大鼠局灶性脑缺血后少突胶质前体细胞激活及髓鞘再生的实验研究

目的探讨脑缺血再灌注大鼠少突胶质前体细胞(oligodendrocyte progenitor cells,OPCs)及髓鞘的表达变化。方法线栓法建立大脑中动脉闭塞(middle cerebral artery occlusion,MCAO)模型,免疫组化方法检测脑缺血再灌注后不同时间点1d、1w和2w不同脑区(梗死中心区、梗死周边区和梗死对侧区)OPCs特异性细胞标志物NG2的阳性细胞数及和髓鞘标志物碱性髓鞘蛋白(myelin basic protein,MBP)的表达变化。结果脑缺血后梗死中心区NG2阳性细胞数和MBP的表达随着再灌注时间延长而逐渐减少;梗死周边区NG2阳性细胞数在1w～2w增加,MBP的表达在24h～1w内降低,2w恢复到正常水平;梗死对侧区NG2阳性细胞数和MBP的表达无明显变化。梗死周边区OPCs细胞呈"单极"或"双极"分裂状,并从梗死灶的外带迁移到内带,提示OPCs细胞激活、增生并发生迁移。结论脑缺血再灌注后梗死周边区NG2细胞增多,使得一度缺失的成熟少突胶质细胞及髓鞘得到补充,提示NG2细胞可能参与缺血损伤的修复过程。     

大鼠局灶性脑缺血模型的建立

急性脑缺血溶栓研究已有20余年的历史,曾因导致出血频繁而停用。随着新型溶栓药物的出现,其安全性逐渐提高,引起人们对溶栓疗法的重新认识。目前适用于溶栓研究的局灶性脑缺血动物模型均有不足之处。我们采用并改良Overgaard K[1]等的颈内动脉注入自体血凝块的方法,制作大鼠局灶性脑缺血模型,此模型具有与人类脑缺血极其相似的特点,最适用于溶栓研究,现报道如下。     

栓线法大鼠局灶性脑缺血模型的研究

采用颈部旁侧手术入路，结扎右侧颈总动脉、颈外动脉及其分支后颈内动脉栓线法制作大鼠大脑中动脉闭塞再灌流损伤模型。闭塞成功率９４．１％，动物偏瘫症状评分在缺血２ｈ再灌流３ｄ后趋于稳定，病理改变以尾壳核损害最重。再灌流７ｄ脑梗塞体积为９７．８±９．４ｍｍ３，动物死亡率５９．４％。缺血２ｈ后再灌流先出现过渡灌注，而后呈持续性低灌注。本文模型勿需开颅，缺血效果可靠，对局灶性脑缺血的研究具有实用价值。     

大鼠局灶脑缺血后钙调神经磷酸酶活性和含量变化

目的 研究大脑中动脉闭塞后钙调神经磷酸酶（Calcineurin,CaN)的活性和含量变化规律。探讨CaN在脑缺血中的作用。方法 制备大鼠大脑中动脉永久性闭塞模型。分别测定缺血后不同时间点病灶侧大脑皮质和海马CA1区CaN的活性和含量。结果 皮质组织在缺血6h及其后各时间点CaN 的含量下降，其活性于缺血后4h,6h和12h短暂性增强。海马CA1区CaN的含量于缺血后24h开始降低且不恢复；CaN的活性在缺血后2h,4h和6h减弱。12h始恢复至正常水平。可见，CaN的活性与含量分离。结论 局灶脑缺血后CaN独特的时间变化规律显示其参与介导缺血性神经元损伤。可能具有毒性作用。     

骨髓基质细胞移植治疗局灶性脑缺血

骨髓基质细胞 (ｍａｒｒｏｗｓｔｒｏｍａｌｃｅｌｌｓ,ＭＳＣｓ)是一种非造血组织干细胞 ,它易粘附、易增殖 ,有自我更新的能力和多向分化的潜能。将一定数量的骨髓基质细胞通过脑立体定向术、静脉或颈内动脉注射移植到脑内后 ,能够明显改善局灶性脑缺血动物的神经行为功能。但是有关骨髓基质细胞移植治疗局灶性脑缺血的机制尚有待研究。一、骨髓基质细胞的生物学特性目前发现至少存在 3种形态的ＭＳＣｓ。Ｗｏｏｄｂｕｒｙ等发现 ,以低密度种植的大鼠ＭＳＣｓ为大而扁平的细胞 ;当接近汇合时 ,它们即转变成小的梭形细胞[1] 。Ｃｏｌｔｅｒ…     

大鼠局灶性脑缺血IL-8变化的研究

目的 探讨ＩＬ－８在脑缺血损伤中的变化。方法 采用改良Ｚｅａ Ｌｏｎｇａ线栓法大鼠ＭＣＡＯ模型,将大脑中动脉（ＭＣＡ）闭塞不同的时间,应用双抗体夹心间接ＥＬＩＳＡ法检测缺血组和对照组大鼠受伤脑组织及血清ＩＬ－８浓度。结果 闭塞３ｈ缺血侧半球湿重大于非缺血侧（Ｐ〈０．０５）,并维持至４８ｈ;缺血组病组织ＩＬ－８含量在ＭＣＡ闭塞３ｈ处于较低水平,随后逐渐升高,至６ｈ达高峰,随后又降低;血清中ＩＬ－８含     

