雌激素在脑缺血诱导的大鼠齿状回神经再生中的作用

为研究雌激素对成年动物局灶性脑缺血诱导成年动物海马齿状回神经元再生的影响，将雄性SD大鼠分为假手术+雌激素组(SE)、假手术+生理盐水替代组(SN)、缺血+雌激素组(ME)和缺血+生理盐水替代组(MN)，右侧大脑中动脉闭塞(MCAO)建立脑缺血模型。在缺血90 min后恢复供血再灌注，分别于再灌注后1 3、12、24和28 h处死老鼠并检测各组大鼠脑梗死体积、细胞凋亡以及脑缺血诱导的成年动物海马齿状回神经元再生的情况。在5个时间点的检测中，ME组脑梗死体积显著小于SE组(P<0.05)；在MCAO大鼠中，海马齿状回区域并未发现有神经元丢失及凋亡的现象。同时，MN组与SN组相比较，损伤侧齿状回新生神经元数目明显增多(P<0.05)，说明这种缺血诱导的神经元再生并不依赖于齿状回区域神经细胞的死亡；ME组与MN组相比较，损伤侧新生神经元数目显著增多(P<005)；SE与SN组相比较，手术侧和对侧的新生神经元数目都显著增加(P<0.05)。结果提示雌激素对局灶性脑缺血后海马齿状回神经元再生具有促进作用，且这种促进作用与海马缺血损伤程度无关。     

自主跑轮运动上调成年小鼠海马齿状回区细胞增殖及BDNF、IGF1和WNT4的表达水平

成年海马神经发生在调控学习记忆和情感过程中有重要作用。外界刺激因素,如自主运动能影响成年海马神经发生过程。自主运动能显著增强海马齿状回区的细胞增殖,并使新生神经元增多,但其具体机制仍不是十分清楚。本研究采用跑轮实验这种啮齿动物自主运动模型,使两月龄的C57BL/6成年小鼠跑轮15天后,用BrdU掺入实验观察海马齿状回区的细胞增殖情况。采用逆转录实时荧光定量PCR法(RT-qPCR)和蛋白免疫印迹法(Western blot)检测海马齿状回区、阿蒙角区和皮层区成年神经发生相关因子的mRNA和蛋白表达水平。结果显示,自主跑轮运动15天后的成年小鼠海马齿状回区增殖细胞数目显著上调;齿状回区Bdnf、Igf1的mRNA和蛋白表达水平显著升高,Wnt4的mRNA表达水平显著升高,提示15天自主运动可能通过特异地增加海马齿状回区BDNF、IGF1和WNT4的表达产生上调成年海马神经发生的作用。     

电针刺激对缺血性脑卒中大鼠内源性神经干细胞激活的影响

目的：探讨电针刺激对局灶性脑缺血大鼠内源性神经干细胞的激活作用以及对大鼠神经功能的影响。方法：将大鼠随机分为2组（n=20）：单纯缺血组和电针刺激组。单纯缺血组大鼠建模成功后不给予任何干预,电针刺激组大鼠给予合谷、足三里电针刺激,然后免疫组化观察两组大鼠海马齿状回、侧脑室室下回神经干细胞的增殖和分化情况,以及两组大鼠神经功能的恢复。结果：电针刺激组大鼠侧脑室室下带、海马齿状回颗粒下层神经干细胞增殖活跃,数目多于单纯缺血组,差异有统计学意义（P〈0．05）,神经功能评分改善好于单纯缺血组（P〈0．05）。结论：电针刺激能够促进局灶性脑缺血大鼠内源性神经干细胞的增生和分化,从而促进大鼠神经功能康复。     

神经干细胞及其对脑缺血损伤的潜在治疗作用

新神经元在成年哺乳动物脑的特定区域出现,起源于海马齿状回和室下带的神经干细胞,神经干细胞可以分化成神经元,星形胶质细胞和少突胶质细胞,采用双标技术可以检测新神经元的发生,神经干细胞具有自我更新和多方向分化的潜能,受内在因素和外部环境的调控,证据显示成年人脑海马齿9状回的颗粒细胞产生新神经元,恒河猴的室下带产生新神经元,迁移到新皮质区分化为成熟神经元,人的神经干细胞已从胚胎前脑获得,缺血损伤可以激活齿状回的神经干细胞增殖,激活或移植神经干细胞对缺血损伤的脑组织具有潜在的治疗作用。     

