产前应激对成年子代大鼠缺血性卒中后细胞凋亡的影响

目的:研究产前应激对雄性子代大鼠大脑中动脉缺血/再灌注后神经功能的影响。方法:SD孕鼠随机进行产前应激处理(孕期每日3次限制活动)和无产前应激处理,并对其雄性子代大鼠采用线栓法制备大脑中动脉局灶性脑缺血(MCAO)模型,共分为假手术组、产前应激+假手术组、MCAO模型组、产前应激+MCAO组(n=10)。于再灌注24 h后进行神经功能评分,并检测脑梗死面积、神经细胞凋亡情况和凋亡相关蛋白表达。结果:产前应激+MCAO组子代大鼠神经功能评分、脑梗死面积百分比、TUNEL阳性细胞、半胱氨酸天冬氨酸蛋白酶3(Caspase3)和活化的Caspase 3蛋白表达均较MCAO组显著增加(P0.05),而B淋巴细胞瘤-2(Bcl-2)蛋白表达较MCAO组减少(P0.05)。结论:产前应激可能通过促进子代大鼠脑缺血/再灌注后神经细胞凋亡,加重神经功能缺损。     

大鼠产前手机辐射对子代小脑白质的影响

目的:探讨大鼠产前850～1 900 MHz手机辐射对成年子代小脑白质的影响。方法:孕鼠随机分为短时产前手机辐射组、长时产前手机辐射组和对照组,短时和长时辐射组于孕期第1～17日分别进行每天6 h和24 h手机辐射,各组雄性子代大鼠(n＝8)于3月龄取小脑组织,进行苏木精-伊红(HE)染色观察小脑皮质细胞形态,免疫组化和Western blot检测髓鞘碱性蛋白(MBP)、神经微丝-L(NF-L)和胶质纤维酸性蛋白(GFAP)表达。结果:与对照组比较,短时程和长时程产前手机辐射组子代大鼠小脑浦肯野细胞出现形态学改变;与对照组比较,长时程辐射组MBP、NF-L表达明显减少(P均＜0.05),而GFAP表达明显增多(P＜0.05);与短时程辐射组比较,长时程组子代大鼠小脑MBP、NF-L表达明显减少(P均＜0.05),而GFAP表达明显增多(P＜0.05)。结论:产前手机辐射会导致雄性子代大鼠小脑髓鞘和轴突的损害,以及星形胶质细胞的活化,且这种改变与产前手机辐射的时程相关。     

异丙酚对局灶性脑缺血／再灌注星形胶质细胞GFAP表达的影响

目的：探讨异丙酚对局灶性脑缺血／再灌注后星形胶质细胞胶质纤维酸性蛋白（GFAP）表达的影响。方法：大脑中动脉插线法制作大鼠局灶性脑缺血／再灌注模型。观察脑缺血2h再灌注24h后神经功能损害改变并评分,并采用免疫荧光组织化学法检测大鼠齿状回GFAP蛋白的表达。结果：缺血／再灌注后可诱导大鼠齿状回GFAP表达明显增强,异丙酚可抑制缺血／再灌注后GFAP的表达,明显改善大鼠神经功能损害（P〈0．05或0.01）。结论：异丙酚通过抑制脑缺血后星形胶质细胞GFAP的过度表达发挥抗脑缺血损伤保护神经元作用。     

