卡马西平对匹罗卡品诱导成年癫间疒大鼠海马齿状回神经前体细胞增殖的影响

目的研究卡马西平对成年癫大鼠海马齿状回内源性神经前体细胞增殖的影响。方法采用氯化锂和匹罗卡品联合诱导大鼠癫模型,将癫大鼠随机分为癫对照组和癫卡马西平组,正常大鼠随机分为正常对照组和正常卡马西平组。癫对照组和正常对照组给以蒸馏水灌胃,同时癫卡马西平组和正常卡马西平组给予卡马西平灌胃。于灌胃后第6d腹腔注射5-溴脱氧尿苷嘧啶(BrdU)标记海马齿状回的内源性神经前体细胞的增殖情况;用免疫组化方法观察各组大鼠在注射BrdU后第1d、第7d齿状回BrdU阳性细胞数量的表达。结果①注射BrdU后第1d,癫对照组大鼠海马齿状回BrdU阳性细胞数较正常对照组明显增加(P<0.01),癫卡马西平组大鼠海马齿状回BrdU阳性细胞数较癫对照组减少(P<0.05);②注射BrdU后第7d,癫对照组大鼠海马齿状回BrdU阳性细胞数较正常对照组明显增加(P<0.01),癫卡马西平组大鼠海马齿状回BrdU阳性细胞数较癫对照组明显减少(P<0.05)。结论卡马西平抑制癫大鼠海马齿状回内源性神经前体细胞增殖。     

实验性脑出血SVZ细胞再生的实验研究

目的明确脑出血后是否存在内源性的神经细胞再生现象。方法 SD大鼠随机分为对照组和脑出血组,每组分为1、3、7、14、21、28d亚组。应用胶原酶脑内立体定向注射制作脑出血模型,应用BrdU腹腔注射在体标记再生的脑室下带(subventricular zone,SVZ)细胞,应用免疫组织化学技术检测BrdU阳性细胞。结果脑出血后7d同侧SVZ和基底节BrdU阳性细胞开始增加,但与对照组比较差异没有显著性(P0.05),脑出血后14d同侧SVZ和基底节BrdU阳性细胞达到高峰,与对照组比较差异具有显著性(P0.01),然后逐渐下降,28d时BrdU阳性细胞虽然仍有增加,但与对照组比较差异没有显著性(P0.05)。结论脑出血后存在内源性神经细胞再生现象。     

2003/成年大鼠脑出血后神经再生的实验研究

目的 观察大鼠实验性脑出血后内源性神经前体细胞的增殖、迁移、分布和在出血灶周边的分化。方法 将成年SD大鼠随机分为正常组、假手术组和脑出血组;脑出血组大鼠通过立体定向术向脑内注入自体动脉血制成脑尾壳核出血模型,并按不同的再喂养时间（1、3、7、14及30 d）分为5个亚组。手术后腹腔注射5-溴脱氧核苷尿嘧啶标记新生的内源性神经前体细胞。采用免疫组化单标观察内源性神经前体细胞（BrdU阳性细胞）的增殖、迁移和分布,免疫荧光双标观察内源性神经前体细胞在出血灶周边的分化情况。结果 与正常组和假手术组相比,脑出血组大鼠的BrdU阳性细胞数显著增加,并在7～14d达高峰;BrdU阳性细胞主要分布于室管膜下层、海马齿状回、脉络丛、胼胝体腹侧、出血灶周边区、外侧隔核、斜角带、缰核和大脑皮层等处。免疫荧光双标显示在脑出血灶周边区可见BrdU/GFAP、BrdU/NSE及BrdU/Nestin三种双标阳性细胞;BrdU/Nestin双标阳性细胞随着脑出血后时间的推移逐渐减少,而BrdU/GFAP、BrdU/NF-200双标阳性细胞则增多。结论 脑出血可诱导内源性神经前体细胞增殖,并向出血灶周边区迁移,进一步分化出神经元和胶质细胞,这可能是脑出血后神经结构重塑和功能恢复的重要物质基础。     

