RT-PCR及Western blotting法观察每组大鼠不问脑区雌激素α受体mRNA及蛋白的表达。结果模型组大鼠脑皮质、基底前脑、海马CA_1区雌激素α受体mRNA及蛋白的表达明显降低,学习、记忆能力下降,与对照组、倍美力组比较有显著差异(P<0.01);对照组与倍美力组比较无明显差异(P>0.05)。结论倍美力可上调大鼠脑皮质、基底前脑及海马CA_1区雌激素α受体mRNA及蛋白的表达,可能参与了学习、记忆能力的改善。     

Alzheimer病患者脑tau、β-tubulin的表达

目的 观察Alzheimer病（AD）患者脑tau、β-tubulin的表达、分布与神经元骨架改变的关系。方法 采用免疫组织化学方法观察tau、β-tubulin,在8例AD、5例非痴呆老人（ND）脑海马、嗅球、脑室周围皮质等部位的表达并进行半定量分析。结果 （1）AD组tau蛋白与ND组相比表达明显增加（P＜0．001）,主要在额叶、颞叶、海马、脑室周及嗅球的表达增高（P＜0．05）;顶叶、枕叶无明显差异。大脑新皮层、海马神经元树突、胞膜均有表达,细胞核膜部分着色,部分神经元呈典型NET样结构;星形胶质细胞也有表达。此外,神经毡细丝、老年斑亦有广泛表达。ND组tau散在表达,无NFT样神经元。（2）AD组的β-tubulin明显减少（P＜0．001）,额叶、颞叶、顶叶、海马及脑室周表达明显下降（P＜0．05）;而枕叶与嗅球无差异。β-tubulin在ND组,主要是海马及皮质神经元树突上广泛表达,白质区轴索结构也有阳性表达。结论 AD患者脑内tau表达增高且存在区域分布差异;而tau的过度产生与β-tubulin的表达减少和晚期AD脑内广泛的神经元丢失、结构破坏相一致,提示AD脑内神经原纤维缠结形成与二者改变的机制值得进一步探讨。     

嗅鞘细胞与神经干细胞共培养后增殖及向神经元的分化

背景：影响神经干细胞增殖分化的外在因素包括细胞因子和微环境,嗅鞘细胞能分泌多种细胞因子,与神经干细胞共培养时改变其微环境。 目的：观察不同浓度嗅鞘细胞对神经干细胞增殖及分化的影响。 方法：体外分别培养Wistar大鼠神经干细胞和嗅鞘细胞,5×107 L-1神经干细胞分别和1×107 L-1,1×109 L-1,1×1011 L-1嗅鞘细胞共培养,同时设立正常对照组,不加嗅鞘细胞。倒置荧光显微镜下观察神经干细胞增殖情况,诱导7 d后行NSE免疫细胞化学染色,计算阳性细胞/细胞总数得出阳性细胞百分比。 结果与结论：①3种浓度嗅鞘细胞与神经干细胞共培养3 d后,均促进了神经干细胞增殖,以1×109 L-1嗅鞘细胞共培养组作用最显著,明显优于1×107 L-1嗅鞘细胞组、1×1011 L-1嗅鞘细胞组。②神经干细胞与1×109 L-1嗅鞘细胞共培养7 d后,神经干细胞分化为神经元样细胞的百分比最高,与正常对照组相比差异有显著性意义(P < 0.01)。     

Adult neurogenesis in the four-striped mice （Rhabdomys pumilio）

In this study, we investigated non-captive four-striped mice （Rhabdomys pumilio） for evidence that adult neurogenesis occurs in the adult brain of animal models in natural environment. Ki-67 （a marker for cell proliferation） and doublecortin （a marker for immature neurons） immunos-taining conifrmed that adult neurogenesis occurs in the active sites of subventricular zone of the lateral ventricle with the migratory stream to the olfactory bulb, and the subgranular zone of the dentate gyrus of the hippocampus. No Ki-67 proliferating cells were observed in the striatum substantia nigra, amygdala, cerebral cortex or dorsal vagal complex. Doublecortin-immunore-active cells were observed in the striatum, third ventricle, cerebral cortex, amygdala, olfactory bulb and along the rostral migratory stream but absent in the substantia nigra and dorsal vagal complex. The potential neurogenic sites in the four-striped mouse species could invariably lead to increased neural plasticity.     

Cortex- and striatum- derived neural stem cells produce distinct progeny in the olfactory bulb and striatum

Neural stem cells can be isolated from the mouse embryonic cortex but do not persist in the adult cortex. In contrast, neural stem cells from the striatal embryonic germinal zone persist in the adult subependyma. Emx1-lineage analysis revealed that cortex-derived neural stem cells survive and migrate ventrally into the subependyma where they intermix with the host striatal neural stem cells [S. Willaime-Morawek et al. (2006)J. Cell Biol. 175, 159-168]. Cortex-derived cells proliferate faster in the subependyma and reach the olfactory bulb earlier than striatum-derived cells. In the olfactory bulb, cortex-derived cells produce more cells and more dopaminergic neurons in the glomerular layer than striatum-derived cells. Cortex-derived cells also give rise to more astrocytes and less neurons in the striatum than striatum-derived cells. Thus, history matters; cortex-derived neural stem cells in the subependyma give rise to progeny in the olfactory bulb and striatum but in different proportions than striatum-derived neural stem cells.     