甘珀酸干预对大鼠脑缺血再灌注损伤的影响

目的观察缝隙连接阻断剂甘珀酸对局灶性脑缺血/再灌注损伤的影响。方法采用大鼠大脑中动脉阻塞再灌流模型（MCAO）,将动物随机分为脑缺血60min再灌注（MCAO）组,脑缺血再灌注加甘珀酸干预（MCAO＋CBX）组和假手术组（sham）。采用尼氏染色显示脑梗死灶并计算梗死灶体积;应用免疫荧光与TUNEL染色法分别观察脑缺血后3d与7d不同时间点缺血边缘区胶质纤维酸性蛋白（GFAP）的表达和细胞凋亡情况。结果（1）缺血后3d、7d MCAO＋CBX组大鼠梗死体积小于MCAO组,3d、7d MCAO＋CBX组大鼠梗死体积较MCAO组分别缩小5%和4.6%;（2）缺血后3d、7d于缺血边缘区可见大量TUNEL阳性染色细胞,且MCAO组大鼠缺血边缘区细胞凋亡数目明显多于MCAO＋CBX大鼠（P〈0.001）;（3）缺血后3d和7d组缺血边缘区GFAP表达明显增强,3d的MCAO组与MCAO＋CBX组大鼠缺血边缘区GFAP的表达均较假手术组强（P〈0.05）,7d的MCAO＋CBX组大鼠缺血边缘区GFAP的表达较假手术组强（P〈0.001）,但明显弱于MCAO组大鼠（P〈0.01）;结论缝隙连接阻断剂甘珀酸可减少大鼠大脑中动脉阻塞后脑梗死体积,其机制可能与阻断缝隙连接后缺血边缘区神经元凋亡降低有关,星型胶质细胞的反应性变化参与了该过程。     

大鼠脑缺血再灌注损伤后神经元和星形胶质细胞的动态变化及Cyclin D1表达的差异

目的研究大鼠局灶性脑缺血再灌注损伤后神经元和星形胶质细胞表达量的动态演变及各自cyclin D1的表达差异。方法建立大鼠大脑中动脉阻塞（MCAO）再灌注模型,随机分为再灌注后1d组,3d组,7d组,14d组和假手术组,应用流式细胞术检测各组再灌注后不同时间点神经元和星形胶质细胞数量变化及各自cyclin D1的表达。结果缺血侧梗死边缘区皮质星形胶质细胞的表达增加,而神经元的表达下降,与假手术组比较有明显差异（P〈0．05）;神经元和星形胶质细胞中各自cyclin D1的表达在再灌注7d、14d后表达上调,且星形胶质细胞中的cyclinD1增加更明显,与假手术组比较有统计学差异（P〈0．05）。结论大鼠脑缺血再灌注后,缺血侧梗死边缘区皮质星形胶质细胞和神经元的cyclinD1表达均有不同程度的上调,星形胶质细胞的cyclin D1表达上调比神经元的更为显著。     

成年大鼠吻侧迁移流的神经发生

目的探讨成年SD大鼠吻侧迁移流(rostral migratory stream,RMS)的神经发生。方法成年6周龄SD大鼠被处死。矢状位切片,免疫组织化学染色观察RMS区微管相关蛋白双皮质素(doublecortin,DCX)及胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)的表达及DCX/GFAP、DCX/p-CREB的共表达情况。结果在RMS区的垂直臂、肘部、水平臂均有DCX阳性细胞和GFAP阳性细胞,RMS区有GFAP/DCX和p-CREB/DCX共表达细胞。结论吻侧迁移流有广泛的神经元前体细胞及星形胶质细胞标记物表达;迁移神经元标记物可表达于星形胶质细胞,且神经元的迁移受到CREB信号通路的调控。     

Ephrin-A3逆向通路对海马星形胶质细胞谷氨酸摄取能力的影响

目的观察EphA4介导的ephrin-A3逆向通路激活对星形胶质细胞谷氨酸摄取能力的影响。方法采用原代培养的大鼠海马星形胶质细胞,使用免疫荧光双标法定位ephrin-A3在海马星形胶质细胞上的表达,Western blot法观察糖氧剥夺(oxygen-glucose deprivation,OGD)后星形胶质细胞ephrin-A3表达水平的变化,随后实验分为三组:空白对照组(不含星形胶质细胞),药物对照组(加入IgG-Fc)和EphA4组(加入ephrin-A3逆向通路激动剂预聚集化的EphA4-Fc),分别在正常及缺血条件下的特定时间点以谷氨酸浓度测定试剂盒检测不同干预组星形胶质细胞谷氨酸摄取能力的变化。结果 ephrin-A3高表达于海马星形胶质细胞,并在缺血后出现蛋白表达水平一过性上调。与对照组相比,EphA4干预组星形胶质细胞谷氨酸摄取能力较对照组明显下降。结论 Ephrin-A3高表达于海马星形胶质细胞并参与调节星形胶质细胞谷氨酸摄取能力。     

