癫痫发作后成鼠及幼鼠海马神经发生的改变

目的:观察幼鼠及成鼠癫痫发作后海马神经发生的变化。方法:选用3周龄和成年雄性SD大鼠,氯化锂-匹罗卡品药物点燃造模,造模成功后根据溴脱氧尿嘧啶核苷(BrdU)注射时间点分为24 h组、2周组,并设相应的对照组。通过免疫荧光染色技术在激光共聚焦显微镜下观察幼鼠及成年鼠海马齿状回颗粒细胞层神经细胞的增殖情况。结果:(1)幼鼠24 h及2周实验组海马齿状回颗粒细胞层BrdU阳性细胞数均显著高于相应对照组(P<0.05),且24 h组较2周组显著增多(P<0.05);(2)成年鼠24 h实验组的BrdU阳性细胞数明显多于相应对照组(P<0.05),而2周组则相反(P<0.05)。结论:癫痫发作可导致海马神经发生的改变,幼鼠癫痫发作后海马齿状回颗粒细胞层神经细胞增殖显著升高,但随着时间延长呈下降趋势。慢性癫痫发作可引起成年鼠海马神经细胞增殖的降低。     

枸杞多糖对癫痫大鼠海马神经细胞增殖的影响

目的:探讨枸杞多糖(Lycium barbarum polysaccharide,LBP)对大鼠癫痫发作后海马齿状回颗粒细胞层神经细胞的影响。方法:选择健康5周龄雄性SD大鼠,随机分为三组,即对照组、癫痫组和LBP干预组。癫痫组腹腔注射氯化锂-匹罗卡品,LBP干预组在注射氯化锂-匹罗卡品前分别灌服25 mg/kg、50 mg/kg、100 mg/kg的LBP 2周,对照组给予等剂量生理盐水。溴脱氧尿嘧啶核苷(Brd U)标记后免疫荧光染色,观察大鼠海马齿状回颗粒细胞层Brd U阳性细胞的增殖情况。结果:与对照组相比较,癫痫组的Brd U阳性细胞明显增多(P0.01);与癫痫组相比较,不同剂量LBP干预组的Brd U阳性细胞均明显减少(P0.01),但各剂量LBP干预组间无显著性差异(P0.05)。结论:LBP可干预大鼠癫痫发作后海马齿状回颗粒层Brd U阳性细胞数的异常增生,具有明显的神经保护作用。     

成年大鼠脑损伤后海马新生神经元功能特点的研究

目的：观察弥漫性脑损伤后海马齿状回新生神经元GAD67和Fos蛋白的表达，探讨海马齿状回新生神经元的功能特点。方法：参考Marmarou方法制作大鼠弥漫性脑损伤模型，采用BrdU标记和免疫荧光组织化学方法并结合激光共聚焦显微镜技术观察海马齿状回中BrdU阳性细胞GAD67的表达和二次致伤后Fos蛋白的表达情况。结果：①成年大鼠弥漫性脑损伤后海马齿状回颗粒细胞层中的BrdU免疫阳性细胞可表达GAD67，而位于亚颗粒增生带的BrdU免疫阳性细胞未见GAD67表达；②在病理刺激下，成年大鼠弥漫性脑损伤后齿状回颗粒细胞层内的BrdU免疫阳性细胞可表达Fos蛋白。结论：弥漫性脑损伤后齿状回颗粒细胞层中的新生神经元不仅可以表达神经活性递质而且能够被病理刺激激活，具有与成熟神经元相似的功能特点。     

MK-801诱发精神分裂症对大鼠探索能力、疼痛反应及海马神经细胞增殖的影响

目的:观察大鼠精神分裂症后探索能力、痛觉及海马齿状回颗粒细胞层神经细胞增殖的改变。方法:MK-801腹腔注射制备精神分裂症动物模型,分别监测给药后第1、5、10、14 d大鼠的探洞次数和甩尾时间。Brdu标记后取材,应用免疫荧光染色和激光共聚焦显微技术观察海马齿状回神经细胞的增殖情况。结果:①洞板试验:实验组大鼠的探洞次数较对照组显著下降(P<0.01),且随着MK-801给药时间的增加而减少(P<0.01);②甩尾试验:实验组大鼠对疼痛刺激的反应时间较对照组缩短(P<0.01),且随着MK-801给药时间的延长痛觉敏感度不断增加(P<0.01);③神经细胞的增殖:停药后第1 d,实验组海马齿状回颗粒层神经细胞增殖数较对照组减少(P<0.05),第16 d两组间无显著差异(P>0.05)。结纶:MK-801诱发精神分裂症后可致大鼠的探索能力减退、痛觉敏感,同时可降低海马齿状回颗粒细胞层神经细胞的增殖。     

