Ⅰ型糖尿病脑病模型大鼠空间学习记忆以及海马齿状回细胞凋亡和Bax与Bcl-2蛋白表达的变化

目的探讨1型糖尿病脑病海马齿状回细胞凋亡、Bax与Bcl-2蛋白表达、空间学习记忆之间的相互关系。方法通过腹腔注射链脲佐菌素(溶于柠檬酸缓冲液)建立1型糖尿病脑病模型大鼠,通过腹腔注射柠檬酸缓冲液建立载体模型大鼠。应用Morris水迷宫、TUNEL技术、Bax与Bcl-2免疫组化方法,观察各组大鼠海马齿状回颗粒下区(SGZ)、颗粒细胞层(GCL)的Bax、Bcl-2、TUNEL阳性细胞和空间学习记忆的变化。结果糖尿病脑病组大鼠的SGZ、GCL的Bax阳性细胞数、TUNEL阳性细胞数、水迷宫的逃避潜伏期和游泳距离均明显高于载体模型组或正常对照组大鼠的对应指标(P<0.01);而糖尿病脑病组大鼠的Bcl-2阳性细胞数则明显低于载体模型组或正常对照组大鼠(P<0.01),但以上各组3类阳性细胞的形态和分布却均无明显差异。结论体内长期缺乏胰岛素可打破凋亡基因Bax与抗凋亡基因Bcl-2的蛋白表达水平之间原有的正常动态平衡状态,致使海马齿状回的细胞倾向于多发生细胞凋亡事件,这可能是引发1型糖尿病脑病神经发生障碍及空间学习记忆异常的一个因素。     

褪黑素对1型糖尿病脑病大鼠学习记忆和海马齿状回细胞凋亡及Bax与Bcl-2蛋白表达的影响

目的探讨褪黑素治疗对1型糖尿病脑病空间学习忆忆、海马齿状回细胞凋亡以及凋亡相关基因Bax与Bcl-2蛋白表达的影响。方法将成年健康雄性Wistar大鼠随机分成4组(每组均为12只大鼠):1型糖尿病脑病组、载体模型治疗组、褪黑素治疗组、正常对照组。通过腹腔注射链脲佐菌素(55mg/kg,溶于柠檬酸缓冲液中),建立1型糖尿病脑病模型;通过对该脑病模型大鼠腹腔注射褪黑素(200μg/kg,溶解于乙醇生理盐水中),建立褪黑素治疗模型;分别通过对大鼠腹腔注射等体积的柠檬酸缓冲液、乙醇生理盐水,建立载体治疗模型;但未对正常对照组大鼠进行过任何处理。应用Morris水迷宫、Bax、Bcl-2、TUNEL反应技术,观察各组大鼠空间学习记忆、海马齿状回的颗粒下区(SGZ)和颗粒细胞层(GCL)的细胞凋亡及Bax与Bcl-2基因的蛋白表达程度。结果1型糖尿病脑病大鼠在水迷宫的逃避潜伏期和游泳距离均比其它各组的对应指标明显延长(P<0.01),该脑病组大鼠海马齿伏回的SGZ和GCL的Bax阳性细胞数、TUNEL阳性细胞数也均比其它各组的对应指标明显升高(P<0.01),但该脑病组SGZ和GCL的Bcl-2阳性细胞数则明显低于其它各组的相应指标(P<0.01)。应用褪黑素治疗该脑病组大鼠30d后,可将1型糖尿病脑病组大鼠的以上各项指标逆转至接近正常水平(P<0.01)。结论褪黑素治疗不仅可纠正因1型糖尿病脑病大鼠海马齿状回凋亡基因Bax和抗凋亡基因Bcl-2蛋白质表达水平比例异常而引发的细胞凋亡,还可改善该脑病的空间学习记忆障碍,提示褪黑素在防治该脑病中具有一定的功效。     

Ⅰ型糖尿病脑病大鼠学习记忆及侧脑室室管膜下区神经发生的变化

目的观察Ⅰ型糖尿病脑病对学习记忆及侧脑室室管膜下区神经发生的影响。方法建立Ⅰ型糖尿病大鼠模型及胰岛素治疗糖尿病模型,用免疫组织化学方法计数侧脑室室管膜下区BrdU阳性细胞数,并用Morris水迷宫测定大鼠的学习记忆能力。结果糖尿病大鼠SVZ的BrdU细胞数以及学习记忆能力明显下降(P<0.01);用胰岛素治疗,可使上述指标明显上升(P<0.01),并接近正常水平。结论胰岛素治疗对糖尿病大鼠神经干细胞增殖及学习记忆能力均有相似的促进作用。     

