大鼠脑缺血再灌注诱导自体神经干细胞原位增殖的研究

目的研究缺血性脑损伤对内源性神经干细胞增殖、迁移的影响。方法参照Pulsinelli-Brierley法制作短暂性全脑缺血动物模型,全脑缺血10min后再灌注,采用SABC免疫组化染色显示5'-溴脱氧尿嘧啶(BrdU)阳性细胞和神经巢蛋白(Nestin)阳性细胞,光镜下观察并统计分析脑缺血损伤后内源性神经干细胞增殖、迁移的变化过程。结果脑缺血再灌流24h后,海马、齿状回和室管膜下区的BrdU阳性细胞和Nestin阳性细胞增多,7～10d达到高峰,术后20d仍有表达;在室管膜下区,BrdU阳性细胞和Nestin阳性细胞有向皮质、海马迁移的现象。结论①成年大鼠全脑缺血后7～10d,内源性神经干细胞的增殖达到高峰。②增殖的内源性神经干细胞存在由增殖区向靶区迁移的现象。     

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。     

大鼠局灶性脑缺血后神经前体细胞增殖迁移的研究

目的研究成年大鼠内源性神经前体细胞在局灶性脑缺血后不同时间窗的增殖分化情况。方法用Longa线栓法制作大鼠脑缺血模型，用免疫组化的方法检测大鼠内源性神经前体细胞最佳增殖时间及最大增殖效果：j结果脑缺血半球室管膜下区、嗅球和头端迁徙渠道(rostral migratory stream，RMS)Brdu阳性细胞数在脑缺血后1d明显增多，3～7d达高峰，14d后开始下降；Brdu阳性细胞数在脑缺血半球室管膜下区和嗅球成止相关；脯缺血侧海马齿状回未见Brdu阳性细胞数明显增多；Brdu阳性细胞在脑缺血缸后第3周数量最大，从第4周开始下降。结论成年大鼠局灶性脑缺血后3～7d内源性神经前体细胞增殖达高峰，住该期进行外源性细胞移植治疗可能更有效。     

糖皮质激素对大鼠内源性神经前体细胞增殖的影响

目的探讨大剂量糖皮质激素(GCs)对成年大鼠内源性神经前体细胞增殖的影响.方法将25只大鼠随机分为对照组和地塞米松(DEX)作用1、3、7、14 d组,应用免疫组化方法检测神经前体细胞标记物巢蛋白(nestin)的表达,并通过5-溴脱氧尿苷(BrdU)观察神经前体细胞的增殖.结果正常大鼠海马齿状回(DG)和室下区(SVZ)存在神经前体细胞,并且其中一些细胞处于分裂增殖状态.应用大剂量GCs作用3、7、14 d组与对照组相比DG的nestin和BrdU阳性细胞数明显减少,SVZ的nestin和BrdU阳性细胞数在DEX作用7、14 d组与对照组相比明显减少,并且DG与SVZ二者阳性细胞数随着作用时间的延长而减低更为明显.结论大剂量GCs持续作用可抑制大鼠脑内的内源性神经前体细胞的增殖,DG区的神经前体细胞对GCs的反应较SVZ更为敏感.     

反义Noggin基因对成年大鼠海马内Nestin及GFAP表达的影响

目的探讨Noggin基因对成年大鼠海马内Nestin及GFAP表达的影响。方法反义寡核苷酸技术封闭内源性Noggin基因的表达,免疫组化法检测成年大鼠海马内Nestin与GFAP的表达。结果侧脑室连续4d注射Noggin基因的反义寡核苷酸后,可见海马齿状回(dentate gyrus,DG)内Nestin阳性细胞数与GFAP阳性细胞数较对照组显著增加;室下区GFAP阳性细胞数亦明显增加。结论Noggin对成年海马干细胞的分化有重要作用,内源性Noggin基因的表达可使神经干细胞向神经元方向分化。     

