丁苯酞对局灶性脑梗死大鼠皮层Rho相关激酶Ⅱ蛋白表达的影响

目的：探索丁苯酞对局灶性脑梗死大鼠模型皮层 Rho相关激酶Ⅱ（ Rho-associated kinase Ⅱ,ROCKⅡ）表达的影响。方法成年Sprague-Dawley 雄性大鼠45只,随机分为假手术组15只,对照组15只,丁苯酞组15只。应用光化学法建立局灶性脑梗死大鼠模型。术后24h行干湿质量法检测各组大鼠脑组织含水量变化,行免疫组织化学和Western Blot检测各组大鼠梗死区周围皮层ROCKⅡ蛋白表达水平。结果与假手术组比较,对照组和丁苯酞组脑组织含水量明显增加,梗死区周围皮层ROCKⅡ蛋白表达明显增加。丁苯酞组脑组织含水量较对照组降低,梗死区周围皮层ROCKⅡ蛋白表达减少。结论丁苯酞可降低局灶性脑梗死大鼠皮层ROCKⅡ蛋白的表达,减轻脑水肿的形成。     

大鼠大脑中动脉供血区梗死后小脑皮质neurocan动态表达

目的观察大鼠大脑中动脉闭塞后小脑皮质neurocan的变化。方法采用Longa的大鼠大脑中动脉内栓线阻断法(MCAO)制备动物模型;用实时定量PCR法检测neurocan表达。结果本研究结果显示持续性大脑中动脉闭塞后,neurocan在小脑皮层有动态表达,第1d和3d较假手术组轻度增高(P0.05),7d、10d和14d较假手术组明显增高(P0.05)。结论大鼠大脑中动脉闭塞后,小脑皮质出现neurocan的动态表达,因此远离梗死灶的相关部位脑组织继发损害机制可能与神经抑制因子有关。     

大鼠大脑中动脉供血区梗死后小脑皮质BDNF mRNA动态表达

目的 观察大鼠大脑中动脉闭塞后小脑皮质BDNF mRNA的变化.方法 采用Longa的大鼠大脑中动脉内栓线阻断法(MCAO)制备动物模型;用实时定量PCR法检测BDNF mRNA表达.结果 该研究结果显示:持续性大脑中动脉闭塞后,BDNF mRNA在小脑皮层有动态表达,第1、10和14天较假手术组明显增高(P<0.05),第3、5和7天明显下降(P<0.05).结论 远离梗死灶的相关部位脑组织继发损害机制可能与神经营养因子障碍有关.     

栓线法制作大鼠局灶性脑缺血再灌注损伤模型

对尼龙线栓塞大鼠大脑中动脉的局灶性脑缺血再灌注模型进行了探讨．结果，假手术组未见神经病学体征，手术组（缺血３ｈ、再灌注３ｈ）神经病学评分为１．５６±０．５１．用２，３，５ 氯化三苯基四氮唑染色，示手术组在大脑皮质、基底节均出现梗死灶，手术组缺血侧脑组织含水量显著高于手术对侧和假手术组两侧脑组织含水量．电子显微镜观察到手术组额顶叶皮质神经细胞的超微结构呈缺血性改变．     

局灶性脑缺血-再灌注损伤大鼠杏仁核和心肌的改变

目的观察局灶性脑缺血-再灌注损伤(CIRI)模型大鼠神经功能缺失和脑组织形态学变化以及杏仁核和心肌的病理改变,探讨其机制。方法线栓法制备大鼠右大脑中动脉闭塞(MCAO/R)模型,缺血2 h/再灌注24 h。神经功能缺失评分观察神经功能缺失症状,TTC染色观察脑梗死面积,AutoCAD图像分析软件计算脑水肿程度,H-E染色观察脑组织、杏仁核和心肌的病理学改变,计数梗死侧相应脑区的正常锥体神经元密度。结果MCAO 2 h/再灌注24 h后,神经功能缺失;脑组织损伤侧出现明显梗死灶和脑水肿,假手术组梗死面积和脑水肿程度均为0,损伤组梗死面积和脑水肿程度分别为(21.95±2.13)%和(33.42±11.30)%;皮质区和杏仁核缺血性坏死、水肿,正常锥体神经元密度明显减少,假手术组皮质区和杏仁核锥体细胞计数分别为(41.56±6.50)和(40.96±4.30),损伤组皮质区和杏仁核锥体细胞计数分别为(10.08±2.67)和(8.56±2.25);心肌细胞出现严重充血及炎性细胞浸润。结论局灶性CIRI后脑组织严重受损,神经功能异常,杏仁核和心肌出现明显病理学改变。     