电针对脑缺血神经元凋亡影响的形态学研究

为了探讨针刺治疗“脑卒中”的机制,本研究以大鼠一侧大脑中动脉栓塞后再灌注为动物模型,分别以ＴＵＮＥＬ法和ＰＩ染色法观察电针改善脑缺血性神经元凋亡的情况。结果显示：①局灶性脑缺血能诱导神经元凋亡：缺血侧凋亡神经元数目明显多于对照侧,差异非常显著;②电针能明显抑制神经元凋亡：电针治疗组缺血侧梗塞区凋亡神经元数目明显减少。本文表明电针能抑制脑缺血性神经元凋亡。     

电针预处理对脑缺血再灌注后小胶质细胞活化状态的影响

目的:观察电针预处理对脑缺血再灌注后小胶质细胞活化状态的影响。方法:成年雄性SD大鼠随机分为假手术组(sham)、缺血组(MCAO)、电针处理组(EA+MCAO)三组。采用大脑中动脉栓塞(MCAO)诱导大鼠局灶性脑缺血再灌注模型。缺血再灌注后6 h、24 h、3 d和7 d取材,运用Western blot及免疫荧光技术检测缺血半暗带小胶质细胞活化状态以及M1/M2型特异性标志分子的表达水平。结果:脑缺血再灌注后,小胶质细胞被激活,数量表达增加(P0.05,vs.sham组),形态从静息状态的分支状转变为圆形的阿米巴状。M1型标志分子i NOS主要表达于缺血再灌注后24小时(P0.05,vs.sham组),M2型标志分子Arginase主要表达于缺血再灌注后7天(P0.05,vs.sham组)。电针预处理上调Arginase的表达水平,下调i NOS的表达水平(P0.05,vs.MCAO组)。结论:缺血再灌注后小胶质细胞被激活,电针预处理促使活化的小胶质细胞由M1向M2转化。     

甘珀酸干预对大鼠脑缺血再灌注损伤的影响

目的观察缝隙连接阻断剂甘珀酸对局灶性脑缺血/再灌注损伤的影响。方法采用大鼠大脑中动脉阻塞再灌流模型（MCAO）,将动物随机分为脑缺血60min再灌注（MCAO）组,脑缺血再灌注加甘珀酸干预（MCAO＋CBX）组和假手术组（sham）。采用尼氏染色显示脑梗死灶并计算梗死灶体积;应用免疫荧光与TUNEL染色法分别观察脑缺血后3d与7d不同时间点缺血边缘区胶质纤维酸性蛋白（GFAP）的表达和细胞凋亡情况。结果（1）缺血后3d、7d MCAO＋CBX组大鼠梗死体积小于MCAO组,3d、7d MCAO＋CBX组大鼠梗死体积较MCAO组分别缩小5%和4.6%;（2）缺血后3d、7d于缺血边缘区可见大量TUNEL阳性染色细胞,且MCAO组大鼠缺血边缘区细胞凋亡数目明显多于MCAO＋CBX大鼠（P〈0.001）;（3）缺血后3d和7d组缺血边缘区GFAP表达明显增强,3d的MCAO组与MCAO＋CBX组大鼠缺血边缘区GFAP的表达均较假手术组强（P〈0.05）,7d的MCAO＋CBX组大鼠缺血边缘区GFAP的表达较假手术组强（P〈0.001）,但明显弱于MCAO组大鼠（P〈0.01）;结论缝隙连接阻断剂甘珀酸可减少大鼠大脑中动脉阻塞后脑梗死体积,其机制可能与阻断缝隙连接后缺血边缘区神经元凋亡降低有关,星型胶质细胞的反应性变化参与了该过程。     