半乳凝素1对脑损伤大鼠室管膜下区内源性神经干细胞原位增殖及迁移的影响

背景：半乳凝素1表达于室管膜下区的星形胶质细胞中并诱导其分化，分化的星形胶质细胞可明显提高脑源性神经生长因子的产生。 目的：观察侧脑室注入半乳凝素1对脑缺血损伤大鼠内源性神经干细胞增殖、迁移的影响。 设计、时间及地点：细胞学体内观察实验，于2007-03/11在佳木斯大学神经科学研究所完成。 材料：纯种清洁级成年Wistar大鼠48只，随机分为模型组、药物组，24只/组。半乳凝素1为北京晶美生物工程公司产品。 方法：两组大鼠均采用线栓法制作局灶性大脑中动脉闭塞模型。造模后24 h，药物组经右侧侧脑室注入10 μL浓度为0.2 g/L的半乳凝素1，模型组注射等量生理盐水。分别于缺血再灌注后3，7，14，28 d处死大鼠，取脑组织制作石蜡切片，处死前1 d腹腔注射BrdU。 主要观察指标：免疫组织化学染色检测大脑室管膜下区BrdU和巢蛋白阳性细胞的表达。 结果：模型组缺血再灌注3 d后大脑室管膜下区BrdU、巢蛋白阳性细胞开始增加，7 d增殖达高峰，14 d后表达开始下降，28 d后下降至最低。药物组缺血再灌注3 d后大脑室管膜下区两种阳性细胞均明显增加；7 d 增殖达高峰，半定量分析BrdU、巢蛋白阳性细胞数分别是模型组的2倍和1.5倍，且BrdU阳性细胞向腹外侧迁移，巢蛋白阳性细胞胞体变大，突起增长，并有向外侧迁移进入脑实质的迹象；14 d后开始下降；28 d降至最低，但仍明显多于模型组（P < 0.05）。 结论：经侧脑室注入半乳凝素1在大鼠脑缺血后可激活内源性神经干细胞原位增殖，并存在由增殖区向外周脑实质迁移的趋势。     

胰岛素样生长因子1在脑梗死大鼠神经干细胞增殖、迁移和分化中的作用*

背景：胰岛素样生长因子1是一种多肽类激素,已证明其对前体细胞增殖有促进作用。 目的：探讨静脉注射胰岛素样生长因子1对大鼠脑缺血后神经干细胞增殖、迁移和分化的影响。 方法：成年雄性SD大鼠80只,随机分为对照组和实验组,40只/组。两组大鼠均采用改良线栓法制备局灶性脑缺血模型,实验组大鼠通过尾静脉注射胰岛素样生长因子1,按100 μg/kg计算,连续注射6 d;对照组给予等剂量的生理盐水。分别于干预后7,14,21,28 d断头去脑,各组分别在处死前1 d腹腔注射BrdU。采用免疫组织化学及其双重染色法检测BrdU阳性细胞、PSA-NCAM阳性细胞、BrdU+PSA-NCAM双阳性细胞、BrdU+MAP2双阳性细胞的表达。 结果与结论：BrdU阳性细胞、PSA-NCAM阳性细胞数均在缺血后7 d最多;BrdU+PSA-NCAM双标阳性细胞在缺血后双侧室管膜下区和海马齿状回区均可以检测到,于7 d计数最多,之后逐渐减少;BrdU+MAP2双阳性细胞却从14 d开始逐渐增多,随BrdU+PSA-NCAM双阳性表达的逐渐降低,BrdU+MAP2双阳性表达逐渐增高,呈现此消彼涨的变化。提示静脉途经给予胰岛素样生长因子1能诱导大鼠缺血性脑损伤后神经干细胞的增殖、分化和迁移。     