次声激活小胶质细胞抑制成年大鼠海马神经干细胞增殖

目的:探讨次声对成年大鼠海马齿状回颗粒细胞下层(subgranular zone,SGZ)神经干细胞增殖抑制作用的细胞学机制。方法:成年雄性Sprague-Dawley大鼠置于次声压力舱,连续暴露于16 Hz、130 dB次声7 d(2h/d)后,给与小胶质细胞抑制剂米诺环素(50 mg/kg,药物组,n=16)或等体积生理盐水(对照组,n=16),同时设立不经次声作用的正常对照组(n=16);分别于1、3、7和14 d处死大鼠,利用免疫组织化学法以Iba1、OX42标记小胶质细胞,BrdU标记增殖的神经干细胞。结果:小胶质细胞在SGZ区分布较为密集;与正常对照组相比,次声暴露后3 d时OX42免疫反应性明显增强、SGZ区BrdU阳性细胞数目减少最为明显(P0.01);米诺环素可显著改善次声暴露后BrdU阳性细胞数目的减少(P0.01)。结论:小胶质细胞活化参与次声抑制成年大鼠海马SGZ区神经干细胞的增殖。     

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.     

胰岛素与尼莫地平对大鼠基底前脑胆碱能神经元及空间学习记忆的影响

目的探讨胰岛素与尼莫地平对基底前脑胆碱能神经元与学习记忆在1型糖尿病脑病发生中的影响。方法将成年雄性Wistar大鼠随机分成糖尿病组、干预组、载体组及正常对照组。用链脲佐菌素成功建立成1型糖尿病模型12周后,对干预组大鼠每日皮下注射长效胰岛素(3 IU)、腹腔注射尼莫地平(20 mg/kg),连续用药6周,在相同条件下对载体组大鼠注射等体积的无药物液体,但对糖尿病组或正常对照组大鼠未进行任何处理。应用Morris水迷宫及胆碱乙酰转移酶(ChAT)免疫组化方法,分别测定各组大鼠的空间学习记忆能力及基底前脑胆碱能神经元的变化。结果糖尿病组大鼠内侧隔核、斜角带核垂直支、斜角带核水平支的ChAT阳性神经元数均明显减少(P<0.05),空间学习记忆能力也明显下降(P<0.05);干预组大鼠以上三个核团的ChAT阳性神经元数与学习记忆能力均明显大于糖尿病组大鼠(P<0.05),但干预组的各项指标仍然明显低于载体组大鼠或正常对照组大鼠(P<0.05)。结论在糖尿病的长期自然发展过程中,若不进行治疗则可累及到基底前脑胆碱能神经元,导致学习记忆障碍,这可能是引发1型糖尿病脑病的一个负性因素;此时联合应用胰岛素与尼莫地平仍可有效遏制该脑病向纵深发展的恶性势头。     

高血糖影响海马GSK3β-β-catenin信号抑制大鼠海马颗粒下区成体神经干细胞再生

目的:探讨持续高血糖对大鼠海马颗粒下区(subgranular zone,SGZ)神经干细胞状态的影响及可能机制。方法:SD大鼠注射链脲佐菌素(streptozotocin,STZ)诱导1型糖尿病模型,腹腔注射5-溴脱氧尿嘧啶核苷(bromodeoxyuridine,Brd U)标记新生细胞。利用免疫组化法检测海马SGZ区Ki67,5-溴脱氧尿嘧啶核/双皮质素(bromodeoxyuridine/doublecortin,Brd U/DCX)标记的阳性细胞。利用免疫印迹法检测海马胰岛素受体β(insulin receptorβ,IRβ),糖元合成酶激酶3β(pho-glycogen synthase kinase-3β,GSK3β),β-链蛋白(β-Catenin)表达水平。结果:STZ注射大鼠胰岛素降低,血糖升高;糖尿病大鼠海马区Ki67阳性细胞,Brd U/DCX阳性细胞和对照组比较明显减少,海马区IRβ,pho-GSK3β,β-Catenin表达水平和对照组比较显著下降(P0.05)。结论:STZ诱导的1型糖尿病大鼠海马SGZ区新生细胞增值能力下降,细胞分化异常,这一变化和海马区胰岛素信号下调,GSK3β活化,β-Catenin降解增加有关。     