大鼠精神分裂症后自发性运动量及海马神经发生的改变

目的:探讨成年大鼠精神分裂症后自发性运动量和海马神经发生的改变。方法:通过连续2周腹腔注射环苯已哌啶(phencyclidine,PCP)建立大鼠精神分裂症模型,利用动物运动分析系统监测大鼠自发性运动量,5-溴-2-脱氧尿苷嘧啶(BrdU)标记新生的神经细胞,用免疫荧光标记法监测海马齿状回BrdU、NeuN、S-100β的表达,利用激光共聚焦显微镜观察海马神经细胞的增殖与分化情况。结果:精神分裂症模型大鼠比对照组大鼠自发性运动量高出2～3倍(P0.05);BrdU阳性细胞数约下降了24%(P0.05);两组BrdU阳性细胞的分化无明显差异性(P0.05),大多分化为神经元。结论:精神分裂症可导致成年大鼠自发性运动量增加,并引起海马神经发生的改变,降低神经细胞的增殖。     

癫痫发作敏感大鼠齿状回苔状纤维侧枝发芽

目的　探讨海马齿状回苔状纤维侧枝发芽与癫痫发作敏感性形成之间的关系。方法　在颈部皮下注射惊厥剂量的海人酸 (KA ,10mg/kg)诱发大鼠出现癫痫发作后 ,采用Timm’s染色法 ,分别在注射KA后3d、7d和 1个月 3个时间点观察致痫大鼠海马齿状回内苔状纤维发芽的情况。结果　Timm’s染色发现 ,注射KA后 7d ,海马齿状回分子层内带和颗粒细胞上层出现苔状纤维的异常发芽 ,注射KA后 1个月海马齿状回内Timm’s染色颗粒颜色加深 ,范围增大。提示海马苔状纤维发芽形成的时间过程与癫痫发作敏感性形成的时间过程一致。结论　海马齿状回分子层内带和颗粒细胞上层出现异常的苔状纤维发芽可能与癫痫发作敏感性形成有关。     

Proliferation and differentiation of neural precursors in hippocampus and dentate gyrus after intracerebral hemorrhage in adult rats

目的:观察成年大鼠脑出血后海马齿状回神经前体细胞的增殖与分化,探讨脑出血后神经前体细胞的变化规律.方法:制作大鼠脑出血模型,5-溴脱氧尿嘧啶核苷(BrdU)腹腔注射标记增殖细胞,用免疫组织化学法检测大鼠海马齿状回BrdU、神经元核抗原(NeuN)、胶质纤维酸性蛋白(GFAP)阳性细胞数的变化.结果:正常组和假手术组大鼠海马齿状回均有少量BrdU阳性细胞,脑出血后大鼠各时间段的BrdU阳性细胞均较正常组和假手术组增加,7d组达到峰值后逐渐下降,28 d组仍高于正常组和假手术组.正常成年大鼠海马齿状回可见少量BrdU/NeuN和BrdU/GFAP双标阳性细胞,脑出血后双标阳性细胞数较正常组增加.结论:脑出血后大鼠海马齿状回神经前体细胞增殖明显,且可以向神经元和神经胶质细胞分化.     