细胞周期抑制剂对大鼠局灶性脑缺血后神经元的保护作用

目的通过观察选择性细胞周期抑制剂olomoucine对局灶性脑缺血边缘区神经元凋亡的影响,以探讨细胞周期调控与神经元细胞凋亡的关系。方法建立光化学法诱导大鼠局灶性脑缺血模型,随机分为脑缺血组(对照组和干预组)和假手术组,采用HE染色显示梗死灶并测定其面积;应用免疫荧光化学法检测梗死灶周围神经元核心抗原(NeuN)的表达及通过TUNEL方法检测神经元凋亡;免疫印迹(Western blot)观察损伤侧皮层NeuN、周期素蛋白A(cyclin A)和周期素蛋白B1(cyclin B1)蛋白的表达。结果缺血后3d对照组梗死灶面积占脑片面积百分比值的平均值明显大于干预组(P<0.05);缺血后缺血边缘区NeuN表达减弱,对照组NeuN表达明显弱于干预组(P<0.05);缺血后梗死灶周围可见大量TUNEL阳性染色细胞,而且对照组数量明显多于干预组(P<0.05);干预组大鼠NeuN(TUNAL双标阳性表达明显弱于对照组大鼠(P<0.05);NeuN的蛋白量的表达,干预组较对照组明显增加(P<0.05),而对照组cyclin A和cyclin B1蛋白量的表达明显高于干预组(P<0.05)。结论通过对细胞周期的调控,可减少神经元凋亡和脑梗死体积,从而为缺血性脑损伤后的神经元提供一个保护作用。     

大鼠脑缺血/再灌注后bFGF和GAP-43的表达与神经再生

目的:观察大鼠脑缺血/再灌注后不同时间段碱性成纤维生长因子(bFGF)和生长相关蛋白43(GAP-43)表达与神经元再生的变化,探讨其与神经再生的有关机制。方法:建立大鼠大脑中动脉阻塞模型(MCAO),并分为缺血再灌注3 d、7 d、14 d和28 d四组(n=6)。以神经损伤严重程度评分(NSS),运动评分测试(SMT)评估神经功能缺损程度,Nissl和TUNEL染色法观察不同时段缺血区周边组织神经元存活和凋亡情况,应用蛋白免疫印迹法和免疫荧光双标法检测缺血/再灌注后不同时段缺血区周边组织bFGF和GAP-43的表达水平和神经元再生的变化情况。结果:大鼠脑缺血/再灌注后3 d,大鼠出现了明显的神经功能缺损及运动功能障碍,缺血区周边组织神经元凋亡亦达到高峰,同时bFGF和GAP-43表达增强,7 d达到高峰,以后逐渐减弱,缺血周边组织可见散在的新生神经元,持续到28 d。结论:大鼠脑缺血/再灌注后内源性bFGF和GAP-43表达水平增加,可能与其神经修复和再生有关。     

人参皂甙Rg1对去卵巢帕金森病模型大鼠黑质多巴胺能神经元的保护作用

目的：探讨人参皂甙Rg1对6-羟基多巴（6-OHDA）制备的去卵巢（OVX）帕金森病（PD）模型大鼠黑质（SN）多巴胺能神经元的保护作用及其可能机制。方法：应用6-OHDA制备的OVX PD模型大鼠,侧脑室给予Rg1或雌激素。免疫组织化学染色酪氨酸羟化酶（TH）阳性神经元和Bcl-2蛋白。Perls’铁染色检测SN铁含量。结果：①Rg1或雌激素可抑制阿朴吗啡诱导的PD大鼠旋转行为;②在损毁侧SN,Rg1或雌激素用药组TH阳性神经元数量较6-OHDA组显著增多;③6-OHDA组损毁侧SN内铁含量较健侧明显升高,应用Rg1或雌激素后,SN铁含量较模型组明显减少;④与6-OHDA模型组相比,Rg1及雌激素均可增加损毁侧大鼠SN内Bcl-2蛋白表达。结论：人参皂甙Rg1具有类雌激素样作用,对OVX PD模型大鼠黑质DA能神经元有明显的保护作用,其作用机制可能与降低铁负载和抗凋亡有关。     

