大鼠脑缺血再灌注后海马CA1区AS与Syp相关性分析

目的研究大鼠脑组织缺血再灌注后星形胶质细胞与Syp变化的关系。方法建立局灶性脑缺血再灌注模型,72只大鼠随机分为假手术组、缺血再灌注组,各时间点处死取脑,应用免疫组化法检测海马CA1区GFAP、Syp的表达。结果不同时间点缺血再灌注组GFAP、Syp表达均高于同时期假手术组(P<0.01);缺血再灌注组GFAP与Syp高度相关(P<0.01)。结论脑缺血再灌注后,海马CA1区星形胶质细胞与Syp变化具有高度相关性。     

缺血后海马高迁移率族蛋白与星形胶质纤维酸蛋白表达及相关性

目的研究大鼠局灶性脑缺血再灌注星形胶质纤维酸蛋白(GFAP)与高迁移率族蛋白(HMGB1)在海马CA1区表达变化,探讨二者之间的关系。方法采用大脑中动脉栓塞2h制备SD大鼠脑缺血模型,60只雄性SD大鼠随机分为假手术组、缺血再灌注组,按1d、3d、7d、14d、28d时间点再分5个亚组,各时间点处死取脑,用免疫组化和荧光双标结合共聚焦扫描的方法来检测高迁移率族蛋白和星形胶质纤维酸蛋白在脑内海马CA1区表达变化。结果不同时间点缺血再灌注组GFAP、HMGB1表达均高于同时期的假手术组(P<0.05)。缺血再灌注组星形胶质细胞1d、3d、7d逐渐激活增生,7d达到高峰,14d开始下降;HMGB1在1d、3d、7d、14d是表达增加,14d达高峰,28d下降(与前一时间点比较P<0.05)。缺血再灌注组GFAP和HMGB1表达具有相关性(P<0.05),存在HMGB1和GFAP共定位细胞。结论脑缺血再灌注后,海马CA1区HMGB1增加与星形胶质细胞激活成正相关,过度表达的HMGB1和增殖的星形胶质细胞可能与缺血再灌注后神经元的迟发性损伤有关。     

大鼠脑缺血后海马CA1区胶质纤维酸性蛋白表达与迟发性神经元死亡的关系

目的观察大鼠大脑缺血再灌注后海马CA1区胶质纤维酸性蛋白(GFAP)的表达与迟发性神经元死亡的关系。方法采用大鼠大脑中动脉阻塞再灌注模型(MCAO),将大鼠随机分为MCAO后3d、7d、30d组及假手术组,应用免疫荧光与TUNEL染色法分别观察脑缺血再灌注后不同时间点缺血侧海马CA1区GFAP表达情况和迟发性神经元死亡(DND)的变化。结果(1)3d组海马DND阳性(DND 组)的MCAO大鼠、海马DND阴性(DND-组)的MCAO大鼠与假手术组大鼠比较,缺血侧海马CA1区GFAP染色的平均光密度无显著性差异(P>0.05),但GFAP阳性细胞的形态发生变化;(2)7d组大鼠缺血侧海马CA1区GFAP阳性细胞大量活化增殖,表现为胞体变大,突起增多;DND( )、DND(-)组海马CA1区GFAP染色的平均光密度较假手术组增高(P<0.01),且DND(-)组的GFAP平均光密度较DND( )组明显增高(P<0.01);(3)30d组大鼠缺血侧海马CA1区GFAP表达呈瘢痕样改变,DND( )、DND(-)组与假手术组比较其GFAP染色的平均光密度明显增高(P<0.05),且DND( )组的GFAP平均光密度较DND(-)组明显增高(P<0.05)。结论大鼠MCAO后星形胶质细胞反应性变化的差异可能与海马CA1区迟发性神经元死亡的发生有关。     