N-甲基-D-天冬氨酸受体亚单位2B特异性拮抗剂对缺氧缺血性脑损伤新生大鼠脑室下区神经干细胞增殖的影响

目的:研究N-甲基-D-天冬氨酸受体(NMDAR)亚单位2B(NR2B)特异性拮抗剂(Ro 25-6981)对缺氧缺血性脑损伤(HIBD)新生大鼠脑室下区(SVZ)神经干细胞(NSCs)增殖的影响。方法:7 d龄新生SD大鼠随机分为Ro 25-6981组(HIBD前2 h,腹腔注射Ro 25-6981 10 mg.kg-1)、HIBD组(HIBD前2 h,腹腔注射等剂量生理盐水)和假手术组(仅游离右侧颈总动脉,不结扎)。采用免疫组化学染色检测SVZ Nestin表达量及BrdU阳性细胞数的变化。结果:HIBD组12h后Nestin表达量开始增多,48 h达峰值,之后缓慢下降;与其相比,Ro 25-6981组在12 h和24 h时下降明显(P<0.05)。HIBD组BrdU阳性细胞数在缺氧缺血3 h后缓慢上升,72 h达高峰;与其相比,Ro 25-6981组在各时间点BrdU阳性细胞表达均有所下降,以24、48和72 h减少明显,尤以72 h为著(P<0.05)。结论:Ro 25-6981能够降低HIBD新生大鼠SVZ Nestin的表达及Brdu阳性细胞数,对SVZ NSCs增殖起抑制作用,提示NR2B参与并促进HIBD引起的SVZ NSCs的增殖。     

成年大鼠脑创伤后脑组织碱性成纤维细胞生长因子表达的变化

目的:研究液压冲击性脑损伤后成年大鼠脑组织碱性成纤维细胞生长因子(bFGF)表达时程和空间分布的变化。方法:制作液压冲击性脑损伤模型,免疫组织化学法动态检测内源性bFGF的变化。结果:在正常脑组织中,bFGF免疫活性低水平表达,且主要位于胶质细胞核及神经元细胞质中。液压冲击伤后bFGF免疫活性增强,聚集核周边。此外,部分bFGF免疫活性聚集于神经元周围间隙中半定量分析显示液压冲击伤后3d,大脑皮质bFGF阳性细胞数量较正常对照组升高4.8倍;伤后7 d,bFGF免疫活性达高峰,伤后30 d消失。结论:液压冲击伤后,损伤的胶质细胞和神经元暂短地合成bFGF,并以旁分泌和自分泌形式起作用。     

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。     

Proliferation and neuronal differentiation of mitotically active cells following traumatic brain injury

Recent studies have identified endogenous neural stem cells in adult rodent brains. The present study characterizes the early response of mitotically active cells in the brain to traumatic brain injury. Animals were subjected to lateral fluid percussion injury and sacrificed at various times after injury. To examine increases in cell proliferation animals were injected with the mitotic marker bromodeoxyuridine (BrdU) 24 h before sacrifice. Increased numbers of mitotically active cells were observed at 2 days in the subgranular zone (SGZ) and the subependymal zone (SEZ) under the injury site. To characterize the differentiation potential of these cells, animals were injected with BrdU 18 and 20 h after injury, then sacrificed at multiple time points after injury. Histologically, co-localization with betaIII-tubulin (neuronal marker) and BrdU was evident at 10 and 15 days postinjury in the SGZ. Flow cytometry analysis was used to quantitatively assess neurogenesis in the SEZ. Animals were sacrificed 1, 5, or 10 days after injury and tissue sections extracted, grown in tissue culture for 24 h, fixed, and stained for nestin and betaIII-tubulin to identify newly formed neurons. The percentage of cells expressing both markers was determined using flow cytometry analysis. There was a significant increase in newly differentiated neurons by 10 days postinjury in the SEZ. Thus, we conclude that traumatic brain injury stimulates an increase in proliferation of endogenous neural stem/progenitor cells and that a significant number of these express a neuronal marker. This response may be the brain's way of trying to heal itself after injury.     