GAD67-GFP精神分裂症小鼠行为学改变及海马齿状回颗粒细胞层GABA能神经元的表达

目的:探讨谷氨酸脱羧酶67-绿色荧光蛋白(GAD67-GFP)基因敲入小鼠制备精神分裂症模型后学习与记忆功能的改变及海马齿状回颗粒细胞层GABA能神经元的表达。方法:利用聚合酶链式反应(PCR)鉴定GAD67-GFP基因敲入小鼠,MK-801连续腹腔注射2周制备精神分裂症动物模型,通过悬尾实验、Morris水迷宫实验、免疫荧光标记技术等,观察GAD67-GFP基因敲入小鼠的学习与记忆功能的改变及GABA能神经元在海马齿状回颗粒细胞层的表达。结果:停药后实验组与对照组比较:(1)实验组体重增加明显低于对照组(P0.05);(2)行为学改变:1悬尾实验:实验组不动时间明显小于对照组(P0.05);2Morris水迷宫实验:定位航行实验中实验组逃避潜伏期,游泳总路程明显长于对照组(P0.05),而其平均游泳速度与对照组没有明显差异(P0.05);空间探查实验中实验组经过平台所在点的次数和在平台所在象限的时间明显小于对照组(P0.05);(3)在海马齿状回颗粒细胞层中实验组的GFP阳性细胞明显多于对照组(P0.05)。结论:通过对GAD67-GFP基因敲入小鼠进行腹腔注射MK-801制备精神分裂症模型后,其学习与记忆功能显著下降,且海马齿状回颗粒细胞层GABA能神经元明显增加。提示精神分裂症后学习记忆功能减退可能与GABA能神经元的表达有关。     

锂-匹鲁卡品诱导大鼠癫痫发作后海马齿状回IL-1 mRNA表达的变化

目的:观察大鼠癫痫发作后海马齿状回细胞因子IL-1(IL-1R、IL-1β、IL-1ra)mRNA的表达变化,探讨IL-1在癫痫发作中的作用.方法:大鼠随机分为对照组和实验组.实验组大鼠腹腔注射氯化锂以及匹鲁卡品,对照组注射生理盐水,观察其行为学特征,并用RT-PCR方法检测大鼠海马齿状回内细胞因子IL-1 mRNA的动态表达变化.结果:大鼠腹腔注射锂-匹鲁卡品后30 min内相继出现严重癫痫持续状态发作,实验组各种细胞因子在严重癫痫持续发作后1 h表达水平开始明显增加(P<0.05),随着时间的延长表达逐渐增多,IL-1β的表达于发作后6 h达高峰,IL-1ra和IL-1 R1则在发作后12 h达高峰,随后表达逐渐下降,各因子到发作后48 h表达降低但仍高于对照组表达水平(P<0.05).结论:癫痫发作后急性期细胞因子IL-1β、IL-1ra及其受体IL-1 R1在不同时间点均有增加,提示炎性因子参与了癫痫发作以及癫痫发作后脑损伤.     

BMP4参与海人酸损伤后成年大鼠海马齿状回颗粒细胞增殖及胶质增生

目的探讨海人酸（KA）侧脑室注射致大鼠海马损伤后骨形成蛋白-4（BMP4）的表达变化及其与颗粒细胞增殖和胶质细胞增生的关系。方法将成年大鼠分为对照组与实验组。侧脑室注射KA7d后,用尼氏染色检测海马神经元丢失,用免疫组织化学与原位杂交的方法检测海马齿状回BMP4mRNA阳性细胞与BrdU标记细胞、GFAP阳性细胞数的变化。结果正常成年大鼠BMP4mRNA阳性细胞主要分布于海马齿状回的门区、颗粒下层、CA3、CAI区。BrdU标记细胞主要分布在齿状回颗粒下层。GFAP阳性细胞主要分布在齿状回、CA3区。在KA侧脑室注射致海马损伤后7d,海马CA3、CA4区神经元丢失明显,BMP4mRNA阳性细胞与BrdU、GFAP阳性细胞均明显增加。结论KA侧脑室注射致海马损伤后,成年大鼠海马齿状回颗粒细胞增殖增强和胶质增生可能与BMP4表达增加有关。     