产前束缚应激子代大鼠海马神经颗粒素表达降低

神经颗粒素（neurogranin,NG）是脑特异性突触后蛋白,参与在学习记忆功能中起核心作用的信号转导通路及突触可塑性。本研究旨在探讨产前束缚应激对子代大鼠海马NG表达的影响。连续7d对孕晚期大鼠进行束缚应激,建立产前束缚应激模型,分为对照雌、雄组,应激雌、雄组。采用免疫组化方法观察NG在产前束缚应激子代大鼠海马不同亚区的分布特点;采用蛋白免疫印迹方法检测产前束缚应激子代大鼠海马NG蛋白的表达。结果显示：各组子代大鼠海马各区均有NG蛋白表达,CA1和CA3区表达高于齿状回（dentate gyrus,DG）;应激组雌、雄子代大鼠海马NG的表达明显低于对照组（P〈0．01）,应激组雌性子代比雄性子代减少更显著,对照组雌、雄子代之间无差异。免疫组化与蛋白免疫印迹方法所得结果一致。上述结果表明,NG在产前束缚应激子代大鼠海马表达降低,并且雌性比雄性降低明显,NG对产前束缚应激子代大鼠有差异性调制,NG表达减少可能与产前束缚应激子代大鼠学习记忆能力下降有关。     

大鼠局灶性脑缺血后星形胶质细胞过度增殖对局部微循环的影响

目的 观察局灶性脑缺血后海马和缺血边缘区星形胶质细胞过度增殖对微循环的影响。方法将大鼠随机分为假手术组、缺血组和干预组,缺血组和干预组采用线栓法制备大脑中动脉栓塞模型,缺血组侧脑室采用ALZET微渗透泵给予0．2％DMSO,干预组给予细胞周期抑制剂roscovitine,假手术组不插入线栓,不给与任何药物干预。缺血7d后于股静脉注入FITC标记的葡聚糖标记血管,小鼠抗大鼠单克隆抗胶质纤维酸性蛋白（glial fibrillary acidic protein,GFAP）抗体标记星形胶质细胞;激光共聚焦三维成像显示胶质细胞于微循环之间的关系。结果缺血同侧海马和缺血边缘区在GFAP阳性细胞增多的同时,局部微血管血流灌注明显减少,应用细胞周期抑制剂roscovitine抑制星形胶质细胞增生可以明显增加局部微血管的血流灌注。结论脑缺血后缺血边缘区和海马的反应性星形胶质细胞增生与微循环灌注减少密切相关。     

模拟高原低氧对大鼠脑内神经胶质细胞的影响

目的：观察模拟高原低氧对成年大鼠脑内胶质细胞的影响。方法：大鼠在模拟海拔4000m高原低压舱内连续暴露1、3、7d,胶质原纤维酸性蛋白（GFAP）与Griffoniasimplicifolia同功凝集素（GSA-IB4）组织细胞化学分别显示星形胶质细胞与小胶质细胞。结果：GFAP与GSA-IB4阳性细胞在低氧后3．7d两个时相显著增多,主要分布于新皮层、海马、纹状体及室管膜下层等区域。结论：模拟高原低氧能引起大鼠脑内星形胶质细胞与小胶质细胞的显著活化。     

Modulation of Behavioral Deficits and Neurodegeneration by Tannic Acid in Experimental Stroke Challenged Wistar Rats

Oxidative stress and inflammatory responses play a critical contributing factor in cerebral ischemia and reperfusion, which lead to lipid peroxidation and neuronal dysfunction that may represent a target for therapeutic intervention. The present study was aimed to elucidate the neuroprotective effect of tannic acid (TA), a natural polyphenol with potential antioxidant and antiinflammatory properties on middle cerebral artery occlusion (MCAO) model in rats. To test this hypothesis, male Wistar rats were pretreated with TA (50 mg/kg b.wt.) and then subjected to 2-h MCAO followed by 22 h of reperfusion. After 2-h MCAO/22-h reperfusion, neurological deficit, infarct sizes, activities of antioxidant enzymes, cytokine level, histology, and immunohistochemistry were used to analyze the expression of glial fibrillary acidic protein (GFAP) in ischemic brain. The pretreatment of TA showed a marked reduction in infarct size, improved neurological function, suppressed neuronal loss, and downregulated the GFAP expression in MCAO rats. A significantly depleted activity of antioxidant enzymes and content of glutathione in MCAO group were protected significantly in MCAO group pretreated with TA. Conversely, the elevated level of thiobarbituric acid reactive species and cytokines in MCAO group was attenuated significantly in TA-pretreated group when compared with MCAO group. The results indicated that TA protected the brain from damage caused by MCAO, and this effect may thorough diminish the oxidative stress and inflammatory responses.     