大鼠脑缺血再灌注损伤后不同部位神经细胞Nestin的表达

目的研究大鼠脑缺血再灌注损伤后神经细胞巢蛋白（Nestin）的表达，为神经干细胞治疗脑损伤提供理论依据。方法成年健康雄性SD大鼠30只，随机分为实验组和对照组。线栓法建立大脑中动脉闭塞再灌注模型，应用免疫组化SABC法观察2组再灌注后各观察部位不同时间Nestin的表达情况。结果缺血再灌注6h Nestin阳性细胞少量表达；1d时数量增多；3d时明显增多，7d时变化最为显著；各实验组与对照组比较差别均极显著。结论Nestin阳性细胞在正常成年脑组织中广泛表达，损伤后各部位Nestin阳性细胞的表达呈一致性增强，各部位阳性细胞数的增加量在不同时间又有所不同。     

Ependymal cell proliferation and apoptosis following acute spinal cord injury in the adult rat

BACKGROUND: Studies have reported that spinal cord injury can induce the reactive proliferation of ependymal cells and secondarily cause the apoptosis of nerve cells. However, there is no generally accepted theory on the apoptotic characteristics of ependymal cells in the injured spinal cord.OBJECTIVE: To observe the reactive proliferation and apoptosis of ependymal cells in adult rats following acute spinal cord injury.DESIGN, TIME AND SETTING: A randomized control study based on neuropathology was performed in the Third Military Medical University of Chinese PLA between 2005 and 2007.MATERIALS: Forty healthy, adult, Wistar rats were included in the present study.METHODS: Moderate spinal cord injury was established in twenty rats using Feeney's method, while the remaining 20 rats served as controls and were only treated with laminectomy. All rats were injected intraperitoneally with 1.25 mL of BrdU solution (10 mg BrdU/mL saline) 3 times at 4 hours intervals during the 12 hours prior to sacrifice.MAIN OUTCOME MEASURES: Ependymal cell proliferation and apoptosis in the rat spinal cord were determined by BrdU and nestin immunofluorescence double-labeling, as well as the TUNEL method, at 1, 3, 7, and 14 days after operation.RESULTS: In the moderate spinal cord injury rats, nestin expression was observed in the cytoplasm of ependymal cells. One day immediately following surgery, ependymal cells were BrdU-labeled. The number of BrdU-positive cells increased at 3 days, reached a peak at 7 days, and gradually reduced thereafter. The ependyma developed ti'om a constitutive monolayer cells to a multi-layer cell complex. Some BrdU/Nestin double-positive ependymal cells migrated out from the ependyma. TUNEL-positive cells were also detected in the ependyma in the central region, as well as ischemic regions of the injured spinal cord. In addition, TUNEL-positive cells were visible in the ependyma. No TUNEL-positive ependymal cells were observed in the normal spinal cord.CONCLUSION: Proliferating ependymal cells induced apoptosis in the central and surrounding region following spinal cord injury.     

Endogenous adult neurogenesis and cognitive function recovery following traumatic brain injury in the rat hippocampus

BACKGROUND:Endogenous neural progenitor cells play a beneficial role for cognitive recovery following traumatic brain injury.However,there are few classification-control studies aimed at varying graded brain trauma.OBJECTIVE:To observe the effects of adult endogenous neurogenesis on cognitive function repair and regeneration of neural progenitor cells following varying graded traumatic hippocampal injury to determine the significance of endogenous neurogenesis in the repair of brain injury.DESIGN,TIME AND SETTING:A randomized,controlled,animal experiment was performed at the Key Laboratory of Injuries,Variations and Regeneration of Nervous System,Tianjin Medical University General Hospital,from February to October 2009.MATERIALS:Mouse anti-rat 5-bromodeoxyuridine (BrdU) and neuronal nuclei (NeuN) monoclonal antibodies were purchased from Millipore Corporation,USA.METHODS:A total of 45 Wistar rats were randomly assigned to three groups.Mild and severe injury groups were respectively subjected to (182 ± 2) kPa and (284 ± 4) kPa lateral fluid percussion to establish models of brain injury,and the control group was subjected to surgery with no lateral fluid percussion.MAIN OUTCOME MEASURES:Cognitive function was estimated using the Morris water maze.Proliferation,survival,and differentiation of newly generated cells in the injured hippocampus were observed through the use of immunofluorescent staining.RESULTS:At 7 days post-injury,the number of BrdU+ cells in the hippocampal dentate gyrus significantly increased in the mild and severe injury groups compared with the control group (P<0.01).At 61 days post-injury,the number of BrdU7NeuN+ cells in the hippocampal dentate gyrus was significantly greater in the mild injury group compared with the severe injury and control groups (P< 0.01).In addition,the control group exhibited the greatest proportion of surviving cells that differentiated into mature neurons compared with the injury groups (P< 0.01).Moreover,at 61 days post-injury,cognitive function in rats with mild injury recovered to normal levels,whereas the severe injury group exhibited cognitive deficits (P< 0.01).CONCLUSION:Traumatic brain injury may be a stimulation factor for proliferation of neural progenitor cells in the adult hippocampus but severe brain trauma does not lead to an increased number of newly generated cells.Endogenous adult neurogenesis repairs neurological functions to an extent.However,recovery of neurological function remains limited following severe traumatic brain injury.     