大鼠脑出血后内源性神经干细胞激活和增殖的实验研究

目的 观察大鼠脑出血模型内源性神经干细胞(NSCs)的激活、增殖情况及其对神经行为学表现的影响.方法 将72只SD大鼠按单双号分为脑出血组和假手术组.每组36只.脑出血组利用立体定向技术,将一定量的Ⅳ型胶原酶用微量进样器分别精确注入大鼠内囊诱导脑出血模型.假手术组注射等量体积的PBS.分别于术后1、7、14、21、28和35 d观察大鼠的神经功能表现.所有大鼠处死前1 d腹腔内注射5.溴脱氧尿嘧啶(BrdU),免疫组织化学方法动态检测大鼠脑内巢蛋白(nestin)和BrdU的表达.结果 假手术组大鼠脑内未见nestin和BrdU的表达.脑出血组血肿周围基底节和脑室下区可见nestin和BrdU的表达.脑出血后7 d后开始明显增加.14 d达高峰,21 d开始下降.28d恢复正常.脑出血后l～35d大鼠神经功能无明显恢复,与内源性NSCs的增殖程度无明显相关.结论 脑出血可导致内源性NSCs的激活和诱导其增殖:然而这种状态下NSCs的增殖能力和内源性NSCs对脑出血后神经功能缺损的修复均有限.     

次声对成年大鼠脑室下区神经前体细胞增殖的影响

目的探讨次声作用对成年大鼠脑室下区（SVZ）神经前体细胞增殖的影响。方法成年雄性Sprague-Dawley大鼠（n=24）随机分为正常对照组、对照组和次声组,次声组动物置于次声压力仓（16Hz、130dB）内连续作用7d（2h/d）。照射结束后,分别于3d、7d、10d和14d处死。处死前各组大鼠腹腔注射5-溴脱氧尿嘧啶尿苷（BrdU）3次,以BrdU免疫组织化学法检测各组SVZ内增殖细胞数目的变化。结果次声照射结束后3d,次声处理组动物SVZ区的BrdU阳性细胞数目较对照组显著减少（P〈0.01）,7d时阳性细胞数继续降低（P〈0.01）,但10d时开始逐渐恢复,14d时达到正常水平（P〉0.05）。结论16Hz、130dB次声可抑制成年大鼠SVZ神经前体细胞增殖。     

凝血酶预处理对大鼠脑出血后内源性神经再生的影响

目的 探讨凝血酶预处理(thrombin preconditioning,TPC)对大鼠脑出血后内源性的神经细胞再生的影响。方法 SD大鼠随机分为对照组、脑出血(Intracerebral hemorrhage,ICH)组和 TPC组,每组分为3、7、14、21、28 d亚组。应用胶原酶脑内立体定向注射制作脑出血模型。TPC组首先将1U凝血酶立体定向注入右侧纹状体,1 d后再制作脑出血模型。所有大鼠应用BrdU腹腔注射在体标记再生的脑室下带( subventricular zone, SVZ)细胞,应用免疫组织化学技术检测BrdU阳性细胞。结果 脑出血后3 d同侧SVZ 和基底节BrdU阳性细胞开始增加,但与对照组比较差异没有显著性(P>0.05),7 d时BrdU阳性细胞数增加明显,与对照组比较有明显差异(P<0.05),14 d达到高峰,然后逐渐下降,28 d时BrdU阳性细胞虽然仍有增加,但与对照组比较差异没有显著性(P>0.05)。TPC组3 d时BrdU阳性细胞数目开始增加,与脑出血组和对照组比较,差异均具有显著性(P<0.01),14 d达高峰,一直持续至21 d,28 d时BrdU阳性细胞虽然略有下降,但与对照组和脑出血组比较,仍有统计学差异(P<0.05)。结论 凝血酶预处理能够增强脑出血后内源性神经再生,可能为脑出血后内源性神经再生的研究提供新的思路。     