大鼠前脑缺血再灌注后GFAP、S-100表达的变化

目的 探讨胶质纤维酸性蛋白（GFAP）和S-100蛋白在大鼠前脑缺血再灌注后反应性星形胶质细胞的活化情况.方法 利用免疫组织化学方法检测前脑缺血再灌注模型的细胞活化情况.结果 脑缺血再灌注后第1d,顶叶皮层和海马可见少量GFAP阳性细胞表达 脑缺血再灌注第3d及第5d后GFAP阳性表达明显增加,并与对照组比较有统计学意义（P＜0.01）.S-100蛋白在脑缺血再灌注后第1d即有增加,并随着时间延长表达明显增强,各时间点与对照组有显著性差异（P＜0.01）.结论 脑缺血再灌注后GFAP、S-100蛋白表达增加,说明反应性星形胶质细胞的活化参与了脑缺血损伤后神经元的修复过程.     

反复前脑缺血大鼠脑白质区胶质纤维酸性蛋白表达变化

目的观察大鼠反复前脑缺血再灌注后不同脑白质区胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP）表达的变化，探讨其规律，为对脑缺血后星形胶质细胞的进一步研究提供实验依据。方法反复夹闭大鼠双侧颈总动脉制备前脑缺血再灌注模型，免疫组化法检测脑缺血再灌注后1周、2周、4周胼胝体、内囊和脑室周围GFAP的表达。结果缺血再灌注后，不同部位各时间点GFAP的表达均高于假手术组水平；在胼胝体、内囊GFAP的表达在1周时增加，2周时持续上升，4周时更明显；而脑室周围则在1周时上升，2周时达高峰，4周时回落但仍高于1周时的水平。结论反复前脑缺血后白质区GFAP表达明显升高，但不同脑区变化的规律和幅度略有差异，说明不同脑区对缺血的敏感性不同，星形胶质细胞的反应性略有差异。     

缺血预处理后海马CA1区反应性星形胶质细胞增生与迟发性神经元缺血耐受性的关系

目的 探讨缺血预处理后海马CA1区反应性星形胶质细胞增生与迟发性神经元缺血耐受性的关系。方法 实验动物被随机分为手术组、缺血组、预缺血组、预缺血后再缺血组。阴断沙土鼠双侧颈总动脉造成前脑缺血模型。采用细胞特异性抗原胶质纤维酸性蛋白（GFAP）免疫组化法标记星形胶质细胞。结果 预缺血后1－7天,海马CA1区GFAP阳性的星形胶质细胞数轻度增加,至28天时增生非常显著（P＜0．01）。预缺血后1－7天再缺血,海马CA1区存活正常神经元数逐渐下降,预缺血后28天再缺血又显著增加（P＜0．01）。结论 缺血预处理后,神经元可出现迟发性缺血耐受,反应性星形胶质细胞增生可能起了重要作用。     

腺苷预处理对脑缺血再灌注脑内星形胶质细胞的影响

目的 探讨腺苷预处理对脑缺血再灌注损伤脑内星形胶质细胞的影响.方法 制作大鼠脑缺血再灌注损伤模型.60只SD大鼠随机分为3组:假手术组(F组)、缺血再灌注组(IR组)、腺苷预处理组(AP组),再按缺血再灌注后不同时间把各组随机分成4个亚组,每组5只大鼠.应用Zeal Longa 5级评分法进行神经功能评分,并通过免疫组织化学法检测脑组织内胶质纤维酸性蛋白(glial fibrillary acid protein,GFAP)的表达.结果 (1)神经功能评分AP组各亚组均小于IR组各亚组(P均＜0.05),但大于F组各亚组(P均＜0.05);(2)F组GFAP阳性表达均较弱,IR组和AP组在脑缺血再灌注后2h开始出现GFAP阳性表达的细胞数量增多,AP组在6h、24h AP组GFAP阳性表达比IR组增强(P均＜0.05),在72h时AP组GFAP阳性表达较IR组减少(P＜0.05).结论 腺苷预处理能在大鼠局灶性脑缺血再灌注损伤早期阶段促进GFAP的表达,72h后抑制GFAP的过度表达.     