妥泰对大鼠局灶脑缺血神经保护的实验研究

目的观察妥泰对大鼠局灶脑缺血的保护作用.方法以腔内线栓法制作大鼠局灶脑缺血模型,将50只Sprague-Dawley大鼠随机分为对照组(A组10只)、40 mg/kg治疗组(B组20只)、80 mg/kg治疗组(C组20只),在缺血30分钟后一次腹腔注射妥泰,对照组注射生理盐水.分别在4、24小时后观察神经功能评分,24小时后干/湿重法测定脑组织水分含量、氯化三苯基四氮唑(TTC)染色观察测定梗死体积百分比.结果①B、C两组在24小时后的神经功能评分明显优于A组(P＜0.05,P＜0.01).②B、C两组在24小时后脑水肿程度均较A组显著改善,C组(P＜0.01)比B组(P＜0.05)更明显.③C组与A组相比能减小梗死体积(P＜0.05).结论妥泰能减轻线栓法大鼠急性局灶脑缺血模型神经功能损害,减轻脑组织水肿程度,大剂量妥泰还能减小梗死体积,妥泰对大鼠脑缺血有一定的神经保护作用.     

大鼠局灶性脑缺血再灌注后CAD表达与细胞凋亡的关系

目的　探讨大鼠局灶性脑缺血再灌注后缺血半暗带 Caspase-3激活的 DNA酶 (CAD)基因的表达变化与细胞凋亡的关系。方法　线栓法建立大鼠大脑中动脉闭塞 (MCAO)及再通模型。应用 RT-PCR技术检测MCAO再通后不同时相缺血半暗带皮质 CAD基因的表达 ,同时利用 TU NEL法观察对应区域细胞凋亡的动态变化规律。结果　脑缺血再灌注 6h,半暗带皮质 CAD m RNA显著升高 ,密度比值为 0 .74± 0 .0 4,再灌注 2 4h达到高峰 (1.13± 0 .11)。对应各时相均可见神经细胞凋亡 ,凋亡细胞以再灌注 48h组为最高 (113 .10± 13 .88)。结论　脑缺血再灌注可致 CAD基因表达上调 ,可能参与了缺血后神经细胞凋亡过程     

缬沙坦对大鼠局灶性脑缺血保护作用的实验研究

缬沙坦(valsartan)是一种新型的血管紧张素Ⅱ(AngⅡ)受体1拮抗剂(AAG)。资料表明，长期应用AAG能够降低高血压患者脑卒中的发生率。为探讨AAG是否对缺血性脑损伤有保护作用，我们取正常血压大鼠，采用大脑中动脉闭塞(MCAO)模型．观察缬沙坦对大鼠脑缺血的保护作用。     

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。     

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.     

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。     

EGF infusion stimulates the proliferation and migration of embryonic progenitor cells transplanted in the adult rat striatum

Immature progenitor cells (generated by in vitro propagation) may provide a useful alternative to primary cells (from dissected embryonic tissue) for transplantation if their migratory and proliferative and differentiation properties can be controlled and directed in vivo. In this study E15 murine EGF-responsive progenitor cells were transplanted to the striatum of adult rats. Simultaneously, these animals received continuous infusion of either epidermal growth factor (EGF) or vehicle, to the lateral ventricle, for 8 days. In animals that received EGF, the transplanted progenitors migrated toward the lateral ventricle and proliferated, as evidenced by bromodeoxyuridine incorporation. Progenitor cells transplanted to rats that received vehicle infusions showed neither of these responses. In all animals, transplanted progenitors expressed an immature astrocyte or oligodendrocyte phenotype, the majority of cells being astrocytes. We conclude that EGF stimulates the migration and proliferation of murine progenitor cells in vivo, either directly or indirectly, but does not influence their phenotypic differentiation.     

Electroacupuncture promotes the proliferation of endogenous neural stem cells and oligodendrocytes in the injured spinal cord of adult rats

A contusive model of spinal cord injury at spinal segment T8-9 was established in rats. Huantiao (GB30) and Huatuojiaji (Ex-B05) were punctured with needles, and endogenous neural stem cells were labeled with 5-bromo-2’-deoxyuridine (BrdU) and NG2. Double immunofluorescence staining showed that electroacupuncture markedly increased the numbers of BrdU+/NG2+ cells at spinal cord tissue 15 mm away from the injury center in the rostral and caudal directions. The results suggest that electroacupuncture promotes the proliferation of endogenous neural stem cells and oligodendrocytes in rats with spinal cord injury.     