次声对成年大鼠齿状回神经前体细胞增殖的影响

目的研究次声对成年大鼠海马齿状回神经前体细胞增殖的影响。方法大鼠随机等分为正常对照组、假次声组和次声组（每组16只）。次声组暴露于8Hz、130dB次声环境7d（2h／d），暴露结束后第1、3、7、14d处死，采用抗5-溴脱氧尿嘧啶尿苷（BrdU）免疫组化方法，观察齿状回BrdU阳性细胞数的变化。结果次声作用结束后第1d，齿状回BrdU阳性细胞数与假次声组和正常对照组相比均无统计学差异；第3d及第7d，BrdU阳性细胞数减少（P〈0．05），第14d恢复正常水平。结论8Hz、130dB次声可抑制正常成年大鼠海马神经前体细胞增殖，可能与次声引起大鼠脑内微环境改变有关。     

雌性和雄性大鼠脑缺血再灌注神经干细胞增殖的比较研究

目的探讨雌性和雄性成年大鼠脑缺血再灌注海马齿状回颗粒下层(subgranular zone,SGZ)、侧脑室室下层(subventricular zone,SVZ)和梗死皮质周边神经干细胞(neural stem cell,NSC)增殖的差异性.方法雌性和雄性成年大鼠各16只,制作线栓法大脑中动脉闭塞2 h后再灌注模型,用免疫组织化学方法动态检测海马齿状回SGZ、SVZ和梗死皮质周边5-溴脱氧尿嘧啶核苷(bromodeoxyuridine,BrdU)标记的阳性细胞数的变化,并采用HPIAS图像分析系统进行对比分析.结果雄性大鼠SGZ、SVZ及梗死周边区域BrdU阳性细胞在缺血再灌注3 d开始增加,7 d达到高峰,11d后开始下降,18 d进一步减少.而雌性大鼠则是3 d开始增加,7 d明显增加,11d达到高峰,18 d才开始减少,且各时间点雌性大鼠的BrdU阳性细胞数均明显高于雄性大鼠.结论脑缺血再灌注雌雄成年大鼠均有NSC增殖,雌性大鼠NSC增殖较雄性大鼠更为明显.     

雌激素对大鼠脑缺血再灌注神经干细胞内源性激活的实验研究

目的探讨雌激素对脑缺血再灌注神经干细胞(neural stem cell,NSC)内源性激活的作用.方法将44只雄性大鼠随机分成假手术组、对照组和实验组.采用传统的线栓法制备大鼠大脑中动脉闭塞再灌注模型,实验组大鼠腹腔注射苯甲酸雌二醇,对照组腹腔注射生理盐水,通过免疫组织化学技术标记大鼠海马齿状回颗粒下层(subgranular zone,SGZ)、侧脑室室下层(subventricular zone,SVZ)以及梗死皮质周边区在缺血再灌注3、7、11、18 d的5-溴脱氧尿嘧啶核苷(bromodeoxyuridine,BrdU)阳性细胞,并采用HPIAS图像分析系统进行分析.结果正常成年大鼠脑组织SGZ和SVZ存在少量Brdu阳性细胞,对照组脑缺血再灌注3 d SGZ、SVZ和梗死皮质周边Brdu阳性细胞的数量开始增多,7 d达到高峰,11d后开始减少,18 d进一步下降.实验组与对照组相比,在各个时间点均能显著提高BrdU阳性细胞的数量(P＜0.01),而且增殖可以持续11d.结论脑缺血再灌注可激活脑内NSC的增殖,雌激素可以促进脑缺血再灌注多个区域NSC的增殖.     

