七叶皂苷钠对脑缺血再灌注损伤大鼠MG、iNOS表达的影响

目的观察七叶皂苷钠对大鼠脑缺血再灌注损伤后小胶质细胞(MG)及诱导型一氧化氮合酶(iNOS)表达的影响,探讨其脑保护作用的可能机制。方法将SD大鼠随机分为假手术组、对照组、七叶皂苷钠组。改良线栓法制备建立局灶性脑缺血-再灌注动物模型(MCAO模型),分别于手术后1d、3d、7d免疫组化法观察大鼠海马一区OX-42及iNOS的表达情况。结果对照组、七叶皂苷钠组3d时OX-42达峰,7d时明显降低;iNOS 1d达峰,3d明显下降,7d时接近正常。七叶皂苷钠组优于对照组(P<0.05)。结论七叶皂苷钠可能通过抑制小胶质细胞的活化和iNOS的表达,对大鼠脑缺血再灌注损伤发挥脑保护作用。     

环孢素A对大鼠急性脑缺血再灌注损伤小胶质细胞的活化及iNOS表达的影响

目的 观察环孢素A(Cyclosporin,CSA)对大鼠脑缺血再灌注损伤后小胶质细胞(microglia,MG)活化及诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS)表达的影响,探讨其脑保护作用的可能机制.方法 将SD大鼠随机分为假手术组、模型组、环孢素A组.改良线栓法制备建立局灶性脑缺血-再灌注动物模型,即大脑中动脉闭塞(MCAO)模型,分别于手术后1 d,3 d,7 d,14 d免疫组化法观察大鼠海马一区OX-42及iNOS的表达情况,同时对大鼠神经行为学进行评分.结果 模型组、环孢素组1 d即有OX-42的大量表达,3 d时OX-42达到高峰,14 d时接近正常;iNOS 1 d达峰,7 d及14 d时接近正常.环孢素组OX-42、iNOS较模型组减少(P<0.05),且神经行为学评分优于模型组(P<0.05).结论 小胶质细胞的激活及iNOS大量表达和脑缺血-再灌注损伤密切相关,环孢素A可显著减少脑缺血再灌注损伤后小胶质细胞的激活和iNOS的表达,对大鼠脑缺血再灌注损伤有一定的保护作用.     

大鼠坐骨神经切断后腰脊髓腹角内胶质细胞和神经元的可塑性变化

目的：探讨大鼠单侧坐骨神经切断后腰脊髓腹角胶质细胞和运动神经元的反应及其相互关系。方法：用免疫组织化学技术、HE染色和Tunnel法，观察坐骨神经切断后1，6，12，24h及3，7和14d腰脊髓腹角胶质原纤维酸性蛋白(GFAP)标记的星形胶质细胞、OX-42标记的小胶质细胞及运动神经元的变化。结果：坐骨神经切断侧腰脊髓腹角可见星形胶质细胞和小胶质细胞活化，星形胶质细胞的活化早于小胶质细胞；后期运动神经元发生凋亡，HE染色显示凋亡细胞周围为反应性OX-42阳性小胶质细胞和GFAP阳性星形胶质细胞包绕。结论：研究结果提示，坐骨神经切断后切断侧腰脊髓腹角活化的胶质细胞与凋亡的运动神经元之间关系密切。     

大鼠坐骨神经切断后腰脊髓腹角内胶质细胞和神经元的可塑性变化

目的:探讨大鼠单侧坐骨神经切断后腰脊髓腹角胶质细胞和运动神经元的反应及其相互关系.方法:用免疫组织化学技术、HE染色和Tunnel法,观察坐骨神经切断后1,6,12,24 h及3,7和14 d腰脊髓腹角胶质原纤维酸性蛋白(GFAP)标记的星形胶质细胞、OX-42标记的小胶质细胞及运动神经元的变化.结果:坐骨神经切断侧腰脊髓腹角可见星形胶质细胞和小胶质细胞活化,星形胶质细胞的活化早于小胶质细胞;后期运动神经元发生凋亡,HE染色显示凋亡细胞周围为反应性OX-42阳性小胶质细胞和GFAP阳性星形胶质细胞包绕.结论:研究结果提示,坐骨神经切断后切断侧腰脊髓腹角活化的胶质细胞与凋亡的运动神经元之间关系密切.     