外源性褪黑素对成年高血压大鼠神经发生的影响及其与学习记忆的关系

目的探讨褪黑素（MT）对成年高血压大鼠脑神经干细胞的影响及其与学习记忆的关系。方法将30只成年健康雄性wistar大鼠随机分为假手术组、肾血管性高血压组及褪黑素治疗组，用Morris水迷宫测定大鼠的学习记忆能力，并用免疫组织化学方法观察侧脑室室管膜下区（SVZ）和齿状回颗粒下区（SGZ）的BrdU阳性细胞数变化。结果高血压大鼠SVZ和SGZ的BrdU细胞数及学习记忆能力均比假手术组明显下降（P〈0．01），用MT治疗可使上述指标有所逆转至假手术组水平。结论褪黑素可能具有促进高血压大鼠神经干细胞增殖和提高大鼠学习记忆能力的作用。     

下调脑源性神经营养因子/酪氨酸受体激酶B信号通路可降低孤独症模型鼠海马神经元过度增殖并改善症状

目的:探讨脑源性神经营养因子(BDNF)/酪氨酸受体激酶B(TrkB)信号通路活性下调对孤独症大鼠模型海马神经干细胞增殖的影响及其治疗作用。方法:采用Wistar妊娠母鼠在孕期12.5 d时腹腔注射丙戊酸法建立子代孤独症大鼠模型,断乳后给予侧脑室注射K252a,以下调BDNF/TrkB通路活性,应用5-溴脱氧尿嘧啶核苷(BrdU)免疫荧光观察海马神经干细胞的增殖变化,并采用水迷宫检测子代孤独症大鼠模型的学习记忆行为。结果:与对照组比较,孤独症大鼠模型海马BDNF/TrkB通路活性上升,表现为其效应分子环磷酸腺苷反应元件结合蛋白(CREB)表达显著增加,模型组海马神经干细胞增殖也显著增加;BrdU免疫荧光显色显示,K252a处理能逆转大鼠模型海马神经干细胞的过度增殖,同时,水迷宫行为学检测显示,K252a处理显著改善大鼠模型的学习记忆能力。结论:下调BDNF/TrkB通路活性可以减轻孤独症模型大鼠海马神经干细胞过度增殖并改善症状,为孤独症的临床治疗提供了实验依据。     

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.     

高原环境对小鼠成体神经发生的影响

目的:探索和研究我国青藏高原高海拔生存环境对于小鼠海马齿状回成体神经干细胞增殖和新生细胞分化的影响。方法:健康成年昆明小鼠分为两组,一组为1519 m海拔高度的兰州对照组,另一组为4547 m海拔高度的青藏高原沱沱河高原环境组。运用BrdU腹腔注射和免疫荧光组织化学相结合的方法研究和比较不同环境中齿状回内成体神经干细胞的增殖和新生细胞的分化。结果:高原环境组的BrdU免疫阳性细胞数与低海拔对照组相比减少了40%(P=0.001),而高原环境组小鼠齿状回中的BrdU/Prox-1标记的双阳性细胞在BrdU标记的阳性细胞中的百分比与对照组相比没有显著的差异(P=0.211),并且两组小鼠齿状回中90%以上的BrdU阳性细胞同时也被Prox-1标记。结论:在青藏高原的高海拔低氧环境中,小鼠海马内成体神经干细胞的增殖明显受抑制,然而新生的细胞向颗粒细胞的分化并没有受到明显的影响。     

Extensive training in a maze task reduces neurogenesis in the adult rat dentate gyrus probably as a result of stress

It has recently been shown that hippocampal neurogenesis can be modulated either directly or indirectly by ascending cholinergic inputs from the basal forebrain. In the present work, we sought to address whether extended training in a spatial navigation task would affect hippocampal neurogenesis in the presence of a severe and selective cholinergic depletion. Young female rats received stereotaxic injections of the immunotoxin 192 IgG-saporin into the basal forebrain nuclei and/or the cerebellar cortex. Starting from 4 to 5 weeks post-lesion, and for the subsequent 2 weeks, the animals were trained on paradigms of reference and working memory in the water maze and received single daily i.p. injections of bromodeoxyuridine (BrdU) at the end of each testing session. In line with previous observations, a dramatic 80% decrease in neuron proliferation was seen in the dentate gyrus of lesioned animals, as compared to vehicle-injected or intact controls. Interestingly, however, rats subjected to maze training over 2 weeks, irrespective of their learning success, exhibited significantly fewer newborn neurons than matched controls with no maze exposure. Thus, at least for the type of task used here, which has previously been shown to impose a certain degree of stress, extended training and learning does not appear to affect proliferation in the dentate gyrus.     