首乌仙海片对缺血性卒中模型大鼠脑组织GFAP、GAP- 43表达的影响

目的 研究补益肝肾中药首乌仙海片对大脑中动脉阻塞(MCAO)大鼠星形胶质细胞活性及神经轴突生长的作用.方法 12周龄雄性Wistar大鼠40只,随机分为假手术组8只、模型组16只和中药组16只.模型组和中药组按Tamura法制成MCAO模型,假手术组大鼠行相同的开颅术而不凝闭大脑中动脉,于造模成功后6 d,中药组灌胃给予首乌仙海片9 g/kg,余2组分别灌胃给予等量生理盐水.假手术组大鼠于灌胃1周后,其余2组大鼠分别于灌胃1周、2周末各处死8只,断头取脑,ABC法染色,采用免疫组化法,进行脑组织胶质纤维酸性蛋白(GFAP)、神经生长相关蛋白质(GAP- 43)的测定.结果 中药组给药1周时,GFAP的表达较同期模型组与假手术组均有显著增强(P<0.01),中药组给药2周时,GFAP的表达较给药1周时及同期模型组与假手术组均显著增强,差别均有高度统计学意义(P<0.01).中药组治疗2周时,梗死灶周围区GAP- 43表达明显增高,与其他2组比较差别有高度统计学意义(P<0.01).相关性分析表明,中药组灌胃2周时,GFAP与GAP- 43在大鼠脑组织中的表达具有相关性,r=0.75,P=0.031 9,GAP- 43表达随GFAP表达的上调而增强.结论 首乌仙海片可诱导缺血性卒中后神经功能重塑,与该药改善星形胶质细胞活性,促进神经轴突的再生有关.     

丁苯酞对急性缺血性脑卒中大鼠的脑保护作用及其机制

目的：探讨丁苯酞对急性缺血性脑卒中大鼠的脑保护作用,阐明其对急性缺血性脑卒中的治疗机制。方法：48只SD大鼠随机分为假手术组、模型组（局灶性脑缺血大鼠模型）和丁苯酞组（局灶性脑缺血大鼠模型+丁苯酞治疗）,每组16只。对各组大鼠进行神经症状评分测定,对大鼠脑组织进行HE染色,采用氯化三苯基四氮唑（TTC）测定脑梗死面积百分率,采用Western blotting法和逆转录-聚合酶链反应（RT-PCR）测定大鼠脑组织中肝细胞生长因子（HGF）、血管内皮细胞生长因子（VEGF）、基质金属蛋白酶2（MMP-2）、基质金属蛋白酶9（MMP-9）、核转录因子κB抑制蛋白α（IκBα）和核转录因子κB（NF-κB）亚单位p65（NF-κB p65）蛋白及mRNA表达水平。结果：假手术组大鼠脑组织无梗死灶形成,无神经功能缺损症状;与模型组比较,丁苯酞组大鼠脑梗死面积百分率和神经功能评分明显降低（P<0.01）。假手术组大鼠脑组织细胞结构完整、分布均匀;模型组大鼠脑组织大部分细胞坏死,胞浆破裂,细胞核破裂、凝缩;丁苯酞组大鼠脑组织少量细胞肿胀和坏死。与假手术组比较,模型组大鼠脑组织中HGF、VEGF、MMP-2、MMP-9和NF-κB p65蛋白及mRNA表达水平均明显降低（P<0.01）,IκBα蛋白及mRNA表达水平明显升高（P<0.01）;与模型组比较,丁苯酞组大鼠脑组织中HGF、VEGF、MMP-2、MMP-9和NF-κB p65蛋白及mRNA表达水平均明显升高（P<0.01）,IκBα蛋白和mRNA表达水平明显降低（P<0.01）。结论：丁苯酞可改善急性缺血性脑卒中大鼠的神经功能,减少脑梗死面积,其机制可能与丁苯酞升高脑组织HGF水平,并通过干扰NF-κB信号通路而促进脑血管形成有关。     