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。     

星形胶质细胞及其炎症因子在CO中毒所致迟发性脑损伤中的作用

目的 探讨大鼠海马组织星形胶质细胞活化水平及炎症因子表达在急性一氧化碳中毒迟发性脑病(DEACMP)中的作用及可能的机制。方法 经Morris水迷宫训练筛选认知功能正常的SD雄性大鼠,随机分为实验组,对照组,实验组大鼠采用静态吸入法置备DEACMP模型。分别于中毒后7d、14d、21d各时间点通过Morris水迷宫比较两组学习记忆能力;苏木精-伊红染色观察海马病理损伤程度;免疫荧光法和Western blot法检测海马区星形胶质细胞特异性标志物(GFAP)的表达情况;ELSIA法检测海马组织中TNF-α、IL-1β、IL-6表达水平。结果①大鼠表现出典型的CO中毒症状;②Morris水迷宫结果表明,实验组第7天大鼠水迷宫逃避潜伏期无明显差异(P>0.05);实验组14d、21d水迷宫逃避潜伏期逐渐延长,与对照组相比差异有统计学意义(t=2.60、t=5.56,P<0.05);③苏木精-伊红染色与对照组相比,实验组海马细胞出现不同程度的细胞损伤;④免疫荧光结果统计显示,实验组第7天、14天、21天的海马CA1区GFAP表达明显增多,与对照组相比差异有统计学意义(t=-5.20...     

巢蛋白阳性星形胶质细胞与癫痫持续状态后海马区胶质瘢痕形成的相关性研究

目的 了解癩痫持续状态(SE)后7d海马各区巢蛋白(nestin)阳性星形胶质细胞表达及数目的变化,探讨其与SE导致海马区胶质瘢痕形成的关系.方法 SD大鼠随机分为SE组和对照组,应用匹罗卡品建立SE大鼠模型;在SE后7d处死大鼠行盲法免疫荧光组化实验,观察大鼠海马各区nestin与胶质纤维酸性蛋白单克隆抗体(GFAP...     

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。     

rTMS促进大鼠局灶性脑缺血海马内源性神经干细胞分化的研究

目的探讨重复经颅磁刺激（rTMS）对局灶性脑缺血大鼠海马内源性神经干细胞分化的影响。方法线栓法制备大鼠大脑中动脉闭塞（MCAO）模型,随机分为脑缺血自然恢复组和rTMS治疗组,用荧光显微镜和共聚焦显微镜观察缺血14d、28d后各组大鼠海马中5-溴脱氧尿核苷（BrdU）与神经元特异核蛋白（NeuN）、神经胶质酸性蛋白（GFAP）共同标记的阳性细胞,并在高倍荧光显微镜下对双标阳性细胞计数。结果脑缺血后14d、28d,rTMS治疗组大鼠海马BrdU／NeuN双标阳性细胞数量分别为17．12±2．91、23．20±5．97,较相应自然恢复组12．96±2．79、15．92±2．52明显增加,两组同一时间点组间比较有统计学差异（P〈0．01）。而脑缺血后14d、28d,rTMS治疗组大鼠海马BrdU／GFAP双标阳性细胞数量分别为30．48±4．58、36．48±4．90,较相应自然恢复组37．44±3．58、43．60±5．96减少,两者相比有统计学差异（P〈0．05）。结论局灶性脑缺血大鼠海马增殖的内源性神经干细胞,可分化为神经元或神经胶质细胞,而rTMS可促进海马内源性神经干细胞向神经元的分化。     

16Hz次声作用对大鼠海马TRPV4、GFAP和fos蛋白表达的影响

目的研究16Hz，90dB和130dB次声作用后，大鼠海马瞬时感受电位香草酸家族4（TRPV4）通道蛋白、胶质纤维酸性蛋白（GFAP）和fos蛋白的表达情况。方法16Hz，90dB和130dB次声作用于大鼠，2h／d，作用7d后采用免疫组织化学染色方法，观察大鼠海马中TRPV4蛋白、GFAP和fos蛋白表达的情况。结果16Hz，130dB次声作用7d后，与对照组相比较大鼠海马中显著表达TRPV4阳性神经元，GFAP阳性星形胶质细胞和fos阳性神经元（P〈0．05），三者分布一致，关系密切；90dB组大鼠的上述三种蛋白表达均较130dB组弱（P〈0．05）。结论16Hz，90dB和130dB次声作用可以引起大鼠海马TRPV4阳性细胞表达增多，且能够激活神经元和星形胶质细胞。     