亚低温联合镁剂对脑缺血再灌注大鼠保护作用的研究

目的:rt-PA溶栓为缺血性卒中最有效的治疗方法,脑血流再通后挽救濒临死亡的神经细胞同时,也可能发生更为严重而持久的脑缺血再灌注损伤。本研究探讨联合应用局部亚低温(32-35℃)及硫酸镁对局灶性脑缺血再灌注大鼠的保护作用及其可能机制。方法:通过线栓法建立大鼠大脑中动脉阻塞(MCAO)及再通模型,将50只雄性Wistar大鼠随机分为假手术组、常温组、亚低温组、硫酸镁组、亚低温+硫酸镁组,每组10例,采用Longa神经功能评分、TTC染色、干湿重法、TUNEL技术,检测和比较各组脑缺血再灌注后大鼠的神经功能、脑梗死体积、脑组织含水量及凋亡细胞数。结果:与常温组相比,亚低温组与亚低温+硫酸镁组的梗死体积、神经功能评分、脑组织含水量、凋亡细胞数均明显降低,差异有显著意义(P0.05);而与亚低温组相比,亚低温+硫酸镁组局灶脑缺血大鼠的脑梗死体积、神经功能评分、脑组织含水量、凋亡细胞数均显著减少,差异有显著意义(P0.05)。结论:与单独应用亚低温相比,局部亚低温与硫酸镁联合应用,对局灶性脑缺血再灌注大鼠可发挥更有效的脑保护作用。其机制可能与抑制脑缺血再灌注后凋亡及减轻脑水肿有关。二者联用可能为缺血性卒中患者提供一种减轻溶栓后再灌注损伤的有效脑保护方法。     

毛蕊异黄酮通过抑制calpain-1的表达发挥抗脑缺血再灌注损伤的研究

目的:探讨毛蕊异黄酮抗脑缺血再灌注损伤的作用是否与抑制calpain-1的表达有关。方法:将SD大鼠随机分为假手术组、模型组以及药物组,采用线栓法建立大鼠大脑中动脉阻断(MCAO)模型,于缺血再灌注前30 min腹腔注射给予20 mg/kg毛蕊异黄酮或等体积的溶剂。再灌注24 h后,行神经功能学评分、脑梗死面积以及神经元凋亡检测;再灌注12 h、24 h时,采用免疫组化和蛋白印迹技术检测大鼠脑皮层calpain-1的表达。结果:与假手术组大鼠比较,MCAO模型组大鼠再灌注24 h后神经功能学评分、梗死面积、神经元凋亡率及calpain-1的表达均明显升高(P0.05),而毛蕊异黄酮能够降低模型组大鼠再灌注24 h后神经功能学评分、梗死面积、神经元凋亡率以及calpain-1的表达(P0.05)。结论:毛蕊异黄酮可能通过抑制calpain-1的表达发挥抗脑缺血再灌注损伤作用。     

缺血后处理对大鼠局灶性脑缺血再灌注损伤时TLR4 信号通路表达的影响

目的:观察缺血后处理对大鼠局灶性脑缺血再灌注损伤后TLR4通路表达的影响。方法:成年健康雄性SD大鼠110只,随机分为假手术组(sham组)(n=10)、缺血再灌注组(I/R组)和后处理组(IP组),后两组又依据缺血再灌注6h、12h、24h、48h、72h不同的时间点再分五个亚组。对各组行神经行为学评分,脑组织梗死体积测量,TUNEL技术检测神经细胞凋亡的情况,免疫组织化学技术观察各组大鼠脑组织TLR4、NF-κB和TNF-α蛋白的表达,原位杂交方法检测各组大鼠脑组织TLR4mRNA、NF-κBmRNA的表达。结果:缺血后处理可下调TLR4、NF-κB、TNF-α细胞炎性因子的表达,抑制细胞凋亡、减少脑梗死体积,改善神经行为。结论:后处理可通过抑制TLR4信号通路表达,减少脑梗死体积,改善神经功能。     