电针对脾虚证大鼠海马区神经干细胞增殖分化的影响

BACKGROUND:Spleen deficiency has varying impact on the immune system and digestive system of the human body, and can also damage the normal function of the central nervous system as there is a close relationship between the spleen and brain. OBJECTIVE:To explore the effect of electroacupuncture on the proliferation and differentiation of neural stem cells in the rat hippocampus of spleen deficiency syndrome. METHODS:Sixty Sprague-Dawley rats were randomly divided into three groups: normal, control and electroacupuncture groups. The animal model of spleen deficiency syndrome was prepared in the control group and electroacupuncture group. Two weeks after modeling, rats in the electroacupuncture group were given electroacupuncture treatment, and the changes of body mass in normal rats and model rats were measured. Five rats from each group were taken to observe the histological changes of neural stem cells in the hippocampus at 1, 2, 3, 4 weeks after treatment. RESULTS AND CONCLUSION:The body mass of rats in the model group was significantly lower than that in the normal group at 2 weeks after modeling (P < 0.05). In the electroacupuncture group, the number of BrdU positive cells was significantly higher than that in the normal group and control group at 2, 3, 4 weeks after modeling (P < 0.05); the number of BrdU/Nestin positive cells was significantly higher than that in the normal group at 2, 3, 4 weeks (P < 0.05); the number of BrdU/glial fibrillary acidic protein positive cells was significantly higher than that in the normal group at 2 weeks (P < 0.05); and the number of BrdU/neuronspecific enolase positive cells was significantly higher than the normal group at 3 weeks (P < 0.05). In the control group, the number of BrdU/Nestin positive cells was significantly lower than that in the normal group and electroacupuncture group at 1 week after modeling, significantly lower than that in the electroacupuncture group at 2 and 4 weeks, and significantly lower than the normal group at 3 weeks (P < 0.05); the number of BrdU/glial fibrillary acidic protein positive cells was significantly lower than that in the normal group and electroacupuncture group at 1, 2, 4 weeks; and the number of BrdU/neuronspecific enolase positive cells was significantly lower than that in the normal group and electroacupuncture group at 1, 2 weeks. Taken together, these experimental findings show that electroacupuncture treatment effectively promotes the proliferation of hippocampal neural stem cells that are induced to differentiate into astrocytes and neurons.     

5'-溴-2'-脱氧尿嘧啶核苷标记人巨细胞病毒感染SD大鼠脑组织神经元模型的建立

目的 用5'-溴-2'-脱氧尿嘧啶核苷(BrdU)标记的人巨细胞病毒(HCMV)进行脑内定位注射,建立SD大鼠脑组织感染模型;研究大鼠脑组织海马部位神经元对HCMV的容许性及HCMV在其内的感染特点. 方法 运用BrdU标记HCMV基因组,并测定病毒滴度.将21只SD大鼠随机分为病毒感染组(n=18)及对照组(n=3),病毒感染组根据感染时间分为感染24、48及96 h组.每个感染时间组再分为2μl及4μl两个剂量组,每个剂量组包括3只动物,均以脑内注射途径将BrdU标记的HCMV直接注入大脑海马部位;对照组大鼠注射不含病毒的细胞培养上清液(4μl).感染大鼠术后分笼饲养,于各感染时间点处理大鼠取脑组织,冰冻切片;对海马周围部位脑组织分别运用抗BrdU及抗微管相关蛋白2(MAP-2)抗体进行免疫荧光双标染色检测标记病毒;抗pp65抗体免疫组化染色及Nissil复染检测病毒蛋白pp65. 结果 病毒感染后24 h,两个剂量组均未在神经元内检测到标记病毒及pp65.病毒感染后48h和96 h,两个剂量组在海马部位神经元中均检测到了标记病毒阳性荧光及pp65的阳性着色;而且2μl组的受感染神经元细胞数较4μl组少,但同-剂量组在镐h和96 h受感染神经元细胞数未见明显变化. 结论 成功建立了BrdU标记HCNV感染SD大鼠脑组织的模型.大鼠脑组织海马部位神经元是HCMV的半容许细胞,HCMV在其内仅表现为潜伏性感染.     

托吡酯对癫痫持续状态大鼠海马神经元损伤的保护作用

为探讨托吡酯(topiramate,TPM)对癫痫持续状态(status epilepticus,SE)大鼠海马神经元损伤的保护作用,将大鼠随机分为正常对照组、海人酸(kanic acid,KA)组和TPM预处理组,观察海马神经元超微结构和bcl-2表达的变化。先将TPM组大鼠用TPM预处理,然后采用KA(10mg/kg)腹膜腔注射制作SE模型,在痫性发作终止后6、24和48h取海马进行研究。结果显示:KA组神经元呈凋亡特征;TPM组神经元结构大致正常,但出现核仁边聚和细胞器增多现象,亦观察到少量凋亡神经元。KA组于SE后6h观察到bcl-2表达增高,与对照组相比差异显著,(P<0.05);24h时开始减弱,48h仅有微弱表达;TPM组在24h时bcl-2呈强表达(P<0.001),并持续至48h。以上结果提示:托吡酯预处理能减轻癫痫大鼠神经元的损伤,其机制可能与上调bcl-2的表达有关。     