Intravenous injection of neural progenitor cells improved depression-like behavior after cerebral ischemia

Poststroke depression (PSD) occurs in approximately one-third of stroke survivors and is one of the serious sequelae of stroke. The onset of PSD causes delayed functional recovery by rehabilitation and also increases cognitive impairment. However, appropriate strategies for the therapy against ischemia-induced depression-like behaviors still remain to be developed. Such behaviors have been associated with a reduced level of brain-derived neurotrophic factor (BDNF). In addition, accumulating evidence indicates the ability of stem cells to improve cerebral ischemia-induced brain injuries. However, it remains to be clarified as to the effect of neural progenitor cells (NPCs) on PSD and the association between BDNF level and PSD. Using NPCs, we investigated the effect of intravenous injection of NPCs on PSD. We showed that injection of NPCs improved ischemia-induced depression-like behaviors in the forced-swimming test and sucrose preference test without having any effect on the viable area between vehicle- and NPC-injected ischemic rats. The injection of NPCs prevented the decrease in the level of BDNF in the ipsilateral hemisphere. The levels of phosphorylated CREB, ERK and Akt, which have been implicated in events downstream of BDNF signaling, were also decreased after cerebral ischemia. NPC injection inhibited these decreases in the phosphorylation of CREB and ERK, but not that of Akt. Our findings provide evidence that injection of NPCs may have therapeutic potential for the improvement of depression-like behaviors after cerebral ischemia and that these effects might be associated with restoring BDNF-ERK-CREB signaling.     

远志皂苷元促进人神经前体细胞系的体外增殖

Senegenin,an effective component of Polygala tenuifolia root extract,promotes proliferation and differentiation of neural progenitor cells in the hippocampus.However,the effects of senegenin on mesencephalon-derived neural progenitor cells remain poorly understood.Cells from a ventral mesencephalon neural progenitor cell line(ReNcell VM) were utilized as models for pharmaceutical screening.The effects of various senegenin concentrations on cell proliferation were analyzed,demonstrating that high senegenin concentrations(5,10,50,and 100 μmo/L),particularly 50 μmol/L,significantly promoted proliferation of ReNcell VM cells.In the mitogen-activated protein kinase signal transduction pathway,senegenin significantly increased phosphorylation levels of extracellular signal-regulated kinases.Moreover,cell proliferation was suppressed by extracellular signal-regulated kinase inhibitors.Results suggested that senegenin contributed to in vitro proliferation of human neural progenitor cells by upregulating phosphorylation of extracellular signal-regulated kinase.     

Injection of neural progenitor cells improved learning and memory dysfunction after cerebral ischemia

Accumulating evidence indicates that stem cells have the ability to improve neurological deficits seen after cerebral ischemia. However, the effects of neural progenitor cells (NPCs) on cerebral ischemia-induced learning and memory dysfunction remain to be clarified. The purpose of the present study was to determine whether the injection of exogenous NPCs could prevent learning and memory dysfunction after cerebral ischemia. Sustained cerebral ischemia was produced by the injection of 700 microspheres into the right hemisphere of each rat. We demonstrated that injection of NPCs into the hippocampus at 10 min after the induction of cerebral ischemia reduced prolongation of the escape latency seen in acquisition and retention tests of the water maze task on Days 12-28 after cerebral ischemia. Injection of NPCs partially attenuated the decrease in viable areas of the ipsilateral hemisphere on Day 28 after the cerebral ischemia. We also demonstrated that injection of NPCs prevented the decrease in the level of BDNF seen at the early period after cerebral ischemia. These results suggest that the injection of exogenous NPCs into the hippocampus can prevent cerebral ischemia-induced learning and memory dysfunction, possibly through maintenance of the BDNF level.     

Transplantation of human neural progenitor cells into the neonatal rat brain: extensive migration and differentiation with long-distance axonal projections.

Here we examined the ability of human neural progenitors from the embryonic forebrain, expanded for up to a year in culture in the presence of growth factors, to respond to environmental signals provided by the developing rat brain. After survival times of up to more than a year after transplantation into the striatum, the hippocampus, and the subventricular zone, the cells were analyzed using human-specific antisera and the reporter gene green fluorescent protein (GFP). From grafts implanted in the striatum, the cells migrated extensively, especially within white matter structures. Neuronal differentiation was most pronounced at the striatal graft core, with axonal projections extending caudally along the internal capsule into mesencephalon. In the hippocampus, cells migrated throughout the entire hippocampal formation and into adjacent white matter tracts, with differentiation into neurons both in the dentate gyrus and in the CA1-3 regions. Directed migration along the rostral migratory stream to the olfactory bulb and differentiation into granule cells were observed after implantation into the subventricular zone. Glial differentiation occurred at all three graft sites, predominantly at the injection sites, but also among the migrating cells. A lentiviral vector was used to transduce the cells with the GFP gene prior to grafting. The reporter gene was expressed for at least 15 weeks and the distribution of the gene product throughout the entire cytoplasmic compartment of the expressing cells allowed for a detailed morphological analysis of a portion of the grafted cells. The extensive integration and differentiation of in vitro-expanded human neural progenitor cells indicate that multipotent progenitors are capable of responding in a regionally specific manner to cues present in the developing rat brain.