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。     

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。     

脑缺血再灌流诱导内源性神经干细胞的增殖、分化

目的 观察脑缺血再灌注后内源性神经干细胞的分布、增殖和分化.方法 阻塞大鼠大脑中动脉2h不同时间的再灌流制成局灶性脑缺血模型,免疫组化单标、双标观察神经干细胞的增殖、分布、分化.结果 正常组和假手术组,多数脑室室管膜细胞Brdu免疫反应阳性,脑实质内有零星散在Brdu阳性细胞.再灌流1d,室管膜及室管膜下层的Brdu阳性细胞显著增多;再灌流3～10d,视前区梗死区周边区、缺血纹状体和额顶皮质的Brdu阳性细胞显著增加.再灌流3d,Brdu阳性细胞出现于海马齿状回的颗粒下层,并随再灌流时间延长,阳性细胞数量增多.免疫组化双标显示再灌流3d视前区有很少量的Brdu/GFAP双阳性细胞,随后增多.再灌流10d内,未检查到Brdu/NF双标阳性细胞.结论 成年哺乳动物的室管膜、脑实质有少量神经干细胞,脑缺血后室管膜、齿状回颗粒下层出现大量的神经干细胞增殖,这些增殖神经干细胞向缺血区迁移、分化,试图补偿丢失的细胞,重塑受损的神经组织.     

嗅球电刺激对成年大鼠室管膜下区神经发生的影响

目的 探讨嗅球电刺激对成年大鼠室管膜下区(SVZ)神经前体细胞(NPC)增殖及其向嗅球迁移的影响,并对其相关机制进行初步探讨. 方法 SD大鼠80只按随机数字表法分为正常对照组、假刺激组、电刺激ld组、电刺激3d组、电刺激1周组、电刺激2周组、电刺激3周组、电刺激4周组,每组10只,后6组大鼠制备嗅球电刺激模型,以5-溴脱氧尿嘧啶(Brdu)标记新生细胞,免疫组化染色观察大鼠SVZ区内源性NPC的增殖,RT-PCR检测嗅球内Prokineticin 2 (prk2)mRNA的表达；另取大鼠15只,按随机数字表法分为正常对照组、假刺激组和电刺激4周组,每组5只,免疫组化染色观察嗅球内Brdu阳性细胞的表达. 结果 8组大鼠SVZ区Brdu阳性细胞数不同,差异有统计学意义(F=51.475,P=0.000).与正常对照组和假刺激组比较,电刺激ld、3d、l周、2周组大鼠刺激侧Brdu阳性细胞增高,差异有统计学意义(P＜0.05)；8组大鼠嗅球内prk2 mRNA的表达不同,差异有统计学意义(F=154.067,P=0.000).与正常对照组和假刺激组比较,电刺激ld、3d、l周、2周、3周、4周组prk2 mRNA增加,差异有统计学意义(P＜0.05)；3组大鼠嗅球内Brdu阳性细胞数不同,差异有统计学意义(F=36.472,P=0.000).电刺激4周组嗅球内Brdu阳性细胞数较正常对照组和假刺激组明显增多,差异有统计学意义(P＜0.05). 结论 电刺激嗅球可促进SVZ区NPC的增殖并向嗅球迁移,这可能与电刺激提高嗅球内Prk 2的表达水平有关.     

成年大鼠脑创伤后神经前体细胞的增殖及迁移

目的 研究液压冲击性脑损伤后成年大鼠神经前体细胞的增殖及迁移。方法 制作液压冲击性脑损伤模型，免疫组织化学方法动态检测巢蛋白（Nestin）和5溴脱氧尿苷（BrdU）的表达。BrdU标记方法确定增列殖的前体细胞；Nestin的表达用于确定神经前体细胞。结果 同正常对照组相比较，伤侧皮层、海马及室下区的Nestin阳性细胞数于伤后1d明显增多，7d达高峰,30d消失；BrdU阳性细胞数于作后3d达高峰，而7d以后逐渐减小，室下区BrdU阳性细胞及Nestin阳性细胞经胼胝体向对侧迁移。结论 液压冲击性脑损伤可激发成年大鼠神经前体细胞增殖及迁移。     

脑缺血后内源性激活神经干细胞的机制研究

1 脑缺血后内源性神经干细胞的自身激活 近年来研究表明,脑缺血后自身的神经干细胞有大量的增殖分化[1,2],说明神经干细胞可能参与脑缺血的病理生理过程.Zhang等通过建立沙鼠大脑中动脉缺血(MCAO)模型,观察缺血再灌注后不同时期的室管膜下层(SVZ)、海马颗粒细胞层(DG)、嗅球以及梗死灶周边皮质的神经干细胞增殖、分化情况,发现7 d后在梗死同侧脑SVZ出现神经干细胞增殖高峰,14 d达最高,而DG区则无增殖,且14 d后远离梗死区的嗅球有大量的神经干细胞增殖,但28 d后,标记Brdu阳性细胞大量减少,说明脑缺血只引起短暂的神经干细胞的增殖,Zhang认为是脑缺血损伤的应急保护反应[3].     