慢性复合应激对大鼠学习和记忆功能及齿状回神经前体细胞增殖的影响

为研究慢性复合应激对成年雄性大鼠学习和记忆功能及齿状回(DG)神经前体细胞增殖的影响,将成年雄性Wistar大鼠随机分为对照组和应激组,应激组动物每天交替暴露于复合应激原中,持续6周。应激结束后,用Morris水迷宫测试大鼠空间学习记忆成绩。同时,采用BrdU标记分裂细胞方法,观察比较各组大鼠DG内BrdU阳性细胞数的变化和差异。结果显示:应激组动物的学习与记忆成绩均优于对照组(P<0.05);与对照组相比,应激组大鼠DG内BrdU阳性细胞数量显著增加(P<0.05)。上述结果表明:慢性复合应激导致大鼠的学习与记忆能力加强,DG内BrdU阳性细胞增多,提示神经细胞数量增加可能是应激所引起的大鼠学习记忆能力增强的原因之一。     

抑郁模型大鼠海马神经元再生变化及加味温胆汤的干预研究

目的:探讨加味温胆汤对抑郁模型大鼠海马神经元再生变化的影响。方法:以孤养结合慢性不可预见性应激抑郁模型大鼠为研究对象,在实验第7、14、21、28 d,采用免疫荧光双标技术检测各组大鼠海马神经元增殖与分化的再生变化情况。结果:在抑郁形成过程中,大鼠海马神经元NeuN/BrdU、β-tubulinⅢ/BrdU双标细胞数逐渐降低,至21、28 d时模型组NeuN/BrdU、β-tubulinⅢ/BrdU双标阳性细胞数明显减少,经中、西药治疗后,大鼠海马神经元NeuN/BrdU、β-tubulinⅢ/BrdU双标阳性细胞数下降趋势得到抑制,与模型组比较增加明显(P<0.05或P<0.01)。结论:抑郁形成过程中海马神经元增殖与分化减少的再生障碍渐次加重,加味温胆汤可逆转此现象。     

Neurogenesis in the dentate gyrus of the rat hippocampus enhanced by tickling stimulation with positive emotion

Hippocampal neurogenesis is influenced by many factors. In this study, we examined the effect of tactile stimulation (tickling), which induced positive emotion, on neurogenesis in the dentate gyrus (DG) of the hippocampus. Four week-old rats were tickled for 5 min/day on 5 consecutive days and received 5-bromo-2′-deoxyuridine (BrdU) administration for 4 days from the second tickling day. Then they were allowed to survive for 18 h or 3 weeks after the end of BrdU treatment. Neurogenesis in the DG was compared between the tickled and untickled rats by using immunohistochemistry with anti-BrdU antibody. The result showed that the number of BrdU- and NeuN (neural cell marker)-double positive neurons on 18 h as well as 3 weeks of the survival periods was significantly increased in the tickled group as compared with the untickled group. The expression of mRNA of brain-derived neurotrophic factor (BDNF) in the hippocampus of the tickled rats was not altered when compared with the control rats. In conclusion, tickling stimulation which induces positive emotion may affect the generation and survival of new neurons of the DG through the BDNF-independent pathway.     