碱性成纤维细胞生长因子、表皮生长因子对脑缺血大鼠内源性神经干细胞增殖的影响

目的探讨碱性成纤维细胞生长因子(bFGF)、表皮生长因子(EGF)对大鼠局灶性脑缺血模型内源性神经干细胞增殖的影响。方法通过大脑中动脉阻塞法建立大鼠的局灶性脑缺血模型,随机分组后分别皮下注射生理盐水、bFGF、EGF以及bFGF EGF;每日1次,共3d,此后每3d1次。采用免疫组化法,以5溴脱氧嘧啶尿苷(Brdu)标记神经干细胞,观察并比较各组大鼠制模后第7d、14d、21d侧脑室室管膜下区(SVZ)和海马齿状回Brdu阳性细胞的表达。结果制模后,各组大鼠双侧SVZ和海马齿状回均出现Brdu阳性细胞,且阳性细胞数随时间递减;与对照组相比,药物干预组Brdu阳性细胞数显著增加(P<0.05～0.01);与单药组相比,bFGF EGF组(联合组)Brdu阳性细胞数增加更明显(P<0.05～0.01);各药物干预组在制模第7dBrdu阳性细胞数最多(P<0.05～0.01)。结论皮下注射bFGF、EGF可促进脑缺血大鼠模型内源性神经干细胞的增殖;bFGF和EGF联合应用对脑缺血大鼠神经干细胞的增殖效应有协同作用。     

Nestin expression and in vivo proliferative potential of tanycytes and ependymal cells lining the walls of the third ventricle in the adult rat brain

There is a disagreement in the literature concerning the degree of proliferation of cells in the walls of the third ventricle (3rdV) under normal conditions in the adult mammalian brain. To address this issue, we mapped the cells expressing the neural stem/progenitor cell marker nestin along the entire rostrocaudal extent of the 3rdV in adult male rats and observed a complex distribution. Abundant nestin was present in tanycyte cell bodies and processes and also was observed in patches of ependymal cells as well as in isolated ependymal cells throughout the walls of the 3rdV. However, we observed very limited ependymal cell or tanycyte proliferation in normal adult rats as determined by bromodeoxyuridine (BrdU) incorporation or the expression of Ki‐67. Moreover, fewer than 13% of the cells that were BrdU‐positive (BrdU+) or Ki‐67‐positive (Ki‐67+) expressed nestin. These observations stand in contrast to those made in the subventricular zone of the lateral ventricle (SVZ) and subgranular zone of the hippocampal formation (SGZ), where cell proliferation measured by BrdU incorporation or Ki‐67 expression is observed frequently in cells that also express nestin. Thus, while ependymal cell or tanycyte cell proliferation can be promoted by the addition of mitogens, dietary modifications or other in vivo manipulations, the proliferation of ependymal cells and tanycytes in the walls of the 3rdV is very limited in the normal adult male rat brain.     

Conditional ablation and recovery of forebrain neurogenesis in the mouse

Forebrain neurogenesis persists throughout life in the rodent subventricular zone (SVZ) and hippocampal dentate gyrus (DG). Several strategies have been employed to eliminate adult neurogenesis and thereby determine whether depleting adult‐born neurons disrupts specific brain functions, but some approaches do not specifically target neural progenitors. We have developed a transgenic mouse line to reversibly ablate adult neural stem cells and suppress neurogenesis. The nestin‐tk mouse expresses herpes simplex virus thymidine kinase (tk) under the control of the nestin 2nd intronic enhancer, which drives expression in neural progenitors. Administration of ganciclovir (GCV) kills actively dividing cells expressing this transgene. We found that peripheral GCV administration suppressed SVZ‐olfactory bulb and DG neurogenesis within 2 weeks but caused systemic toxicity. Intracerebroventricular GCV infusion for 28 days nearly completely depleted proliferating cells and immature neurons in both the SVZ and DG without systemic toxicity. Reversibility of the effects after prolonged GCV infusion was slow and partial. Neurogenesis did not recover 2 weeks after cessation of GCV administration, but showed limited recovery 6 weeks after GCV that differed between the SVZ and DG. Suppression of neurogenesis did not inhibit antidepressant responsiveness of mice in the tail suspension test. These findings indicate that SVZ and DG neural stem cells differ in their capacity for repopulation, and that adult‐born neurons are not required for antidepressant responses in a common behavioral test of antidepressant efficacy. The nestin‐tk mouse should be useful for studying how reversible depletion of adult neurogenesis influences neurophysiology, other behaviors, and neural progenitor dynamics. J. Comp. Neurol. 514:567–582, 2009. © 2009 Wiley‐Liss, Inc.     