局灶性脑缺血再灌损伤时神经细胞内Ca~(2+)时空动态变化的实验研究

本文用插线法制作局灶性脑缺血／再灌损伤模型,利用激光共聚焦扫描显微镜观察活体脑片细胞内Ｃａ２＋的分布及动态变化,结果表明：（１）缺血／再灌时间不同,梗塞面积不同,缺血４小时梗塞面积占同侧半球的１６．３％,缺血４小时再灌２０小时梗塞面积增加到２５．９％,缺血２４小时梗塞面积占同侧半球的６０．４％。（２）本文首次观察到在缺血４小时纹状体区域的Ｃａ２＋变化明显高于皮层,并且再灌后皮层及纹状体区域Ｃａ２＋的含量明显增加     

局部亚低温联合硫酸镁对局灶性脑缺血大鼠的保护作用

目的:探讨联合应用局部亚低温(32-35℃)及硫酸镁对局灶性脑缺血大鼠的保护作用及其可能机制。方法:通过线栓法建立大鼠大脑中动脉阻塞(MCAO)模型,将40只雄性Wistar大鼠随机分为假手术组、常温组、亚低温组、硫酸镁组、亚低温+硫酸镁组,每组8例,采用Longa神经功能评分、TTC染色、TUNEL技术,检测和比较各组脑缺血后大鼠的神经功能、脑梗死体积、凋亡细胞数。结果:与常温组相比,亚低温组与亚低温+硫酸镁组的梗死体积、神经功能评分、凋亡细胞数均明显降低,差异有显著意义(P0.05);而与亚低温组相比,亚低温+硫酸镁组局灶脑缺血大鼠的脑梗死体积、神经功能评分、凋亡细胞数均显著减少,差异有显著意义(P0.05)。结论:与单独应用亚低温相比,局部亚低温与硫酸镁联合应用,对局灶性脑缺血大鼠可发挥更有效的脑保护作用,其机制可能与抑制脑缺血后凋亡有关。     

丹酚酸A对大鼠脑缺血/再灌注损伤及抗氧化酶活性的影响

目的:探讨丹酚酸A对大鼠脑缺血/再灌注(cerebral ischemia/reperfusion,CI/R)损伤及抗氧化酶活性的影响。方法:采用大鼠脑中动脉闭塞(middle cerebral arteryocclusion,MCAO)2 h再灌注24 h模型。实验终末,检测脑梗死面积,脑水肿以及评价神经功能损伤,并进一步分析脑组织中三种抗氧化酶的活性水平。结果:与模型组相比,丹酚酸A组大鼠脑梗死面积显著减少(P0.05),水肿程度显著减轻(P0.05),神经功能学评分显著下降(P0.05)。模型组再灌注24 h后,SOD,GSH-PX及CAT活性显著下降(P0.05);丹酚酸A组SOD,GSH-PX及CAT活性则显著升高(P0.05)。结论:丹酚酸A对大鼠CI/R损伤具有保护作用,可能与CI/R损伤时的脑组织SOD,GSH-PX及CAT活性显著升高相关。     

Catechin Hydrate Ameliorates Redox Imbalance and Limits Inflammatory Response in Focal Cerebral Ischemia