慢性复合应激对大鼠学习和记忆的影响

本文观察慢性复合应激对海马长时程增强(LTP)和一氧化氮合酶1(NOS1)表达变化的影响,并探讨了其在学习与记忆中的作用。将Wistar大鼠随机分为对照组和慢性复合应激组,每组各10只。应激组动物采用4种应激原无规律、交替性地进行长达6周的慢性复合应激试验。实验过程中测定动物血清皮质酮(CORT)水平,采用Morris水迷宫和Y迷宫测试大鼠学习和记忆成绩,记录海马齿状回LTP群体峰电位(PS)幅值的变化,同时观察海马CA1及齿状回区NOS1阳性神经元数目的变化。结果显示:(1)与对照组比较,慢性复合应激组动物在应激第4周和第6周时,血清皮质酮浓度的比值显著增高(P<0.05,P<0.01);(2)高频刺激(HFS)后各时间点的PS幅值增高更为明显(P<0.01);(3)Morris水迷宫和Y迷宫实验后慢性复合应激组动物的学习和记忆成绩优于对照组(P<0.05,P<0.01);(4)慢性复合应激组和对照组大鼠海马齿状回NOS1阳性神经元个数分别为8.80±3.91和5.44±1.14,两者间有统计学差异(P<0.05)。以上结果提示,慢性复合应激对大鼠的学习和记忆有一定的影响,可能与齿状回群体峰电位幅值增强和NOS1表达增强有关。     

不同年龄脑出血大鼠海马齿状回神经干细胞增殖与分化差异的比较

BACKGROUND:Cerebral hemorrhage can activate the proliferation and differentiation of neural stem cells in the dentate gyrus of the hippocampus. Through continuous differentiation and proliferation, endogenous neural stem cells can gradually replace aging and damaged neurons, thus protecting the brain structure. OBJECTIVE:To compare the difference of the proliferation and differentiation of neural stem cells in the dentate gyrus of the hippocampus of rats with different ages. METHODS:Ninety-six adult rats and 96 aged rats were randomly divided into normal group (n=18 per group), sham operation group (n=12 per group) and cerebral hemorrhage group (model group, n=66 per group), respectively. Cerebral hemorrhage models were made in the two model groups in which, the rats were subjected to cerebral hemorrhage for 6, 24, 48, 72 hours and 7 days, respectively. Then, brain tissues were collected to measure brain water content. BrdU/NeuN and BrdU/GFAP double staining were performed at 3, 7, 14, 21, 28 days after surgery to calculate the number of positive cells. RESULTS AND CONCLUSION:For both adult and aged rats, the brain water content was significantly higher than that in the normal group and sham operation group (P < 0.05), while in the normal and sham operation groups, the brain water content was significantly lower in the aged rats than the adult rats (P  < 0.05). The number of bilateral BrdU-positive cells in the adult and aged model groups was significantly higher than that in the corresponding normal and sham operation groups (P  < 0.05), and moreover, the positive cell number at the hemorrhage side was significantly higher than that at the opposite side (P < 0.05). In addition, the number of BrdU-positive cells at the hemorrhage side in the adult rats was significantly higher than that in the aged rats at different time after cerebral hemorrhage (P  < 0.05). Results from immunohistochemical double staining showed that the BrdU/NeuN and BrdU/GFAP expression in the hippocampal dentate gyrus of adult rats with cerebral hemorrhage was significantly higher than that of normal adult rats. All these experimental results show that there are a few neural stem cells proliferating in the hippocampal dentate gyrus of normal rats, and the proliferation ability is stronger in the adult rats than the aged rats. Cerebral hemorrhage can significantly strengthen the proliferation of neural stem cells in the dentate gyrus in the adult rats compared with the aged rats.     

不同时长REM期睡眠剥夺及莫达非尼干预后大鼠下丘脑Orexin A神经元的表达

目的:探讨不同程度快动眼睡眠(REM)期睡眠剥夺(sleep deprivation,SD)及莫达非尼干预后大鼠下丘脑Orexin A神经元的表达。方法:将成年雄性Sprague-Dawley大鼠随机分为和SD组和对照组,SD组又分为用药组(drug group,DG)和非用药组(non-drug group,NDG),每组分SD12,24,48,72,96h共5个小组;对照组(cage control,CC)1个小组,正常饲养于笼中。每小组3只大鼠。采用小平台水环境法建立大鼠REM期SD模型。免疫组化方法观察大鼠下丘脑Orexin A阳性神经元的数量。结果:Orexin A阳性表达在SD12,24h时长的DG组与NDG组表达较CC组均有增加(P0.05)而二者之间差别不明显(P0.05);在SD48,72,96h时长的NDG组的表达较CC组下降(P0.05),而DG组和CC组间无显著差异(P0.05)且明显高于NDG组表达(P0.05)。结论:推测莫达非尼可能是通过活化下丘脑促觉醒肽Orexin A的分泌和表达实现促觉醒作用。