Morris water maze learning in two rat strains increases the expression of the polysialylated form of the neural cell adhesion molecule in the dentate gyrus but has no effect on hippocampal neurogenesis

In the current study, the authors investigated whether Morris water maze learning induces alterations in hippocampal neurogenesis or neural cell adhesion molecule (NCAM) polysialylation in the dentate gyrus. Two frequently used rat strains, Wistar and Sprague-Dawley, were trained in the spatial or the nonspatial version of the water maze. Both training paradigms did not have an effect on survival of newly formed cells that were labeled 7-9 days prior to the training or on progenitor proliferation in the subgranular zone. However, the granule cell layer of the spatially trained rats contained significantly more positive cells of the polysialylated form of the NCAM. These data demonstrate that Morris water maze learning causes plastic change in the dentate gyrus without affecting hippocampal neurogenesis.     

A free-radical scavenger protects the neural progenitor cells in the dentate subgranular zone of the hippocampus from cell death after X-irradiation

It has been elucidated that cognitive dysfunction following cranial radiotherapy might be linked to the oxidative stress-induced impairment of hippocampal neurogenesis that is mediated by proliferating neural stem or progenitor cells. The novel free-radical scavenger edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) has been clinically used to reduce neuronal damage following ischemic stroke. Previously, we reported that the free-radical scavenger, edaravone, which is currently used to treat patients with brain ischemia, protected cultured human neural stem cells (NSCs) from radiation-induced cell death; the protective effect was observed more significantly in NSCs than in brain tumor cells. Here, in animal models, we demonstrate that edaravone protects neurons in the subgranular zone (SGZ) of the dentate gyrus of the hippocampus from cell death after irradiation. Moreover, edaravone protected spatial memory retention deficits as determined by Morris water maze tests. Our study may shed some light on the beneficial effects of free-radical scavengers in impaired neurogenesis following cranial radiation therapy.     

糖尿病降低大鼠海马突触可塑性的研究

目的 研究大鼠在患1型和2型糖尿病后,对在体海马前穿通纤维-齿状回通路(PP-DG)的突触可塑性造成的影响.方法 将70只SD大鼠(180±20)g随机分成3组:对照组、1型糖尿病模型组、2型糖尿病模型组.在水迷宫测试后,在每个模型组中选出空间记忆能力较差的15只大鼠,研究糖尿病引起的海马PP-DC通路双脉冲易化(PP...     

Effect of long-lasting serotonin depletion on environmental enrichment-induced neurogenesis in adult rat hippocampus and spatial learning

The dentate gyrus of the hippocampal formation produces new neurons throughout adulthood in mammalian species. Several experimental statuses and factors regulating to neurogenesis have been identified in the adult dentate gyrus. For example, exposure to an enriched environment enhances neurogenesis in the dentate gyrus and improves hippocampus-dependent spatial learning. Furthermore, serotonin is known to influence adult neurogenesis, and learning and memory. However, the effects of long-lasting depletion of serotonin over the developing period on neurogenesis have not been investigated. Thus, we examined the influence of long-lasting serotonin depletion on environmental enrichment-induced neurogenesis and spatial memory performance. As reported previously, environmental enrichment significantly increased new neurons in the dentate gyrus. However, there was no improvement of the spatial learning test in adult rats in standard and in environmental enrichment housings. Intracisternal administration of the serotonergic neurotoxin, 5,7-dihydroxytryptamine, on postnatal day 3 apparently reduced serotonin content in the adult hippocampus without regeneration. This experimental depletion of serotonin in the hippocampus of rats housed in an enriched environment had no effect on spatial memory performance, but produced significant decreases in the number of bromodeoxyuridine-labeled new cells in the dentate gyrus. These findings indicate that newly generated cells stimulated by environmental enrichment are not critical for improvements in hippocampus-dependent learning. Furthermore, numbers of bromodeoxyuridine-labeled cells in the dentate gyrus of 5,7-dihydroxytryptamine-injected rats did not differ between 1 day and 4 weeks after bromodeoxyuridine injection. These data suggest that survival of newly generated dentate gyrus cells remains relatively constant under long-lasting serotonin depletion.     

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.