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。     

Protective role of melatonin in domoic acid-induced neuronal damage in the hippocampus of adult rats

Domoic acid (DA), a kainite-receptor agonist and potent inducer of neurotoxicity, has been administered intravenously in adult rats in the present study (0.75 mg/kg body weight) to demonstrate neuronal degeneration followed by glial activation and their involvement with inducible nitric oxide synthase (iNOS) in the hippocampus. An equal volume of normal saline was administered in control rats. The pineal hormone melatonin, which protects the neurons efficiently against excitotoxicity mediated by sensitive glutamate receptor, was administered intraperitoneally (10 mg/kg body weight), 20 min before, immediately after, and 1 h and 2 h after the DA administration, to demonstrate its role in therapeutic strategy. Histopathological analysis (Nissl staining) demonstrated extensive neuronal damage in the pyramidal neurons of CA1, CA3 subfields and hilus of the dentate gyrus (DG) in the hippocampus at 5 days after DA administration. Sparsely distributed glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes were observed in the hippocampus at 4-24 h after DA administration and in the control rats. Astrogliosis was evidenced by increased GFAP immunoreactivity in the areas of severe neuronal degeneration at 5 days after DA administration. Along with this, microglial cells exhibited an intense immunoreaction with OX-42, indicating upregulation of complement type 3 receptors (CR3). Ultrastructural study revealed swollen or shrunken degenerating neurons in the CA1, CA3 subfields and hilus of the DG and hypertrophied astrocytes showing accumulation of intermediate filament bundles in the cytoplasm were observed after administration of DA. Although no significant change could be observed in the mRNA level of iNOS expression between the DA-treated rats and controls at 4-24 h and at 5-day time intervals, double immunofluorescense revealed co-expression of induced iNOS with GFAP immunoreactive astrocytes, but not in the microglial cells, and iNOS expression in the neurons of the hippocampal subfields at 5 days after DA administration. Expression of iNOS was not observed in the hippocampus of control rats. DA-induced neuronal death, glial activation, and iNOS protein expression were attenuated significantly by melatonin treatment and were comparable to the control groups. The results of the present study suggest that melatonin holds potential for the treatment of pathologies associated with DA-induced brain damage. It is speculated that astrogliosis and induction of iNOS protein expression in the neurons and astrocytes of the hippocampus may be in response to DA-induced neuronal degeneration.     

Enhanced neurogenesis during trimethyltin-induced neurodegeneration in the hippocampus of the adult rat

The occurrence of neurogenesis in the hippocampus of the adult rat during trimethyltin (TMT)-induced neurodegeneration was investigated using bromodeoxyuridine (BrdU). Fifteen days after TMT intoxication, BrdU-labeled cells were significantly more numerous in the hippocampus of treated animals, gradually decreasing towards the control value 21 days after intoxication in the dentate gyrus (DG), while in the CA3/hilus region BrdU-labeled cells were still more numerous in TMT-treated rats. In order to investigate the fate of newly-generated cells double labeling experiments using neuronal or glial markers were performed. Colocalization of the neuronal marker NeuN was detected in many BrdU-positive cells in the DG, while in the CA3/hilus region no colocalization of NeuN and BrdU could be observed. No colocalization of BrdU and the astroglial marker GFAP or the microglial marker OX-42 was detected either in the DG and or in the CA3/hilus region. The results indicate an enhancement of endogenous neurogenesis in the hippocampus during TMT-induced neurodegeneration, with the development of a subpopulation of regenerated cells into neurons in the DG, while in the CA3/hilus region the population of newly-generated cells should be regarded as undifferentiated.     