脑室注射痴呆大鼠海马胶质纤维酸性蛋白表达与学习记忆能力的关系

目的探讨星形胶质细胞在老年性痴呆大鼠海马中的表达与老年性痴呆大鼠学习记忆能力减退的关系。方法雄性SD大鼠20只,随机分为痴呆组与假手术组;用Morris水迷宫检测大鼠的学习、记忆能力;用免疫组化技术定量检测大鼠海马CA1区胶质纤维酸性蛋白(GFAP)的表达;分析海马星形胶质细胞变化与学习、记忆能力的关系。结果假手术组海马CA1区锥体细胞排列紧密有序,细胞核大而圆、染色浅、核仁明显、未见明显胞浆浓染、核固缩等神经元变性受损征象。而痴呆大鼠海马CA1锥体神经细胞排列疏松、数目减少、细胞形态异常,许多细胞出现体积缩小、核浓染、核固缩;痴呆鼠海马CA1区GFAP阳性细胞数目明显增多,胞体肥大,突起增粗、变长现象明显,而假手术组海马CA1区仅见少量GFAP阳性细胞,突起较少、短,染色较淡。计数和测量海马CA1区GFAP阳性细胞数目、总面积、平均光密度,痴呆组与假手术组相比均明显增加,有显著意义(P<0.05);水迷宫测试显示痴呆组大鼠隐藏平台获得时间比假手术组明显延长,空间探索时间明显缩短,具有显著意义(P<0.05);显示痴呆组大鼠学习记忆能力与假手术组比较均明显下降;将痴呆大鼠学习成绩与海马CA1区GFAP表达数目之间进行相关分析,两者间存在负相关关系,认为星形胶质细胞参与了学习记忆过程。结论提示海马星形胶质细胞的过度表达可能影响痴呆大鼠的学习记忆能力。     

脑缺血再灌注损伤后GAP-43蛋白的表达和意义

目的 探讨脑缺血再灌注损伤后生长相关蛋白-43（GAP-43）的表达对神经元轴突再生的可塑性变化.方法 成年健康雄性Wistar大鼠40只,随机分为正常对照组、假手术组和缺血1h再灌注2h、6h、12h、24h、48h、3d、7d、14d组,每组各4只（n=4）.应用线栓法制备大鼠脑中动脉闭塞再灌注模型（MCAO）,采用免疫组织化学方法检测GAP-43的表达并观察神经元轴突再生的变化,并进行计算机图像分析.结果 缺血再灌注2h,海马、皮质区及纹状体区GAP-43呈基础表达,6h、12h、24h、48h表达逐渐增高,7d达高峰,P＜0.05,14d达最低表达,P＜0.05.与假手术组比较有显著性差异,P＜0.05.正常对照组无表达.缺血再灌注48h～7d损伤区域神经元轴突呈出芽征,发出突触纤维.结论 脑缺血再灌注损伤后GAP-43呈非特异性表达,并促进神经元的修复和再生.     