Temporal and spacial changes of BrdU immunoreactivity in amygdala kindling development

To investigate the proliferation of neural stem cells (NSC) in epileptic brain, spaciotemporal expression of immunoreactive bromodeoxyuridine (BrdU) was examined in kindling development of rat. Amygdaloid kindling in partial seizure (PS) was effective in proliferation of NSC detected with BrdU-labeling in subventricular zone (SVZ), but not in dentate gyrus (DG). In SVZ, however, the BrdU-labeling cells decreased at stage generalized seizure (GS). These facts indicate that proliferation of NSC increased with PS and decreased with more severe seizures of GS in SVZ, that such a proliferation did not occur in DG with PS or GS. Thus, the proliferation of NSC was spacially and temporally different between brain regions depending on different kindling stages.     

Proliferation of progenitor cells in the adult rat brain correlates with the presence of vimentin-expressing astrocytes

It is well established that proliferation of progenitor cells persists within the hippocampal dentate gyrus (DG) and the subventricular zone of the lateral ventricle (SVZ) in the adult brain. The aim of the present study was to determine whether the rate of cell proliferation within these germinative zones could be correlated to the occurrence of a particular glial environment. The cell proliferation marker bromodeoxyuridine (BrdU) was administrated to rats under different physiological and experimental conditions known to modify the rate of progenitor cell proliferation. Within both germinative zones, BrdU-labeled nuclei were associated with cell bodies immunostained for the neuronal marker polysialylated neural cell adhesion molecule, but not for the glial markers glial fibrillary acidic protein (GFAP) or vimentin (VIM). In all the rats examined, however, proliferating (BrdU-labeled) cells always exhibited close relationships with immature-like astrocytes that expressed both GFAP and VIM. There was a dramatic decrease of cell proliferation in the DG from both the aged rats and the corticosterone-treated adult rats that was correlated with a decreased expression of vimentin by the astrocytes present in this region. In contrast, both cell proliferation and vimentin expression were only slightly affected in the SVZ from these two treatment groups. Conversely, after either adrenalectomy or a surgical lesion through the lateral hippocampus, the increase in cell proliferation observed in the DG was correlated to the occurrence of an increased number of GFAP and VIM double immunostained structures in these regions. All together, these data suggest that immature-like astrocytes present in the germinative zones may provide a microenvironment involved in sustaining the proliferation of progenitor cells.     

Temporal changes of neurogenesis in the mouse hippocampus after experimental subarachnoid hemorrhage

Recent studies indicate the existence of progenitor cells and their potential for neurogenesis in the subventricular zone (SVZ) and the hippocampus dentate gyrus (DG) of normal adult mammalian brain. Increased neurogenesis has been shown following cerebral ischemia and traumatic brain injury; however, the involvement of neurogenesis in subarachnoid hemorrhage (SAH) has not been examined. Adult male CD-1 mice were subjected to SAH by endovascular perforation of the left anterior cerebral artery. Mice received intraperitoneal injections of the cell proliferation-specific marker 5'-bromodeoxyuridine (BrdU) after SAH induction. BrdU incorporation was examined from 1 to 30 days after SAH by immunohistochemistry. The BrdU-positive cells were detected in SVZ and DG of normal control brain, and were significantly decreased in both areas three days after SAH. The number of these cells had recovered to its control level seven days after SAH. Double staining with BrdU and NeuN indicated that the majority of the BrdU-positive cells migrating into the granular cell layer of the DG became NeuN-positive 30 days after SAH. In conclusion, temporal changes of the neurogenesis as shown in the present study suggest that neurogenesis in the hippocampus may affect functional outcome after SAH. The induction of the neurogenesis can provide therapeutic value against SAH.     