人胚额叶皮层和海马干细胞的自主分化研究

目的 研究人胚胎额叶皮层和海马组织神经干细胞的自主分化特性。观察额叶皮层神经干细胞和海马神经干细胞特性的异同。方法 从人胚胎额叶皮层和海马组织分别分离提出神经干细胞，经无血清体外培养、扩增，形成神经球。神经球贴壁进行不加诱导剂的自主分化。采用细胞生长曲线检测神经干细胞的增殖能力。使用5-溴脱氧尿嘧啶核苷（BrdU）标记分裂增生的细胞，观察细胞的分裂增殖情况。免疫细胞化学法鉴定神经干细胞的自主分化能力，比较额叶皮层和海马神经干细胞的分化特点。结果 从人胚胎额叶皮层和海马分离的神经干细胞具有增殖能力，额叶皮层神经干细胞的细胞倍增时间为3．9d，海马神经干细胞的细胞倍增时间为3．2d。细胞贴壁分化后出现Nestin、GFAP、Tuj-1表达阳性的细胞。皮层和海马神经干细胞分化产生的Tuj-1阳性细胞分别是40．7％和19．3％；皮层和海马神经干细胞分化产生的GFAP阳性细胞分别是59．3％和80．7％。结论 分离培养的额叶皮层和海马神经干细胞具有自我更新和增殖能力，可以向神经元、胶质细胞分化。额叶皮层神经干细胞与海马神经干细胞的倍增时问、自主分化特点和分化为神经细胞和胶质细胞的比率各有不同。     

PACAP promotes neural stem cell proliferation in adult mouse brain

In recent years, it has become evident that neural stem cells in the adult mammalian brain continuously generate new neurons, mainly in the hippocampus and olfactory bulb. Although different growth factors have been shown to stimulate neurogenesis in the adult brain, very little is known about the role of neuropeptides in this process. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with pleiotropic effects acting through three receptors to which it has high affinity, namely, PACAP receptor 1 (PAC1), vasoactive intestinal peptide (VIP) receptor 1, and VIP receptor 2. We show that PAC1 is expressed in the neurogenic regions of the adult mouse brain, namely the ventricular zone of the lateral ventricle and the hippocampal dentate gyrus. Cultured neural stem cells isolated from the lateral ventricle wall of adult mice express PAC1 and proliferate in vitro in response to two PAC1 agonists, PACAP and Maxadilan, but not VIP at physiologic concentrations, indicating PAC1 as a mediator of neural stem cell proliferation. Pharmacologic and biochemical characterization of PACAP-induced neural stem cell proliferation revealed the protein kinase C pathway as the principal signaling pathway, whereas addition of epidermal growth factor synergistically enhanced the proliferating effect of PACAP. Further in vitro characterization of the effect of PACAP on neural stem cells showed PACAP capable of stimulating ex novo in vitro formation of multipotent neurospheres with the capacity to generate both neuronal and glial cells. Finally, intracerebroventricular infusion of PACAP increases cell proliferation in the ventricular zone of the lateral ventricle and the dentate gyrus of the hippocampus. We conclude that PACAP, through PAC1, is a potent mediator of adult neural stem cell proliferation.     

Effects of olfactory ensheathing cells on the proliferation and differentiation of neural stem cells

BACKGROUND:Olfactory ensheathing cells can promote oriented differentiation and proliferation of neural stem cells by cell-secreted neural factors. OBJECTIVE:To observe the effect of olfactory ensheathing cells on the differentiation and proliferation of neural stem cells. DESIGN,TIME AND SETTING:Cytology was performed at the Department of Neurology,Tongji Medical College,Huazhong University of Science and Technology,China,from September 2007 to October 2008. MATERIALS:Mouse anti-nestin polyclonal antibo...     

Quantitative immunogold study of glucose transporter (GLUT-1) in five brain regions of scrapie-infected mice showing obesity and reduced glucose tolerance

Distribution of glucose transporter (GLUT-1) in the microvascular endothelium of scrapie-infected SJL/J hyperglycemic mice showing clinical signs of scrapie, obesity and reduced glucose tolerance was studied in five brain regions: cerebral cortex, hippocampus, thalamus, cerebellum and olfactory bulb. Uninfected normoglycemic SJL/J mice showing normal glucose tolerance were used as a control. Ultrathin sections of brain samples embedded at low temperature in the hydrophilic resin Lowicryl K4M were exposed to anti-GLUT-1 antiserum followed by gold-labeled secondary antibodies. Labeling density was recorded over luminal and abluminal plasma membranes of microvascular endothelial cells. Ultrastructural observations revealed attenuation of the microvascular endothelial lining in numerous vascular profiles from brain samples of diabetic mice. Morphometric analysis revealed significant decreases of the labeling density for GLUT-1 in the microvasculature of the thalamus, cerebellum and, to a lesser degree, the hippocampus of diabetic mice. No significant differences between diabetic and non-diabetic, control mice were observed in the microvessels supplying cerebral cortex and olfactory bulb. These findings suggest that abnormal glucose metabolism, manifested by reduced glucose tolerance and hyperglycemia, leads to impaired transvascular glucose transport in some brain regions but not in others, presumably disturbing the function of those brain regions supplied by the affected blood microvessels.     