一氧化氮供体及前体对大鼠脑缺血再灌注损伤保护机制的探讨

目的 观察介入给药一氧化氮（NO）供体硝酸甘油（Nitroglycerine，NG）及前体L-精氨酸（L-Arginine，ARG）对大鼠脑缺血再灌注后海马区星形胶质细胞表达的胶质纤维酸性蛋白（GFAP）的影响，探讨NG及ARG的脑保护机制。方法 采用大鼠大脑中动脉阻塞（MCAO）法建立局灶性脑缺血模型。将大鼠随机分为假手术组、MCAO组、NG组和ARG组。MCAO组、NG组和ARG组于缺血2 h再灌注同时分别局部介入给予生理盐水、NG和ARG，于再灌注3 h或24 h时，荧光法检测血清NO含量。并在3 h或24 h时处死大鼠，病理分析脑梗死体积以及免疫组织化学法检测海马区GFAP表达情况。结果 缺血再灌注后3 h血清NO升高（P <0.01），治疗组较MCAO组明显（P <0.01），GFAP表达阳性细胞数增加，但治疗组较MCAO组减少（P <0.01），各组大鼠脑组织未出现肉眼可见梗死灶；缺血再灌注后24 h，血清NO治疗组较3 h降低，而MCAO组较3 h升高（P <0.05），GFAP表达阳性细胞数较3 h增加（P <0.01），治疗组较MCAO组减少（P <0.01），TTC染色显示脑梗死体积治疗组较MCAO组减小（P <0.05）。结论 脑缺血再灌注后海马区脑组织GFAP表达增强，通过局部介入给予NG、ARG增加NO合成，抑制GFAP高表达，减小脑梗死体积。提示NG、ARG抗脑缺血性损伤的保护机制可能与抑制星形胶质细胞过度表达有关。     

银杏叶提取物对大鼠脑缺血再灌注后GFAP和细胞凋亡的影响

目的 研究银杏叶提取物(EGb761)对大鼠局灶性脑缺血再灌注后神经细胞凋亡和星形胶质细胞表达的变化.方法 雄性SD大鼠随机分为假手术组、单纯缺血组、EGb761干预组,线栓法建立大脑中动脉缺血再灌注模型(MCAO),原位末端标记(TUNEL)法观察神经细胞的凋亡情况,免疫组化法观测星形胶质细胞的表达.结果 与单纯缺血...     

Expression changes of growth-associated protein-43 (GAP-43) and mitogen-activated protein kinase phosphatase-1 (MKP-1) and in hippocampus of streptozotocin-induced diabetic cognitive impairment rats

Diabetes mellitus (DM) may give rise to cognitive impairment, but the pathological mechanism involved was still unknown. We employed streptozotocin (STZ)-induced diabetic rats and test their capacity for learning and memory by three-arm radial maze. We determined the expression level of growth-associated protein-43 (GAP-43) and mitogen activated protein kinase phosphatase-1 (MKP-1) in the hippocampus by immunohistochemistry. MKP-1 mRNA level in the CA1 and dentate gyrus (DG) Hippocampal area is further determined by RT-PCR method. We also observed the ultrastructures of Hippocampal neurons by transmission electron microscopy (TEM). All data were analyzed by the independent samples t-test. Four weeks after STZ induction, the diabetic rats showed decreased capacity for learning and memory as indicated by the increase in the error number and reaction time in three-arm radial maze test. TEM results showed the ultrastructures of diabetic hippocampus, including area CA1 and DG, neurons were characterized by swollen mitochondria, increased heterochromatin accumulation and reduced synaptic contacts. The optical density as well as the positive neuron number for GAP-43 and MKP-1 decreased significantly in the CA1 and DG Hippocampal area in diabetic rats (P<0.01). RT-PCR results also showed MKP-1 mRNA in the CA1 and DG Hippocampal area was decreased in the diabetic rats. These results indicated that DM could down-regulate GAP-43 and MKP-1 expression in Hippocampal area that is in charge of memory and cognition. As indicated by our study, the changes in GAP-43 and MKP-1 expression in hippocampus may play a role in the pathogenesis of diabetic dementia.     