Blockade of L-type voltage-gated Ca channel inhibits ischemia-induced neurogenesis by down-regulating iNOS expression in adult mouse

Neurogenesis in the adult mammalian hippocampus may contribute to repairing the brain after injury. The signals that regulate neurogenesis in the dentate gyrus following ischemic stroke insult are not well known. We have previously reported that inducible nitric oxide synthase (iNOS) expression is necessary for ischemia-stimulated neurogenesis in the adult dentate gyrus. Here, we show that mice subjected to 90 min of middle cerebral artery occlusion (MCAO) significantly increased the number of new neurons and up-regulated iNOS expression in the dentate gyrus. Blockade of the L-type voltage-gated Ca(2+) channel (L-VGCC) prevented neurogenesis in the dentate gyrus and subventricular zone (SVZ), and down-regulated iNOS expression in the dentate gyrus after cerebral ischemia. This study suggests that Ca(2+) influx through L-VGCC is involved in ischemia-induced neurogenesis by up-regulating iNOS expression.     

Regional differences in enhanced neurogenesis in the dentate gyrus of adult rats after transient forebrain ischemia

Neurogenesis in the dentate gyrus occurs throughout life. We observed regional differences in neurogenesis in the dentate gyrus of adult rats following transient forebrain ischemia. Nine days after ischemic-reperfusion or sham manipulation, rats were given 5-bromo-2'-deoxyuridine-5'-monophosphate (BrdU), a marker for dividing cells. They were killed 1 or 28 days later to distinguish between cell proliferation and survival. Neurogenesis was evaluated by BrdU incorporation as well by identifying neuronal and glial markers in six regions of the dentate gyrus: rostral, middle and caudal along the rostrocaudal axis, each further divided into suprapyramidal and infrapyramidal blade subregions. In control rats BrdU-positive cells in the rostral subregions were significantly lower in the suprapyramidal than in the infrapyramidal blades at both 1 and 28 days after BrdU injection. One day after injection, BrdU-positive cells had increased more in five of the subregions in the ischemic rats than in the controls, the exception being the suprapyramidal blade of the rostral subregion. At 28 days after BrdU injection, numbers of BrdU-positive cells were higher in four subregions in the ischemic group, the exceptions being the rostral suprapyramidal and middle infrapyramidal blades. At 28 days after BrdU injection, the percentages of BrdU positive cells that expressed a neuronal marker (NeuN) were the same in the dentate granule cell layers of ischemic and control rats. Our data thus demonstrate regional differences in enhanced neurogenesis in the dentate gyrus of adult rats after transient forebrain ischemia.     

Reduced neuronal nitric oxide synthase is involved in ischemia-induced hippocampal neurogenesis by up-regulating inducible nitric oxide synthase expression

Nitric oxide (NO), a free radical with signaling functions in the CNS, is implicated in some developmental processes, including neuronal survival, precursor proliferation, and differentiation. However, neuronal nitric oxide synthase (nNOS) -derived NO and inducible nitric oxide synthase (iNOS) -derived NO play opposite role in regulating neurogenesis in the dentate gyrus after cerebral ischemia. In this study, we show that focal cerebral ischemia reduced nNOS expression and enzymatic activity in the hippocampus. Ischemia-induced cell proliferation in the dentate gyrus was augmented in the null mutant mice lacking nNOS gene (nNOS−/−) and in the rats receiving 7-nitroindazole, a selective nNOS inhibitor, after stroke. Inhibition of nNOS ameliorated ischemic injury, up-regulated iNOS expression, and enzymatic activity in the ischemic hippocampus. Inhibition of nNOS increased and iNOS inhibitor decreased cAMP response element-binding protein phosphorylation in the ipsilateral hippocampus in the late stage of stroke. Moreover, the effects of 7-nitroindazole on neurogenesis after ischemia disappeared in the null mutant mice lacking iNOS gene (iNOS−/−). These results suggest that reduced nNOS is involved in ischemia-induced hippocampal neurogenesis by up-regulating iNOS expression and cAMP response element-binding protein phosphorylation.     