单侧液压脑损伤对大鼠双侧海马GFAP表达和CA1区突触传递的影响

目的研究单侧液压脑损伤(FPI)对大鼠双侧海马区胶质纤维酸性蛋白(GFAP)表达和CA1区突触传递的影响。方法建立大鼠单侧液压脑损伤模型,脑标本分为对照组(包括正常对照和假手术对照)、FPI损伤同侧组和FPI损伤对侧组。免疫组化法检测海马水平切片GFAP表达,对海马CA1区锥体神经元进行细胞内记录。结果FPI大鼠双侧海马齿状回门区和CA1区GFAP表达均比对照组明显增强。FPI损伤同侧组兴奋性输入-输出关系曲线的斜率比其他两组显著增大(P<0.05);FPI损伤同侧组和对侧组双脉冲易化(PPF)比值和抑制性突触后电位(IPSP)幅值均比对照组显著减小(P<0.05);FPI损伤同侧组和对侧组双脉冲抑制(PPD)比值均比对照组显著增大(P<0.05)。结论大鼠单侧液压脑损伤对双侧海马均可产生影响,导致双侧海马CA1区兴奋性突触传递增强,抑制性突触传递减弱。     

Spreading depression and reverberatory seizures induce the upregulation of mRNA for glial fibrillary acidic protein

The present study evaluates the relative roles of seizure activity and spreading depression in upregulating glial fibrillary acidic protein (GFAP) mRNA expression. Stimulating electrodes were placed bilaterally in the angular bundle, and recording electrodes were placed bilaterally in the dentate gyrus of adult rats. Intense electrographic seizures were induced by delivering stimulus trains through one stimulating electrode. In some cases, spreading depression accompanied the seizures, while in other cases the seizures occured in the absence of spreading depression. Animals were killed 24 h following the last stimulus train, and the forebrains were prepared for quantitative in situ hybridization. Seizure activity and spreading depression led to significant increases in GFAP mRNA levels in the hippocampal formation. Seizure activity alone (without spreading depression) induced a 4-fold increase in GFAP mRNA levels in the hilus and molecular layer of the dentate gyrus and in stratum lacunosum-moleculare of the hippocampus. When seizure activity was accompanied by spreading depression, there was a 10-fold increase in GFAP mRNA levels in these same regions. Regional differences within the hippocampal formation in glial cell response were evident. While GFAP mRNA levels in stratum lacunosum-moleculare of the hippocampus were upregulated by seizure activity and spreading depression, levels in hippocampal stratum radiatum of the hippocampus remained unchanged. The results suggest that abnormal neuronal activity can influence glial cell gene expression and that spreading depression is a stronger signal than seizure activity in upregulating GFAP mRNA levels.     

Effects of age on gene expression during estrogen-induced synaptic sprouting in the female rat

Age and estrogen treatment influenced fiber outgrowth and compensatory neuronal sprouting after unilateral entorhinal cortex lesions (ECL) which model Alzheimer disease-like deafferentation in the dentate gyrus of the hippocampus. In young F344 rats (3 months old), ovariectomy (OVX) decreased reactive fiber outgrowth by 60%. Sprouting in middle-aged rats (18 months old) was reduced in intact females; no further reduction was caused by OVX. Several astrocyte mRNAs were measured in the dentate gyrus of young and middle-aged female rats in three different estrogen states (sham OVX, OVX, or OVX + estradiol) 1 week after ECL. Glial fibrillary acidic protein (GFAP) mRNA was twofold greater in middle-aged rats than young, although both ages showed threefold increases in response to ECL. In prior studies GFAP was found to be decreased by estradiol treatment 3-4 days after ECL; in this study GFAP mRNA had returned to sham OVX levels in young rats by 7 days post-ECL. Surprisingly, estradiol treatment increased GFAP mRNA levels by 25% above OVX in middle-aged rats. Apolipoprotein E (apoE) mRNA was decreased 20% by age in the dentate, although both age groups showed a 25% increase in apoE mRNA in response to ECL. Apolipoprotein J (apoJ) mRNA was increased 20% in the dentate gyrus of middle-aged rats, and both age groups responded to ECL with a 65% increase in apoJ mRNA. The estrogen state did not alter levels of either apolipoprotein mRNA in the deafferented dentate. The data suggest that the estrogen-induced decrease of GFAP in response to lesions does not persist at 7 days post-ECL during sprouting. Overall effects of age on the dentate gyrus include elevated GFAP mRNA and decreased apoE mRNA. The cortical wound site showed consistent enhancement of GFAP mRNA in both age groups by estradiol above sham OVX and greater responses in middle-aged rats.     