Growth-associated protein GAP-43 in the frontal cortex and in the hippocampus in Alzheimer's disease: an immunohistochemical and quantitative study

Summary. We studied the growth-associated protein, GAP-43 (also called neuromodulin and B-50) in post-mortem brain tissue using immunohistochemistry and quantitative Western blotting, from patients with Alzheimer's disease (AD) and age-matched control subjects. By immunohistochemistry, we found a clear reduction of GAP-43 in the frontal cortex, while in the hippocampus, there was a marked reduction in some areas (dentate molecular layer, stratum moleculare and radiale of CA1 and CA4), while not in other areas (stratum lacunosum, pyramidale and oriens of CA1). Moreover, in the hippocampus, neuritic staining was prominent, and was often associated with senile plaques. Quantitative analysis showed that GAP-43 was significantly reduced in AD, both in the frontal cortex (70% of the control value, p < 0.01) and in the hippocampus (81% of the control value, p < 0.05). In the frontal cortex, there was a significant negative correlation between GAP-43 and duration of dementia (r = −0.58; p < 0.02) and a positive correlation between GAP-43 and the synaptic vesicle-specific protein rab3a (r = 0.62; p < 0.05), while no such correlation were found in the hippocampus. In contrast, a significant positive correlation was found between GAP-43 and the number of senile plaques in the hippocampus (r = 0.64; p < 0.05), but not in the frontal cortex. GAP-43 is known to be involved in maintenance of synapses and in neuritic regeneration. Our findings may suggest that in the frontal cortex, GAP-43 levels decline as a consequence of the synaptic degeneration, while in the hippocampus, sprouting processes, involving GAP-43, are active. Received March 29, 1999; accepted September 2, 1999     

大鼠杏仁核电刺激癫痫持续状态后颞叶癫痫模型海马区神经生长相关蛋白43的表达

目的 探讨神经生长相关蛋白43(GAP-43)在大鼠杏仁核电刺激癫痫持续状态后颞叶癫痫(SE)模型海马区的表达及意义.方法 健康雄性Wistar大鼠60只,分成4组:空白对照组、未刺激组、未发作组和发作组.空白对照组:不植入电极；未刺激组:植入电极未行电刺激；发作组:植入电极电刺激后15d内、30 d内能观察到稳定的自发性反复发作(SRS)的大鼠归为发作组,其余归为未发作组.分别在15d、30 d时,将标本应用RT-PCR及免疫荧光检测方法检测大鼠海马GAP-43的表达.结果 致痫15d,发作组、未发作组和未刺激组的GAP-43表达高于空白对照组,并依次降低(P＜0.05).致痫30 d,发作组和未发作组GAP-43表达仍高于空白对照组,差异有统计学意义(P＜0.05)；未刺激组与空白对照组水平相当,二者差异无统计学意义(P＞0.05).结论 GAP-43参与了神经元损伤修复和突触重塑,其可能是颞叶癫痫的病理基础——突触重塑的重要分子机制.     

大鼠全脑缺血再灌注损伤后海马自噬相关蛋白Beclin 1的表达变化

目的研究自噬相关蛋白Beclin 1在大鼠全脑缺血-再灌注损伤后海马中的表达情况,并探讨其意义。方法使用四动脉结扎法制作大鼠全脑缺血-再灌注损伤模型,实验动物随机分为：假于术组和缺血再灌注组。存全脑缺血15min后,分别再灌注0min、30min、3h、6h、12h、24h、1d、3d,使用Western blot检测各个时阃点大鼠全脑缺血-再灌后海马自噬相关蛋白Beclin1的表达情况。结果与假手术组比较,大鼠全脑缺血-再灌注损伤后1d,海马区Beclinl蛋白的表达最强（P〈0．05）。结论大鼠全脑缺血-再灌注损伤后海马自噬相关蛋白Beclinl表达上调,表明脑缺血-再灌注损伤后海马区域的自噬活性上调．     