胎次、性别对成年小鼠海马齿状回神经发生及学习记忆的影响

目的探讨胎次、性别是否对成年小鼠海马齿状回神经发生及学习记忆产生影响。方法运用Morris水迷宫系统检测第1～3胎成年小鼠的学习记忆能力,腹腔注射BrdU,标记神经干细胞,检测不同胎次、性别小鼠海马齿状回中的神经发生的差异。结果 （1）在同性别、不同胎次成年小鼠间,第2胎的学习记忆能力（LMA）均显著地高于第1、3胎的,其影响规律为LMA2〉LMA1〉LMA3,且P〈0.05;在同胎次、不同性别成年小鼠间,雌性小鼠的LMA均高于雄性小鼠的,但其差异无显著性（P〉0.05）。（2）在同性别、不同胎次成年小鼠间,第2胎海马DG新生神经细胞的数量（N）均高于第1、3胎的,其影响规律分别为NF2〉NF3〉NF1和NM2〉NM1〉NM3,但其差异无显著性（P〉0.05）;在同胎次、不同性别成年小鼠间,雌性小鼠的N均高于雄性小鼠的,但其差异无显著性（P〉0.05）。结论胎次、性别对实验动物神经发生及学习记忆等方面产生的影响是肯定的。因此,在使用实验动物时,应予以充分考虑,尽量使用胎次、性别相同的。     

人参皂甙对老龄大鼠海马结构BDNF及Trk B蛋白表达的影响

目的探讨老龄大鼠海马结构BDNF及Trk B蛋白表达的老龄性变化,同时对比观察人参皂甙对其改变的影响。方法雌性Wistar大鼠39只,分为青年组、老龄组、给药组(第17个月始饲以人参皂甙至27月龄)。采用免疫组化及Western blot方法对海马结构BDNF反应产物及Trk B蛋白进行定性、定量分析。结果老龄组CA3、CA1区BDNF含量分别较青年组下降13.3%、10.4%(P<0.05);其给药组和老龄组相比变化不大(P>0.05)。齿状回从青年到老年变化不明显(P>0.05),但给药组比老龄组增加16.7%(P<0.01)。老龄组海马结构Trk B蛋白表达较青年组下调了99.7%;给药组较老龄组上调了78.5%(P<0.01)。结论老龄组海马结构BDNF及Trk B蛋白表达较青年组明显降低,而人参皂甙可明显上调齿状回BDNF含量和海马结构Trk B蛋白的表达。     

Treadmill exercise increases cell proliferation without altering of apoptosis in dentate gyrus of Sprague-Dawley rats

The effects of forced treadmill exercise on cell proliferation and apoptosis in the hippocampal dentate gyrus in Sprague-Dawley rats were investigated. The animals were classified into three groups: the control group, the easy exercise group, and the moderate exercise group. In the control group, rats were left on the treadmill without running for 30 min per day, while rats in the exercise groups were made to run on the treadmill for the same duration. All rats were injected intraperitoneally with 5-bromo-2'-deoxyuridine (BrdU) one-hour prior to exercise once a day for 7 consecutive days beginning at the start of the exercise regimens. Each of the rats was sacrificed 2 h after the last exercise. Both the easy and moderate exercise groups revealed increased number of BrdU-positive cells in the dentate gyrus compared to the control group. The terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay revealed very few apoptotic cells, with no statistically significant differences among the groups. These results showed that treadmill exercise increases cell proliferation without altering of apoptosis in the dentate gyrus of the hippocampus.     

