镁对兔全脑缺血后神经保护作用的实验研究

[目的]观察硫酸镁预处理及硫酸镁治疗对兔全脑缺血后的神经保护作用. [方法]40只兔随机分为4组:对照组、缺血组、镁预处理组和镁治疗组,每组10只.阻断4血管(双侧颈总动脉及椎动脉),诱导全脑缺血,缺血时间为6min.测定缺血再灌注30min海马氨基酸含量.检测缺血再灌注3d海马CA1区神经元密度及凋亡神经元密度. [结果](1)镁预处理组及镁治疗组海马CA1区神经元密度显著高于缺血组(P＜0.01),凋亡神经元密度显著低于缺血组(P＜0.01). (2)镁预处理组及镁治疗组海马天冬氨酸和甘氨酸含量显著低于缺血组(P＜0.01);而γ-氨基丁酸含量显著高于缺血组(P＜0.05). (3)镁预处理组与镁治疗组比较各指标差异无统计学意义(P＞0.05). [结论]硫酸镁预处理及硫酸镁治疗对兔全脑缺血有显著的神经保护作用.     

氯化锂对大鼠海马CA1区神经元的作用

目的探讨氯化锂对SD大鼠脑缺血再灌注后海马CA1区神经元的作用。方法建立SD大鼠全脑缺血动物模型,假手术组仅仅做手术而不缺血;缺血对照组不给任何试剂;溶剂对照给在相同的时间给生理盐水;实验组大鼠在缺血前10min给药1次,之后复灌6d,每天给药1次。应用焦油紫染色方法检测氯化锂对大脑海马CA1区神经元的作用。结果假手术海马CA1区神经元几乎没有死亡;缺血对照组几乎全部死亡;溶剂对照几乎全部死亡;给药处理组神经元存活大约48%。结论氯化锂对大鼠脑缺血复灌后海马CA1区神经元具有保护作用。     

全氟辛烷磺酸亚慢性暴露对小鼠学习记忆及海马神经元的影响

目的 探讨全氟辛烷磺酸(perfluorooctane sulphonate,PFOs)暴露对小鼠的学习记忆能力及海马神经元的影响.方法 将32只健康清洁级8周龄雄性ICR小鼠按体重随机分为4组,分别为对照(含2％吐温-80的生理盐水)组和5、20、40 mg/kg PFOS暴露组,每组8只.采用腹腔注射方式进行染毒,染毒容量为10 ml/kg,每周3次,连续染毒6周.采用Morris水迷宫实验检测小鼠空间学习、记忆能力,并进行海马神经元形态学的定量分析.结果 训练第1天20 mg/kgPFOS暴露组小鼠的逃避潜伏期长于对照组(P＜0.05),而40 mg/kg PFOS暴露组小鼠训练第1、2天和第5天的逃避潜伏期均较对照组延长(P＜0.05,P＜0.01).与对照组相比,5、20 mg/kg PFOS暴露组小鼠海马CA1和CA3区神经元密度、细胞面积和核黑度差异无统计学意义(P＞0.05);而40 mg/kg PFOS暴露组小鼠海马CA1区神经元密度下降,胞面积减小,核黑度值增大,且CA3区神经元密度也下降,差异均有统计学意义(P＜0.01).结论 一定剂量的PFOS暴露可使小鼠学习记忆能力减弱,可能与海马区神经元损伤有关.     

Montelukast神经保护作用及机制研究进展

半胱氨酰白三烯(cysteinylleukoriencs,CysLTs)是中枢神经系统的重要炎症介质,通过激动CysLT受体而产生炎症效应.目前研究表明,CysLT1受体拮抗剂Montelukast对体外和体内实验性脑损伤,特别是对脑缺血以及阿尔茨海默病(Alzheimer'sdisease,AD)和帕金森病(Parkinson's disease,PD)等中枢退行性疾病具有良好的神经保护作用.本文对Montelukast在脑缺血,中枢退行性疾病以及其他脑损伤中的保护作用进行综述,并讨论Montelukast神经保护作用的分子机制.     