Basic fibroblast growth factor-enhanced neurogenesis contributes to cognitive recovery in rats following traumatic brain injury

Stem/progenitor cells reside throughout the adult CNS and are actively dividing in the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus. This neurogenic capacity of the SVZ and DG is enhanced following traumatic brain injury (TBI) suggesting that the adult brain has the inherent potential to restore populations lost to injury. This raises the possibility of developing strategies aimed at harnessing the neurogenic capacity of these regions to repair the damaged brain. One strategy is to enhance neurogenesis with mitogenic factors. As basic fibroblast growth factor (bFGF) is a potent stem cell mitogen, we set out to determine if an intraventricular administration of bFGF following TBI could affect the levels of injury-induced neurogenesis in the SVZ and DG, and the degree to which this is associated with cognitive recovery. Specifically, adult rats received a bFGF intraventricular infusion for 7 days immediately following TBI. BrdU was administered to animals daily at 2-7 days post-injury to label cell proliferation. At 1 or 4 weeks post-injury, brain sections were immunostained for BrdU and neuronal or astrocytic markers. We found that injured animals infused with bFGF exhibited significantly enhanced cell proliferation in the SVZ and the DG at 1 week post-TBI as compared to vehicle-infused animals. Moreover, following bFGF infusion, a greater number of the newly generated cells survived to 4 weeks post-injury, with the majority being neurons. Additionally, animals infused with bFGF showed significant cognitive improvement. Collectively, the current findings suggest that bFGF-enhanced neurogenesis contributes to cognitive recovery following TBI.     

The Alzheimer's disease drug memantine increases the number of radial glia‐like progenitor cells in adult hippocampus

New neurons are continuously generated in the hippocampus of the adult mammalian brain, and N‐methyl‐D ‐aspartate receptor (NMDA‐R) antagonists have been found to increase the number of newly generated neurons in the dentate gyrus (DG) of the adult hippocampus. In this study, we examined the effect of memantine, an NMDA‐R antagonist that is clinically used for the treatment of Alzheimer's disease, on primary progenitor cells exhibiting a radial glia‐like (RGL) morphology in the DG. We injected 3‐month‐old mice with memantine (50 mg/kg body weight, intraperitoneally [i.p.]); 3 days later, we injected the mice with 5‐bromo‐2‐deoxyuridine (BrdU; 75 mg/kg body weight, i.p.). We then counted the number of BrdU‐labeled RGL progenitor cells in the DG 1 or 7 days after the BrdU‐injection. The number of BrdU‐labeled RGL progenitor cells had increased significantly by 5.1‐fold on day 1 and by 13.7‐fold on day 7 after BrdU‐injection. Immunohistochemical staining revealed that the BrdU‐labeled RGL progenitor cells expressed two primary progenitor cell marker proteins, nestin and Sox2. These results clearly demonstrated that memantine promotes the proliferation of RGL progenitor cells. We also found that memantine increased the ratio of horizontally aligned RGL progenitor cells, which are probably produced by symmetric division. These findings suggest that memantine increases the proliferation of primary progenitor cells and expands the primary progenitor cell pool in the adult hippocampus by stimulating symmetric division. © 2008 Wiley‐Liss, Inc.     

Temporal changes of neurogenesis in the mouse hippocampus after experimental subarachnoid hemorrhage

Abstract

Recent studies indicate the existence of progenitor cells and their potential for neurogenesis in the subventricular zone (SVZ) and the hippocampus dentate gyrus (DG) of normal adult mammalian brain. Increased neurogenesis has been shown following cerebral ischemia and traumatic brain injury; however, the involvement of neurogenesis in subarachnoid hemorrhage (SAH) has not been examined. Adult male CD-1 mice were subjected to SAH by endovascular perforation of the left anterior cerebral artery. Mice received intraperitoneal injections of the cell proliferation-specific marker 5 ′ -bromodeoxyuridine (BrdU) after SAH induction. BrdU incorporation was examined from 1 to 30 days after SAH by immunohistochemistry. The BrdU-positive cells were detected in SVZ and DG of normal control brain, and were significantly decreased in both areas three days after SAH. The number of these cells had recovered to its control level seven days after SAH. Double staining with BrdU and NeuN indicated that the majority of the BrdU-positive cells migrating into the granular cell layer of the DG became NeuN-positive 30 days after SAH. In conclusion, temporal changes of the neurogenesis as shown in the present study suggest that neurogenesis in the hippocampus may affect functional outcome after SAH. The induction of the neurogenesis can provide therapeutic value against SAH.