Neuronal differentiation following transplantation of expanded mouse neurosphere cultures derived from different embryonic forebrain regions

In vitro, expanded neurospheres exhibit multipotent properties and can differentiate into neurons, astrocytes and oligodendrocytes. In vivo, cells from neurospheres derived from mouse fetal forebrain have previously been reported to predominantly differentiate into glial cells, and not into neurons. Here we isolated stem/progenitor cells from E13.5 lateral ganglionic eminence (LGE), medial ganglionic eminence (MGE) and cortical primordium, of a green fluorescent protein (GFP)-actin transgenic mouse. Free-floating neurospheres were expanded in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) and implanted after five to six passages into the striatum, hippocampus and cortex of neonatal rats. Cell suspensions of primary LGE tissue were prepared and grafted in parallel. Grafted cells derived from the primary tissue displayed widespread incorporation into all regions, as visualized with the mouse-specific antibody M2, or mouse satellite DNA in situ hybridization, and differentiated into both neurons, astrocytes and oligodendrocytes. Grafts of neurosphere cells derived from the LGE, MGE and cortical primordium differentiated primarily into astrocytes, but contained low but significant numbers of GFP-immunoreactive neurons. Neurons derived from LGE neurospheres were of three types: cells with the morphology of medium-sized densely spiny projection neurons in the striatum; cells with interneuron-like morphologies in striatum, cortex and hippocampus; and cells integrating into SVZ and migrating along the RMS to the olfactory bulb. MGE- or cortical primordium-derived neurospheres differentiated into interneuron-like cells in both striatum and hippocampus. The results demonstrate the ability of in vitro expanded neural stem/progenitor cells to generate both neurons and glia after transplantation into neonatal recipients, and differentiate in a region-specific manner into mature neurons with morphological features characteristic for each target site.     

C57BL／6-gfp小鼠嗅球NSC培养及定位注射方法的建立

目的建立一套稳定的绿色荧光蛋白转基因小鼠（C57BL／6-gfp）嗅球神经干细胞（NSC）体外培养的方法,并初步应用于定位注射试验,为干细胞移植治疗神经性耳聋的实验研究奠定基础。方法培养C57BL／6-gfp小鼠胚胎嗅球NSC,传代并进行分化实验,鉴定后将其立体定位注射于大鼠耳蜗核。结果所培养的NSC生长良好,可稳定传代,能够分化为三种神经细胞。定位注射后可在局部见到绿色荧光阳性的细胞团。结论该方法培养的C57BL／6-gfp小鼠胚胎嗅球NSC可稳定传代并可作为荧光标记细胞进行移植试验。     

Age-related expression of sigma1 receptors and antidepressant efficacy of a selective agonist in the senescence-accelerated (SAM) mouse

The sigma1 receptor is a unique intracellular receptor whose activation results in an efficient modulation of several neurotransmitter responses. Its role as a target for the rapid nongenomic effects of neuro(active)steroids and the age-related diminutions in steroid levels suggested that targeting the sigma1 receptor might allow alleviation of age-related neuronal dysfunctions. We examined here the expression and behavioral efficacy of sigma1 receptors in the senescence-accelerated (SAM) mouse model. The sigma1 receptor mRNA expression was measured by using comparative RT-PCR in the olfactory bulb, hippocampus, hypothalamus, cortex, or cerebellum of senescence-prone SAMP/8 and senescence-resistant SAMR/1 control animals. No difference was observed between substrains in 6-, 9-, and 12-month-old (m.o.) mice. The sigma1 protein expression was analyzed by using immunohistochemical techniques. Labeling was intense in the olfactory bulb, hippocampus, hypothalamus, and midbrain of both SAMR/1 and SAMP/8 mice, and the distribution appeared unchanged in 6-, 9-, and 12-m.o. animals. The receptor's in vivo availability was examined by using in vivo [3H](+)-SKF-10,047 binding. No age-related difference was observed in the olfactory bulb, hippocampus, hypothalamus, cortex, cerebellum, and brainstem of 6- or 12-m.o. SAMR/1 or SAMP/8 mice. The antidepressant efficacy of the selective agonist igmesine was examined in the forced-swimming test. The compound decreased significantly the immobility duration at 60 mg/kg in 6- and 12-m.o. SAMR/1 and in 6-m.o. SAMP/8 mice. In 12-m.o. SAMP/8 mice, the drug efficacy was facilitated; a significant effect was measured at 30 mg/kg. Decreased neurosteroid levels, particularly of progesterone, were seen in 12-m.o. SAMP/8 mice that might explain the enhanced efficacy of igmesine. Preserved sigma1 receptor expression and enhanced behavioral efficacy of sigma1 agonists were measured in SAM animals, confirming the therapeutic opportunities for selective ligands against age-related mood disorders.