兔脑缺血再灌注后PPARγ表达对神经元凋亡的影响

目的观察脑缺血再灌注损伤后过氧化物酶体增殖物激活受体γ(PPARγ)表达对神经元凋亡的影响。方法日本大耳白兔72只,参照Pulsinelli四动脉阻断法建立兔全脑缺血90min/再灌注6h,1d,3d,7d,14d和28d的动物模型。TUNEL法染色观察脑皮质、海马和纹状体区神经元凋亡,免疫组织化学法检测PPARγ阳性神经元在各区的表达。结果脑缺血90min再灌注6h,1d,3d,7d,14d皮质、海马和纹状体区凋亡神经元明显增加(t皮质=3.86,P<0.01;t海马=4.96,P<0.01;t纹状体=2.91,P<0.05)。脑缺血再灌注6h,1d,3d,7d,14d,28d,PPARγ阳性神经元在各区明显增加(t皮质=7.98,P<0.01;t海马=7.65,P<0.01;t纹状体=6.98,P<0.01),其中再灌注6h,7d,14d,28d阳性神经元呈高值表达。结论脑缺血再灌注损伤后PPARγ表达能有效抑制神经元凋亡。     

大鼠脑缺血预处理后脑内c-fos基因FOS表达及意义

目的：探讨脑缺血预处理后脑组织原癌基因c-fos基因表达蛋白FOS变化及其与脑缺血耐受产生之间的关系。方法：通过建立大鼠全脑缺血模型,采用兔抗c-fos原癌基因抗体SABC免疫组化技术对脑缺血再灌注后的海马神经元进行检测FOS表达。结果：实验鼠四血管阻断（预处理）3min后,12h组、24h组及72h组FOS表达均显著高于对照组（假手术组）,P值均＜0．01,72h组最为显著。结论：大鼠短暂非损伤性3min缺血再灌注72h后脑内海马神经元中FOS表达显著增高。FOS表达增高是脑缺血耐受保护机制的一部分。     

十溴联苯醚致大鼠学习记忆及海马神经元氧化损伤和凋亡研究

目的 观察十溴联苯醚(BDE-209)对成年大鼠学习记忆及海马神经元氧化应激和凋亡的影响.方法 将96只SD雄性大鼠随机分为溶剂对照组、BDE-209低(250 mg/kg)、中(500 mg/kg)、高(1 000 mg/kg)剂量组,每天灌胃1次,持续30 d.用Morris水迷宫检测学习记忆功能,化学比色法测定大鼠海马组织总抗氧化能力(T-AOC)、谷胱甘肽转移酶( GST)、超氧化物歧化酶(SOD)、丙二醛(MDA)的水平,考马斯亮蓝法测定蛋白水平,末端标记(TUNEL)法检测海马CA1区神经元凋亡.结果 与对照组比较,BDE-209染毒组大鼠的平均逃避潜伏期明显增加、空间搜索有效策略百分比和穿越平台的次数明显减少(P＜0.05);且随染毒剂量的增加,BDE-209各组的平均逃避潜伏期明显增加、空间搜索有效策略百分比和穿越平台的次数明显减少(P＜0.05).BDE-209染毒组海马T-AOC、GST和SOD的活力明显低于对照组,MDA水平明显高于对照组(P＜0.05);并随染毒剂量的增加各染毒组T-AOC、GST和SOD的活力明显降低,MDA水平明显增加(P＜0.05).TUNEL法检测结果显示BDE-209染毒组较对照组染色深,凋亡细胞数明显增加(P＜0.05);BDE-209染毒组随染毒剂量的增加染色加深,凋亡细胞增加(P＜0.05).结论 BDE-209致成年大鼠学习记忆能力下降可能与海马神经元细胞氧化损伤和凋亡有关.     