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。     

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。     

电刺激嗅球对脑缺血再灌注大鼠内源性神经干细胞增殖、迁移及分化的影响

目的：探讨嗅球电刺激对脑缺血再灌注大鼠脑内神经发生的影响。方法将健康雌性SD大鼠随机分为假手术组、脑缺血再灌注组、电刺激组、假刺激组,其中脑缺血再灌注组、电刺激组,以线栓法建立大鼠右侧大脑中动脉缺血（middle cerebral artery occlusion ,MCAO）/再灌注模型,假手术组仅分离出颈内动脉;电刺激组在右侧嗅球内埋置双极电极,于MCAO再灌注后对嗅球进行电刺激,假刺激组只埋置电极,不进行电刺激;以5－溴脱氧尿嘧啶（Brdu）标记内源性神经干细胞（NSC ）。假手术组于术后48 h、1周,脑缺血再灌注组于缺血再灌注后48 h、1周,电刺激组及假刺激组于刺激后48 h、1周分别行Brdu免疫组化染色,观察各组脑室下区（SVZ）区NSC增殖情况;假手术组、缺血再灌注组、电刺激组、假刺激组4组大鼠在电刺激组行电刺激后腹腔注射Brdu 50 mg/kg ,2次/d ,连续2 d ,4周后处死,分别取脑切片,进行免疫组化和免疫荧光双标染色,观察各组NSC迁移和分化情况。结果缺血再灌注组（37.67±1.97）、假刺激组（36.5±2.35）和电刺激组（43.67±1.63）大鼠48 h后SVZ区Brdu阳性NSC细胞数较假手术组（15.5±1.52）明显增多（F＝57.21,P＜0.05）,缺血再灌注组（41.17±2.94）、假刺激组（41.83±2.14）和电刺激组（47.67±2.34）1周后Brdu阳性细胞数较假手术组（15.5±1.52）增多（F＝73.62,P＜0.01）,电刺激组在各时间点均较其他组Brdu阳性 NSC细胞数增多（P＜0.01）。缺血并注射Brdu 4周后缺血侧皮质区电刺激组Brdu阳性NSC细胞数（57.17±2.4）较缺血再灌注组（46.83±2.48）和假刺激组（45.83±2.14）增多（F＝161.50,P＜0.01）;免疫荧光双标染色示电刺激组Brdu阳性NSC细胞中Brdu/神经元核抗原（Neun ）双阳性细胞比例约为（74.67±3.61）％,Brdu/胶质纤维酸性蛋白（GFAP）双阳性细胞比例约为（38.83±3.36）％,与缺血再灌注组和假刺激组比较差异均无统计学意义（均 P＞0.05）。结论电刺激嗅球可能促进脑缺血大鼠SVZ区NSC增殖,并可能促进新生NSC向缺血皮质区迁移,增加缺血皮质新生神经元数量,但SVZ区新生NSC的分化不受影响。     

缺血性脑损伤诱导大鼠巢蛋白表达的实验研究

目的探讨缺血性脑损伤对内源性神经干细胞增殖、迁移的影响。方法选用健康雄性SD大鼠62只，随机分为正常组（6只）、脑缺血10min再灌流1、3、5、7、10、15、20d组（简称手术组，每时间点6只）、假手术对照组（14只，每时间点2只），参照Pulsinelli—Brierley方法制作短暂性全脑缺血动物模型：用SABC免疫组化法显示巢蛋白（nestin）阳性细胞；光镜下观察nestin阳性细胞的形态学变化并计数，半定量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。结果手术组的nestin阳性细胞在缺血再灌流24h后表达增多，7～10d到高峰，15d时仍有显著表达；在室管膜下区的nestin阳性细胞有向皮质、海马迁移的迹象。结论缺血性脑损伤能诱导内源性神经干细胞增殖，这可能对脑损伤后的修复发挥作用。     

Human neural stem cells promote proliferation of endogenous neural stem cells and enhance angiogenesis in ischemic rat brain