Paradoxical effects of learning the Morris water maze on adult hippocampal neurogenesis in mice may be explained by a combination of stress and physical activity

Studies in rats that assessed the relation of hippocampus-dependent learning and adult hippocampal neurogenesis suggested a direct regulatory effect of learning on neurogenesis, whereas a similar study in mice had not found such causal link. We here report a substantial decrease of BrdU-positive cells and other measures of adult hippocampal neurogenesis in mice trained in the hidden (HID) or cued version (VIS) of the Morris water maze as compared to untrained animals (CTR). Particularly, cells on advanced stages of neuronal development contributed to this decrease, whereas earlier progenitors (type 2 cells) were not diminished in HID, but were diminished in VIS as compared to CTR. The differential regulation of type 2 cells in HID and VIS may have been caused by a different degree of physical activity, given that a time-yoked control group did not differ from HID, and type 2 cells reportedly constitute the proliferative dentate gyrus population that primarily responds to physical activity. The decrease of hippocampal neurogenesis by water maze training was reversible by pre-exposing animals to the water maze prior to training, suggesting that stress associated with training may have caused the acute downregulation of adult neurogenesis. We propose that in mice the Morris water maze does not provide a pure enough learning stimulus to study the presumed effects of 'learning' on adult neurogenesis. In addition, however, our data show that physical activity that is intricately linked to many cognitive tasks in rodents might play an important role in explaining effects of learning on cellular hippocampal plasticity.     

Testosterone and dihydrotestosterone, but not estradiol, enhance survival of new hippocampal neurons in adult male rats

Past research suggested that androgens may play a role in the regulation of adult neurogenesis within the dentate gyrus. We tested this hypothesis by manipulating androgen levels in male rats. Castrated or sham castrated male rats were injected with 5-Bromo-2'deoxyuridine (BrdU). BrdU-labeled cells in the dentate gryus were visualized and phenotyped (neural or glial) using immunohistochemistry. Castrated males showed a significant decrease in 30-day cell survival within the dentate gyrus but there was no significant change in cell proliferation relative to control males, indicating that androgens positively affect cell survival, but not cell proliferation. To examine the role of testosterone on hippocampal cell survival, males were injected with testosterone s.c. for 30 days starting the day after BrdU injection. Higher doses (0.5 and 1.0 mg/kg) but not a lower dose (0.25 mg/kg) of testosterone resulted in a significant increase in neurogenesis relative to controls. We next tested the role of testosterone's two major metabolites, dihydrotestosterone (DHT), and estradiol, upon neurogenesis. Thirty days of injections of DHT (0.25 and 0.50 mg/kg) but not estradiol (0.010 and 0.020 mg/kg) resulted in a significant increase in hippocampal neurogenesis. These results suggest that testosterone enhances hippocampal neurogenesis via increased cell survival in the dentate gyrus through an androgen-dependent mechanism.     

Stroke-induced progenitor cell proliferation in adult spontaneously hypertensive rat brain: effect of exogenous IGF-1 and GDNF

Progenitor cells in the dentate gyrus of hippocampus (DG) and the subventricular zone of lateral ventricles (SVZ) generate new neurons throughout the life of mammals. Cerebral ischemia increases this basal progenitor cell proliferation. The present study evaluated the time frame of proliferation, length of survival and the phenotypes of the new cells formed after transient middle cerebral artery occlusion (MCAO) in adult spontaneously hypertensive rats. Compared to sham controls, ischemic rats showed a significantly higher number of newly proliferated cells (as defined by BrdU immunostaining) in both the DG (by fourfold, p < 0.05) and the SVZ (by twofold, p < 0.05). DG showed increased proliferation only in the first week of reperfusion and 49% of the cells formed in this period survived to the end of third week. Whereas, SVZ showed a continuous proliferation up to 3 weeks after MCAO, but the cells formed survived for less than a week. In both DG and SVZ, at the end of the first week of reperfusion, majority of the BrdU-positive (BrdU+) cells were immature neurons (DCX positive). In the DG, 28% of the cells formed in the first week after MCAO mature into neurons (NeuN positive). The ischemic cortex and striatum showed several BrdU+ cells which were ED-1 positive microglia/macrophages. At 1 week of reperfusion, MCAO-induced progenitor cell proliferation in the ipsilateral DG was significantly increased by i.c.v. infusion of IGF-1 (by 127 +/- 14%, p < 0.05) and GDNF (by 91 +/- 5%, p < 0.05), compared to vehicle. In the growth factor treated rats subjected to transient MCAO, several BrdU+ cells formed in the first week survived up to the third week.