Safety aspects of chronic low-frequency transcranial magnetic stimulation based on localized proton magnetic resonance spectroscopy and histology of the rat brain

Because repetitive transcranial magnetic stimulation (rTMS) is capable of inducing lasting alterations of cortical excitability, it represents a promising therapeutic tool in several neuropsychiatric disorders. However, rTMS, especially when applied chronically, may cause harmful effects in the stimulated tissue. To study the safety of chronic rTMS we used a novel small stimulation coil, which was specially designed to treat rats, and investigated brain tissue using in vivo localized proton magnetic resonance spectroscopy (MRS) and post mortem histological analysis. Histology was based on a modified stereology method in combination with immunohistochemistry applying antibodies against OX-6, OX-42, ED, and GFAP to detect any microglial and/or astrocytic activation 48 h after the last TMS session. Conscious rats were treated with a daily suprathreshold rTMS regimen of 1000 stimuli applied on 5 consecutive days at a frequency of 1 Hz. In comparison with control animals receiving magnetic stimulation over the lumbar spine, quantitative evaluations of cerebral metabolite concentrations by proton MRS revealed no significant alterations of N-acetyl-aspartate, creatine and phosphocreatine, choline-containing compounds, myo-inositol, glucose and lactate after chronic rTMS. Similarly to the in vivo results, post mortem histology revealed no changes in microglial and astrocytic activation after rTMS. In conclusion, these data provide support for the safety of chronic rTMS. However, they do not exclude acute changes on neurotransmitters systems or other physiologic responses during or directly after the rTMS treatment.     

Differential expression of S100beta and glial fibrillary acidic protein in the hippocampus after kainic acid-induced lesions and mossy fiber sprouting in adult rat

The expression of S100beta and glial fibrillary acidic protein (GFAP) was analyzed following bilateral injection of kainic acid (KA), a glutamate derivative, into the CA3 region of the adult rat hippocampus. This treatment produces a progressive degeneration of the pyramidal neurons of the hippocampus while sparing the granule cells of the dentate gyrus which undergo sprouting of their axons in the supragranular layer. Messenger RNA and protein levels were measured, by Northern blot and ELISA, in the hippocampus of lesioned and sham-operated rats 1, 7, and 30 days after KA injection. A significant increase of GFAP and its mRNA was demonstrated at each time point, whereas S100beta mRNA levels were significantly enhanced only 30 days after the KA injection and the levels of S100beta protein remained unchanged at all time points. However, when analyzed by immunohistochemistry the S100beta showed clear changes in its expression and distribution depending on the region considered. One month after KA injection, S100beta immunoreactivity was considerably reduced in the stratum radiatum of CA3 region, but there was increased S100beta immunoreactivity in the stratum moleculare. In particular, a notable band of S100beta positive, hypertrophic astrocytes appeared in the supragranular layer of the dentate gyrus where the sprouting of mossy fiber collaterals was detected by Timm's staining. These data show for the first time that an increase in S100beta expression in subpopulations of reactive astrocytes may be involved in the structural reorganization of the hippocampus following KA-induced neurodegeneration.     

Chronic repetitive transcranial magnetic stimulation increases hippocampal neurogenesis in rats

Aim: While the underlying therapeutic mechanisms of repetitive transcranial magnetic stimulation (rTMS) treatment for depression remain unclear, recent animal studies have suggested that hippocampal neurogenesis might be required for the effects of antidepressant treatments including antidepressant drugs and electroconvulsive therapy. The aim of this study was to examine chronic rTMS effects on hippocampal neurogenesis in rats. Methods: Using a 70‐mm figure‐of‐eight coil, the stimulating parameters were set to 25 Hz and 70% of the rTMS device's maximum power. For 14 consecutive days, bromodeoxyuridine (BrdU) and 1000 pulses of rTMS were administered daily. Cell proliferation in the dentate gyrus was examined with immunohistochemistry. Results: In the rTMS‐treated group, BrdU‐positive cells were significantly increased in the dentate gyrus. Conclusion: Our results suggest that hippocampal neurogenesis might be involved in the antidepressant effects of chronic rTMS.