bFGF对大鼠缺血性脑损伤CaMKⅡmRNA表达影响

目的 研究大鼠脑缺血再灌注后Ca2 >/钙调蛋白依赖的蛋白激酶Ⅱ(CaMK(Ⅱ)mRNA的表达,探讨碱性成纤维细胞生长因子(bFGF)对脑组织缺血再灌注神经元CaMK Ⅱ mRNA的调节作用及机制.方法 应用线栓法制作大鼠局灶性脑缺血再灌注模型,大脑中动脉阻塞1 h再灌注损伤24 h,采用TUNEL法、逆转录聚合酶链反应(RT-PCR)检测海马及皮质内神经元凋亡和CaMK Ⅱ mRNA的表达.结果 大鼠缺血再灌注海马及顶叶皮质内神经元凋亡数增加,而CaMKⅡ mRNA表达较假手术组减少,注射bFGF后神经元凋亡数减少,CaMK Ⅱ mRNA表达明显高于缺血再灌注组.结论 bFGF抑制缺血神经元凋亡,参与CaMK Ⅱ mRNA的调节,对缺血神经元有保护作用.     

bFGF对脑缺血大鼠脑组织HSP70 mRNA表达影响

目的 研究大鼠脑缺血再灌注后热休克蛋白70(HSP70)mRNA的表达,探讨碱性成纤维细胞生长因子(bFGF)对脑组织缺血再灌注神经元HSP70 mRNA的调节作用及机制.方法 应用线栓法制作大鼠局灶性脑缺血再灌注模型,大脑中动脉阻塞1h再灌注损伤24h,采用原位缺口末端标记法(TUNEL)、RT-PCR法检测海马及皮质内神经元凋亡和HSP70 mRNA的表达.结果 大鼠缺血再灌注海马及顶叶皮质内神经元凋亡数增加而HSP70mRNA表达较正常组增加.注射bFGF后神经元凋亡数减少,HSP70mRNA表达明显高于缺血再灌注组.结论 bFGF抑制缺血神经元凋亡,参与HSP70 mRNA的调节,对缺血神经元有保护作用.     

低剂量鱼藤酮诱导BV2细胞IL-6产物及montelukast的作用

目的：在体外帕金森病（Parkinson's disease,PD）模型观察低剂量鱼藤酮诱导的BV2小胶质细胞白介素6（IL-6）生成及montelukast的作用。方法以PD诱导因子鱼藤酮处理小鼠小胶质细胞系BV2细胞。 Western blot方法检测BV2细胞前炎症细胞因子 IL-6蛋白表达, ELISA 方法检测 BV2细胞 IL-6释放。观察 CysLT1R 拮抗剂montelukast对鱼藤酮诱导的小胶质细胞IL-6的影响。结果低剂量鱼藤酮（0．3～3 nM）浓度依赖性地增加IL-6蛋白表达;以鱼藤酮（3 nM）处理细胞1～24h,时间依赖性的诱导IL-6释放增加。 CysLT1R拮抗剂montelukast降低鱼藤酮增高的BV2细胞IL-6水平。结论低剂量鱼藤酮诱导小胶质细胞前炎症细胞因子IL-6增加,montelukast抑制鱼藤酮诱导BV2细胞效应。     

急性脑缺血大鼠海马CA1区细胞凋亡与Caspase-3及Caspase-10蛋白表达的相关性

目的探讨急性脑缺血大鼠海马CA1区细胞凋亡与Caspase-3及Caspase-10蛋白表达的关系。方法线栓法制作大鼠大脑中动脉缺血(MCAO)再灌注模型。应用原位末端标记法检测细胞凋亡,免疫组化法及Western-blotting蛋白印记技术分别检测Caspase-3及Caspase-10蛋白表达。结果①脑缺血大鼠海马CA1区3h出现凋亡阳性细胞,48 h达到高峰,72 h凋亡阳性细胞的数量开始下降。②脑缺血大鼠海马CA1区3 h可见Caspase-3及Caspase-10蛋白阳性细胞,48 h表达达高峰,72 h表达开始减少。③脑缺血大鼠海马CA1区Caspase-3及Caspase-10蛋白表达的变化与细胞凋亡数呈正相关(r=0.976,P﹤0.01;r=0.986,P﹤0.01)。结论脑缺血大鼠海马CA1区存在细胞凋亡,且与Cas-pase-3及Caspase-10蛋白表达的变化一致,Caspase-3及Caspase-10蛋白可促进细胞凋亡发生。     