Transplantation of human neural stem cells into the dentate gyrus or ventricle of rodents has been reportedly to enhance neurogenesis. In this study, we examined endogenous stem cell proliferation and angiogenesis in the ischemic rat brain after the transplantation of human neural stem cells. Focal cerebral ischemia in the rat brain was induced by middle cerebral artery occlusion. Human neural stem cells were transplanted into the subventricular zone. The behavioral performance of human neural stem cells-treated ischemic rats was significantly improved and cerebral infarct volumes were reduced compared to those in untreated animals. Numerous transplanted human neural stem cells were alive and preferentially localized to the ipsilateral ischemic hemisphere. Furthermore, 5-bromo-2′-deoxyuridine-labeled endogenous neural stem cells were observed in the subventricular zone and hippocampus, where they differentiated into cells immunoreactive for the neural markers doublecortin, neuronal nuclear antigen Neu N, and astrocyte marker glial fibrillary acidic protein in human neural stem cells-treated rats, but not in the untreated ischemic animals. The number of 5-bromo-2′-deoxyuridine-positive ? anti-von Willebrand factor-positive proliferating endothelial cells was higher in the ischemic boundary zone of human neural stem cells-treated rats than in controls. Finally, transplantation of human neural stem cells in the brains of rats with focal cerebral ischemia promoted the proliferation of endogenous neural stem cells and their differentiation into mature neural-like cells, and enhanced angiogenesis. This study provides valuable insights into the effect of human neural stem cell transplantation on focal cerebral ischemia, which can be applied to the development of an effective therapy for stroke.     

Insulin-like growth factor-1 is an endogenous mediator of focal ischemia-induced neural progenitor proliferation

The adult mammalian brain contains resident neural progenitors in the subgranular zone of the dentate gyrus (DG) and the subventricular zone (SVZ) of the lateral ventricles. The proliferation of neural progenitors increases after focal cerebral ischemia in both of these regions, but the mechanisms that promote ischemia-induced neural progenitor proliferation are not yet understood. We hypothesize that diffusible factors from the ischemic area play a role in this process as the DG is remote from the area of infarction. In this study, we observed that the peak of neural progenitor proliferation in the ipsilateral DG was between day 2 and day 4 of reperfusion after transient middle cerebral artery occlusion in adult spontaneously hypertensive rats. GeneChip and real-time PCR analysis showed a three- to 102-fold increase in the expression of 15 diffusible, mitogenic factors in the ischemic cortex at 3 days of reperfusion. Of these, insulin-like growth factor-1 (IGF-1) showed increased protein expression in the activated astrocytes in the ischemic penumbra. In addition, the progenitors in both the SVZ and DG showed IGF-1 receptor expression. Inhibiting IGF-1 activity by introcerebroventricular infusion of IGF-1 antibody significantly prevented the ischemia-induced neural progenitor proliferation. These results indicate that IGF-1 formed in the ischemic penumbra might be one of the diffusible factors that mediate post-ischemic neural progenitor proliferation.     

Early expressions of hypoxia-inducible factor 1alpha and vascular endothelial growth factor increase the neuronal plasticity of activated endogenous neural stem cells after focal cerebral ischemia

Endogenous neural stem cells become "activated" after neuronal injury, but the activation sequence and fate of endogenous neural stem cells in focal cerebral ischemia model are little known. We evaluated the relationships between neural stem cells and hypoxia-inducible factor-1α and vascular endothelial growth factor expression in a photothromobotic rat stroke model using immunohistochemistry and western blot analysis. We also evaluated the chronological changes of neural stem cells by 5-bromo-2′-deoxyuridine(BrdU) incorporation. Hypoxia-inducible factor-1α expression was initially increased from 1 hour after ischemic injury, followed by vascular endothelial growth factor expression. Hypoxia-inducible factor-1α immunoreactivity was detected in the ipsilateral cortical neurons of the infarct core and peri-infarct area. Vascular endothelial growth factor immunoreactivity was detected in bilateral cortex, but ipsilateral cortex staining intensity and numbers were greater than the contralateral cortex. Vascular endothelial growth factor immunoreactive cells were easily found along the peri-infarct area 12 hours after focal cerebral ischemia. The expression of nestin increased throughout the microvasculature in the ischemic core and the peri-infarct area in all experimental rats after 24 hours of ischemic injury. Nestin immunoreactivity increased in the subventricular zone during 12 hours to 3 days, and prominently increased in the ipsilateral cortex between 3–7 days. Nestin-labeled cells showed dual differentiation with microvessels near the infarct core and reactive astrocytes in the peri-infarct area. BrdU-labeled cells were increased gradually from day 1 in the ipsilateral subventricular zone and cortex, and numerous BrdU-labeled cells were observed in the peri-infarct area and non-lesioned cortex at 3 days. BrdU-labeled cells rather than neurons, were mainly co-labeled with nestin and GFAP. Early expressions of hypoxia-inducible factor-1α and vascular endothelial growth factor after ischemia made up the microenvironment to increase the neuronal plasticity of activated endogenous neural stem cells. Moreover, neural precursor cells after large-scale cortical injury could be recruited from the cortex nearby infarct core and subventricular zone.     