Epidemiologic studies have shown that foods rich in polyphenols, such as flavonoids, can lower the risk of ischemic disease; however, the mechanism of protection has not been clearly investigated. In this study, we hypothesized that pretreatment effect of catechin hydrate (CH) on functional outcome, neuronal damage and on secondary injuries in the ischemic brain of rats. To test this hypothesis, male Wistar rats were pretreated with CH (20 mg/kg b.wt) for 21 days and then subjected to 2 h middle cerebral artery occlusion (MCAO) followed by 22 h of reperfusion. After 2 h MCAO/22 h reperfusion, neurological deficit, infarct sizes, activities of antioxidant enzymes and cytokines level were measured. Immunohistochemistry and western blot were used to analyse the expression of glial fibrillary acidic protein (GFAP), inducible nitric oxide (iNOS) and NF-kB in ischemic brain. The administration of CH showed marked reduction in infarct size, reduced the neurological deficits, suppressed neuronal loss and downregulate the iNOS, GFAP and NF-kB expression in MCAO rats. A significantly depleted activity of antioxidant enzymes and content of glutathione in MCAO group were protected significantly in MCAO group pretreated with CH. Conversely, the elevated level of thiobarbituric acid reactive species and cytokines in MCAO group was attenuated significantly in CH pretreated group when compared with MCAO group. The results indicated that CH protected the brain from damage caused by MCAO, and this effect may be through downregulation of NF-kB expression.     

Synergistic Effect of Selenium and Melatonin on Neuroprotection in Cerebral Ischemia in Rats

The synergistic scavenger effects of selenium and melatonin collectively we called Se-Mel was studied on the prevention of neuronal injury induced by ischemia/reperfusion. Male Wistar rats were treated with sodium selenite (0.1 mg/kg, i.p.) and melatonin (10 mg/kg, i.p.) 30 min before the middle carotid artery occlusion (MCAO) and immediately after MCAO to male Wistar rats and was continued for 3 days once daily at the interval of 24 h. Behavioral activity (spontaneous motor activity and motor deficit) was improved in Se-Mel-treated rats as compared to MCAO group rats. The level of glutathione and the activity of antioxidant enzymes was depleted significantly while the content of thiobarbituric acid reactive substances, protein carbonyl, and nitric oxide radical (NO^·) was increased significantly in MCAO group. Systemic administration of Se-Mel ameliorated oxidative stress and improves ischemia/reperfusion-induced focal cerebral ischemia. Se-Mel also inhibited inducible nitric oxide synthase expression in Se-Mel+MCAO group as compared to MCAO group rats. Thus, Se-Mel has shown an excellent neuroprotective effect against ischemia/reperfusion injury through an anti-ischemic pathway. In conclusion, we demonstrated that the pretreatment with Se-Mel at the onset of reperfusion, reduced post-ischemic damage, and improved neurological outcome following transient focal cerebral ischemia in male Wistar rat.     

Continuous oral administration of atorvastatin ameliorates brain damage after transient focal ischemia in rats

Aims

Pre-treatment with statins is known to ameliorate ischemic brain damage after experimental stroke, and is independent of cholesterol levels. We undertook pre- vs post-ischemic treatment with atorvastatin after focal cerebral ischemia in rats.

Main methods

Male Sprague–Dawley rats underwent transient 90-min middle cerebral artery occlusion (MCAO). Atorvastatin (20 mg/kg/day) or vehicle was administered orally. Rats were divided into vehicle-treated, atorvastatin pre-treatment, atorvastatin post-treatment, and atorvastatin continuous-treatment groups. In the pre-treatment, rats were given atorvastatin or vehicle for 7 days before MCAO. In the post-treatment, rats received atorvastatin or vehicle for 7 days after MCAO. Measurement of infarct volume, as well as neurological and immunohistochemical assessments, were done 24 h and 7 days after reperfusion.

Key findings

Each atorvastatin-treated group demonstrated significant reductions in infarct and edema volumes compared with the vehicle-treated group 24 h after reperfusion. Seven days after reperfusion, infarct volumes in the post-treatment group and continuous-treatment group (but not the pre-treatment group) were significantly smaller than in the vehicle-treated group. Only the continuous-treatment group had significantly improved neurological scores 7 days after reperfusion compared with the vehicle group. Post-treatment and continuous-treatment groups had significantly decreased lipid peroxidation, oxidative DNA damage, microglial activation, expression of tumor necrosis factor-alpha, and neuronal damage in the cortical ischemic boundary area after 7 days of reperfusion.

Significance

These results suggest that continuous oral administration (avoiding withdrawal) with statins after stroke may reduce the extent of post-ischemic brain damage and improve neurological outcome by inhibiting oxidative stress and inflammatory responses.