Feeding rats diets enriched in lowbush blueberries for six weeks decreases ischemia-induced brain damage

Oxidative stress is an important element in the etiology of ischemic stroke. Lowbush blueberries (Vaccinium angustifolium Aiton) have a high antioxidant capacity and thus we determined whether consumption of lowbush blueberries would protect neurons from stroke-induced damage. Rats were fed AIN-93G diets containing 0 or 14.3% blueberries (g fresh weight/100 g feed) for 6 weeks. Stroke was then simulated by ligation of the left common carotid artery (ischemia), followed by hypoxia. One week later, plasma and urine were collected, and neuronal damage in the hippocampus was determined histologically. In control rats, hypoxia-ischemia resulted in 40 +/- 2% loss of neurons in the hippocampus of the left cerebral hemisphere, as compared to the right hemisphere. Rats on blueberry-supplemented diets lost only 17 +/- 2% of neurons in the ischemic hippocampus. Neuroprotection was observed in the CA1 and CA2 regions, but not CA3 region, of the hippocampus. The blueberry diet had no detectable effects on the plasma or urine oxygen radical absorbance capacity (ORAC) or plasma lipids. We conclude that consumption of lowbush blueberries by rats confers protection to the brain against damage from ischemia, suggesting that inclusion of blueberries in the diet may improve ischemic stroke outcomes.     

Dose-related effects of chronic resveratrol administration on neurogenesis,angiogenesis, and corticosterone secretion are associated with improved spatial memory retention following global cerebral ischemia

Objectives: The polyphenol resveratrol has shown regulatory effects on hippocampal neurogenesis, which according to the neurovascular niche hypothesis, is likely to involve stimulation of angiogenesis. In rodents, global cerebral ischemia leads to selective CA1 neuronal damage, spatial memory impairments, lasting changes in corticosterone (CORT) secretion, and increased neurogenesis. This study examined dose-related effects of 21-day RSV treatment on markers associated with neurogenesis (DCX, PSA-NCAM) and angiogenesis (CD31) in the hippocampus at short (7-day) and long-term (85-day) intervals following global ischemia. In parallel, post-ischemic and stress-induced CORT levels and spatial memory in the Morris water maze were determined.

Methods: Five groups of male Wistar rats were included: sham/saline, ischemia/saline, ischemia/1?mg/kg RSV, ischemia/10?mg/kg RSV, and sham/10?mg/kg RSV. Changes in expression of plasticity markers were paralleled by assessment of basal- and stress-induced CORT secretions, and spatial memory performance.

Results: Our findings revealed a significant attenuation of ischemia-induced DCX/PSA-NCAM expression in RSV-treated rats, whereas RSV treatment increased angiogenesis in the injured CA1 region. RSV attenuated CORT levels 3?days post-ischemia and a trend toward attenuating CORT secretion in response to 15?minutes restraint stress. Increased swimming latencies in the target quadrant during the MWM probe trial in RSV-treated ischemic rats support beneficial effects of RSV on spatial memory retention.

Discussion: Our findings uncover time- and dose-related effects of RSV and global ischemia on the regulation of hippocampal plasticity. Changes in neuro- and angiogenesis are consistent with RSV neuroprotective effects, but appear independent of RSV regulatory effects on corticosterone secretion.     

Feeding Rats Diets Enriched in Lowbush Blueberries for Six Weeks Decreases Ischemia-induced Brain Damage

Abstract

Oxidative stress is an important element in the etiology of ischemic stroke. Lowbush blueberries (Vaccinium angustifolium Aiton) have a high antioxidant capacity and thus we determined whether consumption of lowbush blueberries would protect neurons from stroke-induced damage. Rats were fed AIN-93G diets containing 0 or 14.3% blueberries (g fresh weight/100 g feed) for 6 weeks. Stroke was then simulated by ligation of the left common carotid artery (ischemia), followed by hypoxia. One week later, plasma and urine were collected, and neuronal damage in the hippocampus was determined histologically. In control rats, hypoxia-ischemia resulted in 40±2% loss of neurons in the hippocampus of the left cerebral hemisphere, as compared to the right hemisphere. Rats on blueberry-supplemented diets lost only 17±2% of neurons in the ischemic hippocampus. Neuroprotection was observed in the CA1 and CA2 regions, but not CA3 region, of the hippocampus. The blueberry diet had no detectable effects on the plasma or urine oxygen radical absorbance capacity (ORAC) or plasma lipids. We conclude that consumption of lowbush blueberries by rats confers protection to the brain against damage from ischemia, suggesting that inclusion of blueberries in the diet may improve ischemic stroke outcomes.     