Monocyte chemoattractant protein-1 plays a critical role in neuroblast migration after focal cerebral ischemia.

Transient focal ischemia is known to induce proliferation of neural progenitors in adult rodent brain. We presently report that doublecortin positive neuroblasts formed in the subventricular zone (SVZ) and the posterior peri-ventricle region migrate towards the cortical and striatal penumbra after transient middle cerebral artery occlusion (MCAO) in adult rodents. Cultured neural progenitor cells grafted into the non-infarcted area of the ipsilateral cortex migrated preferentially towards the infarct. As chemokines are known to induce cell migration, we investigated if monocyte chemoattractant protein-1 (MCP-1) has a role in post-ischemic neuroblast migration. Transient MCAO induced an increased expression of MCP-1 mRNA in the ipsilateral cortex and striatum. Immunostaining showed that the expression of MCP-1 was localized in the activated microglia and astrocytes present in the ischemic areas between days 1 and 3 of reperfusion. Furthermore, infusion of MCP-1 into the normal striatum induced neuroblast migration to the infusion site. The migrating neuroblasts expressed the MCP-1 receptor CCR2. In knockout mice that lacked either MCP-1 or its receptor CCR2, there was a significant decrease in the number of migrating neuroblasts from the ipsilateral SVZ to the ischemic striatum. These results show that MCP-1 is one of the factors that attract the migration of newly formed neuroblasts from neurogenic regions to the damaged regions of brain after focal ischemia.     

Activated neural stem cells contribute to stroke-induced neurogenesis and neuroblast migration toward the infarct boundary in adult rats.

Stroke increases neurogenesis. The authors investigated whether neural stem cells or progenitor cells in the adult subventricular zone (SVZ) of rats contribute to stroke-induced increase in neurogenesis. After induction of stroke in rats, the numbers of cells immunoreactive to doublecortin, a marker for immature neurons, increased in the ipsilateral SVZ and striatum. Infusion of an antimitotic agent (cytosine-beta-D-arabiofuranoside, Ara-C) onto the ipsilateral cortex eliminated more than 98% of actively proliferating cells in the SVZ and doublecortin-positive cells in the ipsilateral striatum. However, doublecortin-positive cells rapidly replenished after antimitotic agent depletion of actively proliferating cells. Depleting the numbers of actively proliferating cells in vivo had no effect on the numbers of neurospheres formed in vitro, yet the numbers of neurospheres derived from stroke rats significantly (P<0.05) increased. Neurospheres derived from stroke rats self-renewed and differentiated into neurons and glia. In addition, doublecortin-positive cells generated in the SVZ migrated in a chainlike structure toward ischemic striatum. These findings indicate that in the adult stroke brain, increases in recruitment of neural stem cells contribute to stroke-induced neurogenesis, and that newly generated neurons migrate from the SVZ to the ischemic striatum.     

Galectin-1 regulates neurogenesis in the subventricular zone and promotes functional recovery after stroke

Galectin-1 (Gal-1) has recently been identified as a key molecule that plays important roles in the regulation of neural progenitor cell proliferation in two neurogenic regions: the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus. To test the hypothesis that Gal-1 contributes to adult neurogenesis after focal ischemia, we studied the temporal profile of endogenous Gal-1 expression and the effects of human recombinant Gal-1 on neurogenesis and neurological functions in an experimental focal ischemic model. In the normal brain, Gal-1 expression was observed only in the SVZ. In the ischemic brain, Gal-1 expression was markedly upregulated in the SVZ and the area of selective neuronal death around the infarct in the striatum. The temporal profile of Gal-1 expression was correlated with that of neural progenitor cell proliferation in the SVZ of the ischemic hemisphere. Double-labeling studies revealed that Gal-1 was localized predominantly in both reactive astrocytes and SVZ astrocytes. Administration of Gal-1, which is known to have carbohydrate-binding ability, into the lateral ventricle increased neurogenesis in the ipsilateral SVZ and improved sensorimotor dysfunction after focal ischemia. By contrast, blockade of Gal-1 in the SVZ by the administration of anti-Gal-1 neutralizing antibody strongly inhibited neurogenesis and diminished neurological function. These results suggest that Gal-1 is one of the principal regulators of adult SVZ neurogenesis through its carbohydrate-binding ability and provide evidence that Gal-1 protein has a role in the improvement of sensorimotor function after stroke.     