脑出血大鼠脑内嗅鞘细胞移植后生长相关蛋白-43及层粘连蛋白的表达变化

目的研究嗅鞘细胞移植对大鼠脑出血后脑内生长相关蛋白-43及层粘连蛋白表达的影响。方法取新生3d的Wistar乳鼠嗅球,差速贴壁法培养获得嗅鞘细胞。40只Wistar大鼠造模后(胶原酶Ⅶ注入尾状核)随机分为嗅鞘细胞移植组和脑出血模型组。嗅鞘细胞移植组(20只)术后3d,移植嗅鞘细胞;脑出血模型组(20只)注射等量的生理盐水。两组大鼠分别在术后1d、7d、14d及28d用Bederson方法进行评分。各时间点于每组随机取3只大鼠处死,取脑组织做成石蜡切片,免疫组化法观察生长相关蛋白-43及层粘连蛋白表达的变化。结果运动功能评分显示,移植组与对照组均出现运动功能恢复,移植组明显优于对照组(P<0.05);生长相关蛋白-43及层粘连蛋白免疫组化结果表明,除术前1d外,其它各时间点生长相关蛋白-43及层粘连蛋白阳性表达值,移植组较对照组均强,差异有统计学意义(P<0.05)。结论嗅鞘细胞移植可提高生长相关蛋白-43及层粘连蛋白的表达,改善脑出血后神经再生微环境,促进神经再生。     

Dopaminergic DA1 signaling couples growth-associated protein-43 and long-term potentiation in guinea pig hippocampus

The basic goal of the project was to determine whether dopaminergic DA1 receptor (DA1R) signaling couples growth-associated protein 43 (GAP-43; a putative "plasticity" protein) and long-term potentiation (LTP; an enduring form of synaptic plasticity). Thus, guinea pigs were prepped to stimulate the CA3 and evoke population spikes in the CA1 neurons in the hippocampus in vivo. Animals were injected with either saline or SCH23390 (a selective DA1R antagonist), 1-2 h prior to recordings. It was found that tetanic stimulation (100 Hz, 1 s, three trains at 15 s intervals) readily produced early-LTP and late-LTP in the saline group. In contrast, none of the guinea pigs pre-treated with SCH23390 developed late-LTP, though early-LTP had been present. Furthermore, both GAP-43 mRNA and protein were up-regulated after LTP induction in the saline group. However, GAP-43 protein up-regulation was blocked in animals treated with SCH23390. Anti-GAP-43 immunoreactivity was intense in CA3/CA1 synaptic regions, whereas GAP-43 mRNA hybridization was localized to somatic layers in the hippocampus. Altogether, our results suggest that dopaminergic DA1 signaling partly couples GAP-43 and LTP.     

Expression of GAP-43 in the Granule Cells of Rat Hippocampus After Seizure-induced Sprouting of Mossy Fibres: In Situ Hybridization and Immunocytochemical Studies

The axonal growth-associated protein GAP-43 is believed to play some role in the synaptic remodelling that takes place in the hippocampus of adult rats after certain experimental lesions. GAP-43 mRNA is highly expressed in adult CA3 pyramidal cells but almost absent in the dentate granule cells. We analysed whether the sprouting of granule cell axons, the mossy fibres of the hippocampus, caused by kainic acid-induced seizures in adult rats was associated with any induction of GAP-43 mRNA in granule cells and with any changes in the immunostaining pattern of GAP-43 in the hippocampus. Increased GAP-43 mRNA expression was found to be induced in granule cells 18, 24 and 30 h after a systemic injection of kainic acid which induced generalized seizures in adult rats, and returned to control levels by 48 h post-treatment. No effect was observed in other regions of the hippocampus. However, when kainic acid was injected into 15-day-old rats, which responded with generalized seizures but no sprouting of mossy fibres, there was no induction of GAP-43 mRNA in the granule cells, suggesting a close relation between GAP-43 expression and sprouting of these cells. Seven days after kainic acid injections, GAP-43 immunostaining was decreased in the inner molecular layer of the dentate gyrus except for a thin supragranular band, whereas 30 days after treatment all animals showed increased GAP-43 immunoreactivity in the whole inner molecular layer. Since collaterals of mossy fibres grow in the inner molecular layer after kainic acid-induced seizures, these results support the theory that GAP-43 plays a role in synaptic remodelling in the adult central nervous system.