谷胱甘肽抑制新生大鼠脑缺氧缺血海马CA1区细胞凋亡的研究

[目的]研究谷胱甘肽对缺氧缺血脑损伤引起的神经细胞凋亡的作用.[方法]利用新生大鼠缺氧缺血脑损伤模型,通过末端转移酶介导的原位缺口标记(TUNEL)法检测并比较缺氧缺血状态下用药与否对海马CA1区神经细胞凋亡的影响.[结果]谷胱甘肽(还原性)治疗可显著减少海马CA1区凋亡的阳性细胞数.[结论]谷胱甘肽可有效抑制缺氧缺血损伤引起的海马部位神经细胞凋亡,可有效对抗氧化张力在缺氧缺血损伤中引起的细胞凋亡.     

Neuroprotective effect of oral choline administration after global brain ischemia in rats

Abstract

Choline – now recognized as an essential nutrient – is the most common polar group found in the outer leaflet of the plasma membrane bilayer. Brain ischemia-reperfusion causes lipid peroxidation triggering multiple cell death pathways involving necrosis and apoptosis. Membrane breakdown is, therefore, a major pathophysiologic event in brain ischemia. The ability to achieve membrane repair is a critical step for survival of ischemic neurons following reperfusion injury. The availability of choline is a rate-limiting factor in phospholipid synthesis and, therefore, may be important for timely membrane repair and cell survival. This work aimed at verifying the effects of 7-day oral administration with different doses of choline on survival of CA1 hippocampal neurons following transient global forebrain ischemia in rats. The administration of 400 mg/kg/day divided into two daily doses for 7 consecutive days significantly improved CA1 pyramidal cell survival, indicating that the local availability of this essential nutrient may limit postischemic neuronal survival.     

Long-term treatment with fish oil prevents memory impairments but not hippocampal damage in rats subjected to transient,global cerebral ischemia

Cerebral ischemia leads to neurodegeneration and cognitive impairment. Fish oil (FO) constitutes a rich dietary source of ω-3 polyunsaturated fatty acids especially docosahexaenoic acid (DHA). The objective of the present study was to investigate whether long-term treatment with commercial, high concentration DHA-containing FO could be effective in alleviating both the cognitive and neurodegenerative deficits caused by transient, global cerebral ischemia (TGCI) in rats. Naive rats were trained for 10 days in an 8-arm radial maze task and then subjected to TGCI for 15 minutes (4-VO model) 3 days later (day 13). Retention of the previously acquired cognition (ie, memory) was assessed weekly on days 20, 27, 34, 41, 48, and 55 and measured by 3 behavioral parameters as follows: (i) latency to find the goal box, (ii) number of reference memory errors, and (iii) number of working memory errors. The extent of pyramidal cell death in the hippocampus was examined at the end of the behavioral analysis on day 43. Fish oil (300 mg/kg DHA, gavage) administration occurred once daily beginning 3 days before TGCI (the last day of training) and continued until day 41. Transient, global cerebral ischemia markedly disrupted memory performance measured by all 3 parameters (P < .0001 vs sham). This amnesic effect of ischemia persisted until the end of the behavioral analysis. Treatment with FO progressively reversed the TGCI-induced retention deficit until rats achieved control levels. This protective effect of FO on learning/memory function was clearly observed after both daily and cumulative data analysis (P < .001-0.01 vs vehicle). Such memory improvements remained statistically significant, even after cessation of FO treatment, indicating a sustained effect of FO. In contrast, FO failed to prevent ischemia-induced hippocampal damage in areas CA1, CA2, or CA4. Therefore, the present findings suggest that long-term FO treatment is able to facilitate functional recovery after ischemic brain damage, an effect that was distinct from hippocampal damage.     