Regulation of nestin expression after cortical ablation in adult rat brain

During embryogenesis, transient expression of nestin in proliferating neuroepithelial stem cells signals the commitment of progenitor cells to differentiate. Although adult mammalian brain contains very little nestin, significant upregulation of nestin has been reported following cerebral injury, leading to speculation that nestin may be involved in brain repair. In this study, we assessed the temporal profile of nestin expression following ablation injury of the sensory barrel cortex and investigated the influence of contralateral whisker stimulation on nestin expression. Since the adult mammalian brain contains proliferating neuronal progenitor cells that can be labeled with bromodeoxyuridine (BrdU), we also determined the association of nestin reexpression with BrdU-labeled cells. Nestin reexpression was detected predominantly in the ipsilateral cortex 3 days post-ablation. There was no significant nestin upregulation in the subcortical region. Nestin reexpression was most marked surrounding the lesion, but also extended throughout the entire lateral cortex. Nestin in the ipsilateral cortex subsided by day 7, although perilesional nestin expression was still apparent 28 days post-injury. Western blot analysis of nestin expression 3 days post-ablation confirmed a significant two-fold increase in nestin expression (p<0.05). Double immunofluorescence labeling demonstrated that the majority of nestin expression occurred in astrocytes. We were unable to detect any colocalization with neuronal makers. However, BrdU-labeled cells, which were readily detected in the subventricular zone prior to injury, were readily detected in the perilesional area 3 days post-ablation, concomitant with nestin in this area. Confocal microscopy detected several BrdU-positive cells expressing nestin. Taken together, the data support a potential role for nestin reexpression in brain repair.     

人脐带间充质干细胞移植治疗大鼠创伤性脑损伤

背景：脐带间充质干细胞体内移植治疗脑损伤的效果目前尚较少见报道。 目的：观察人脐带间充质干细胞移植对大鼠液压冲击脑损伤的治疗作用。 方法：从新生儿脐带中分离、培养间充质干细胞。制作中度打击大鼠脑损伤模型。实验分为4组：①脐带间充质干细胞移植组：损伤后原位移植脐带间充质干细胞。②对照组：损伤后原位注射等量DMEN/F12培养基。③单纯损伤组：仅施行损伤。④假损伤组：仅切开头皮及颅骨,不实施机械性损伤。 结果与结论：脐带间充质干细胞移植后1~3周,动物神经功能评分较对照组明显改善;4周后,各组动物神经功能评分均恢复正常。免疫组织化学检测表明少部分移植细胞表达神经元特异性烯醇化酶,胶质纤维酸性蛋白。与对照组相比,移植组损伤区血管内皮生长因子表达明显增加,凋亡细胞减少。提示脐带充间质干细胞脑内移植有助于促进创伤性脑损伤后的早期功能恢复,这种治疗效果是通过刺激宿主细胞分泌血管内皮生长因子,增加损伤区微血管密度,抑制宿主细胞凋亡等实现的。