TRPC1沉默对脑缺血大鼠神经干细胞迁移的影响

目的探讨瞬时受体电位通道1(TRPC1)沉默对脑缺血大鼠内源性神经干细胞增殖、迁移和分化的影响。方法 SD大鼠分为假手术组、脑缺血组、TRPC1沉默组(脑缺血+TRPC1沉默)和阴性对照组,以脑室内注射siRNA沉默TRPC1,以线栓法制作大鼠大脑中动脉脑缺血(MCAO)模型,脑缺血模型制作成功后,腹腔内注射Brdu标记内源性神经干细胞,分别于48 h、4 w后处死大鼠,行Brdu免疫组化染色、免疫荧光双标染色(Brdu/GFAP、Brdu/Neun)观察NSC的增殖、迁移和分化情况。结果脑缺血后48 h,脑缺血组和TRPC1沉默组SVZ区Brdu阳性表达均较假手术组明显增多(P0.01),但TRPC1沉默组Brdu阳性细胞数较缺血组低(P0.01)。脑缺血4 w后,缺血组与假手术组相比,皮质区具有更多的Brdu阳性细胞(P0.01),同样TRPC1沉默组Brdu阳性细胞数多于假手术组,但明显低于脑缺血组(P0.01);免疫荧光双标染色发现,脑缺血各组Brdu/GFAP、Brdu/Neun双阳性细胞的数量均比假手术组明显增高,但TRPC沉默组双阳性细胞显著少于脑缺血组和阴性对照组(P0.01)。结论 TRPC1沉默显著影响脑缺血大鼠SVZ区NSC的增殖、向脑缺血区迁移及向成熟细胞的分化。     

Endogenous neurogenesis and neovascularization in the neocortex of the rat after focal cerebral ischemia

The present study was designed to examine whether endogenous neurogenesis and neovascularization occur in the neocortex of the ischemic rat brain after unilateral middle cerebral artery occlusion (MCAO). Sprague-Dawley rats were divided into six groups (n = 29): one control group (n = 4) and five groups composed of animals sacrificed at increasing times post-MCAO (2 days and 1, 2, 4, and 8 weeks; n = 5 per group). To determine the presence of neurogenesis and neovascularization in the ischemic brain, nestin, Tuj1, NeuN, GFAP, Tie2, RECA, and 5-bromo-2'-deoxyuridine (BrdU) were analyzed immunohistochemically. In addition, nestin, GFAP, and Tie2 expression was determined by Western blotting. Triple-labeling of nestin, BrdU, and laminin was performed to visualize the interaction between endogenous neurogenesis and neovascularization. The number of BrdU- and nestin-colabeled cells increased markedly in the neocortex and border zone of the ischemic area up to 1 week after MCAO and decreased thereafter. Western blot analysis revealed that the expression of nestin, Tie-2, and GFAP was amplified in the ipsilateral hemisphere 2 days after MCAO and peaked 1 week after MCAO, compared with that in the normal brain. After ischemic injury, nestin- and BrdU-colabeled cells were observed in the vicinity of the endothelial cells lining cerebral vessels in the ipsilateral neocortex of the ischemic brain. Endogenous neurogenesis and neovascularization were substantially activated and occurred in close proximity to one other in the ipsilateral neocortex of the ischemic rat brain.     

Buyang Huanwu Decoction regulates neural stem cell behavior in ischemic brain

The traditional Chinese medicine Buyang Huanwu Decoction has been shown to improve the neu-rological function of patients with stroke. However, the precise mechanisms underlying its effect remain poorly understood. In this study, we established a rat model of cerebral ischemia by middle cerebral artery occlusion and intragastrically administered 5 g/kg Buyang Huanwu Decoction, once per day, for 1, 7, 14 and 28 days after cerebral ischemia. Immunohistochemical staining revealed a number of cells positive for the neural stem cell marker nestin in the cerebral cortex, the subven-tricular zone and the ipsilateral hippocampal dentate gyrus in rat models of cerebral ischemia. Buyang Huanwu Decoction significantly increased the number of cells positive for 5-bromodeoxyuridine (BrdU), a cell proliferation-related marker, microtubule-associated protein-2, a marker of neuronal differentiation, and growth-associated protein 43, a marker of synaptic plasticity in the ischemic rat cerebral regions. The number of positive cells peaked at 14 and 28 days after intragastric administration of Buyang Huanwu Decoction. These findings suggest that Buyang Huanwu Decoction can promote the proliferation and differentiation of neural stem cells and hance synaptic plasticity in ischemic rat brain tissue.