血红素加氧酶-1在HIBD新生大鼠海马神经元的表达研究及纳洛酮的干预作用

目的:研究新生大鼠脑缺氧缺血后不同时间点血红素加氧酶-1(hemeoxygen-ase,HO-1)、Caspase-3在海马神经元中的动态变化及纳洛酮注射液的干预作用,进一步探讨HO-1、Caspase-3在新生大鼠缺氧缺血后神经损伤过程中的可能机制及纳洛酮注射液对神经系统保护的作用。方法:新生7天龄SD大鼠随机分为3组:假手术组(S)、生理盐水对照组(C)、纳洛酮干预组(N),每组按照观测的时间点不同分为3、6、12、24 h和3、7天6个亚组,每个亚组8只,用Rice法制备新生大鼠HIBD模型。在每个时间点将大鼠断头取右侧海马脑组织匀浆,用Western blot方法测不同时间点HO-1、Caspase-3动态变化;用TUNEL染色法检测相应时间点脑海马CA1区细胞凋亡。结果:①Western蛋白印迹S组海马HO-1表达很弱,各时间点表达无差异(P>0.05);C组和N组右侧海马HO-1在HI后3h即明显增加(P<0.01),HI后24 h海马HO-1蛋白表达达到峰值,3天后明显下降,7天接近S组,但仍较正常偏高(P<0.01);N组在12、24 h和3、7天海马HO-1表达均较C组高(P<0.01)。②在HI后3 h,C组和N组右侧海马Caspase-3即明显增加(P<0.01),HI后24 h海马Caspase-3蛋白表达达到峰值,3天后明显下降,7天接近S组(P=0.519);N组在12、24 h、3天海马HO-1表达均较C组低(P<0.01)。③Tunel显示S组各时间点右侧海马CA1区仅见少量凋亡细胞,HI后3 h C组和N组右侧海马神经元凋亡细胞数即明显增加(P<0.01),24 h达到高峰,3天开始下降,7天时仍高于S组(P<0.01);N组凋亡数在24 h、3、7天这3个时间点上均较C组明显下降(P<0.01)。结论:HI后脑海马组织细胞中HO-1蛋白、Caspase-3表达均是增加的,两者表达趋势相一致,在时间上吻合,表明HO-1、Caspase-3参与了新生大鼠HI后细胞凋亡的病理过程;纳洛酮注射液能够上调HO-1的表达,抑制Caspase-3活化,减少神经元凋亡,从而起到对HIBD新生大鼠保护作用。     

脯氨酸二硫代氨基甲酸酯减轻新生大鼠缺氧缺血脑细胞凋亡的作用及机制

目的:细胞凋亡在新生儿缺氧缺血性脑病(HIE)的发病机制中起重要作用,通过观察缺氧缺血后凋亡通路上关键成分核因子-κappaB(NF-κB)表达的变化,探讨脯氨酸二硫代氨基甲酸酯减轻新生大鼠脑细胞凋亡的作用及机制。方法:72只7日龄SD大鼠随机分为假手术组、HIBD组、PDTC预处理组(n=24)。各组再随机分为HI 6 h、24 h组(n=12)。结扎大鼠左颈总动脉后行8%低氧暴露制备新生大鼠HIBD模型。PDTC组HI前腹腔注射PDTC(50μg.g-1.wt-1)。采用免疫组化检测NF-κB p65的表达;TUNEL法检测脑细胞凋亡。结果:假手术组左侧海马神经元NF-κB活化和凋亡阳性细胞数极少;缺氧缺血性脑损伤后海马神经元NF-κB活化和凋亡阳性细胞持续增加至HI 24 h(P<0.01或P<0.001);PDTC干预组NF-κB活化和凋亡细胞数均较HIBD组下降(P<0.05或P<0.01)。结论:PDTC预处理可能通过抑制NF-κB过度活化来减少脑细胞凋亡,对新生大鼠缺氧缺血性脑细胞产生保护作用。