脑缺血再灌注损伤模型大鼠大脑皮质细胞色素C与Bax蛋白的表达

目的制备局灶性脑缺血再灌注损伤大鼠模型,并观察大脑皮质细胞色素C(CytC)与Bax蛋白表达的变化。方法采用随机数字表法将22只雄性SD大鼠分为正常组(6只)、假手术组(6只)和模型组(10只)。采用线栓法闭塞大脑中动脉2h后进行再灌注3d,制备局灶性脑缺血再灌注损伤模型。采用神经缺失评分观察大鼠的行为学表现;TTC染色检查脑组织梗死情况;HE染色观察大鼠脑组织形态结构;Western blot技术检测大鼠大脑皮质CytC与Bax蛋白的表达。结果假手术组大鼠无神经功能障碍表现,脑组织未见梗死灶,脑组织神经细胞形态规则;大脑皮质CytC与Bax蛋白的相对表达量,与正常组相比差异无统计学意义(P>0.05)。与假手术组相比,模型组大鼠出现神经功能障碍,左侧半球可见梗死灶,梗死侧脑组织形态学观察可见神经细胞大量坏死脱落,胞质呈空泡变性、疏松,胞核浓缩深染;大脑皮质CytC与Bax蛋白表达量明显增加(P<0.05)。结论大脑中动脉闭塞2h后进行再灌注3d可造成脑缺血再灌注损伤,可能与大脑皮质CytC与Bax蛋白的表达增加有关。     

莫诺苷对脑缺血再灌注大鼠皮层肝细胞生长因子及血管性血友病因子表达的影响

目的研究莫诺苷对脑缺血再灌注7 d大鼠患侧皮层肝细胞生长因子(hepatocyte growth factor,HGF)及血管性血友病因子(von willebrand factor,v WF)表达的影响。方法 25只健康成年雄性Sprague-Dawley(SD)大鼠采用改良Zealonga线栓法制备大鼠大脑中动脉阻塞(middle cerebral artery occlusion,MCAO)模型,术后随机分为假手术组、模型组、莫诺苷小剂量组、中剂量组及大剂量组,每组5只。造模后3 h按照30,90,270 mg/kg剂量每天一次灌胃给予莫诺苷。免疫印迹法和免疫荧光法分别检测莫诺苷对脑缺血再灌注7 d大鼠患侧皮层HGF及v WF表达的影响。结果脑缺血再灌注7 d后,免疫印迹法显示,与假手术组相比,模型组HGF蛋白表达显著升高(P0.01);同模型组相比,莫诺苷给药组(30 mg/kg、90 mg/kg、270 mg/kg)HGF蛋白表达水平均显著升高(P0.05,P0.01,P0.001)。免疫荧光法显示,与假手术组相比,模型组v WF表达显著升高(P0.001);同模型组相比,莫诺苷中、大剂量组(90 mg/kg、270 mg/kg)v WF表达显著升高(P0.01,P0.001)。结论莫诺苷可以上调HGF及v WF在脑内的表达,促进局灶性脑缺血大鼠血管新生。     

补阳还五汤对脑缺血大鼠梗死灶周围脑区新生神经细胞增殖、分化及Wnt/β-catenin信号通路的影响

目的 观察补阳还五汤对脑缺血再灌注大鼠梗死灶周围皮层新生细胞增殖、分化及Wnt/β-连环蛋白(β-catenin)信号通路关键蛋白在新生细胞表达的影响.方法 将SD大鼠按随机数字表法分为假手术组、模型组、补阳还五汤组,每组9只.采用线栓法制备大脑中动脉缺血再灌注模型.造模后24 h,补阳还五汤组灌胃补阳还五汤水煎液16.1 g/kg,每日1次,连续给药30 d.于造模后第3天开始腹腔注射5-溴脱氧尿嘧啶核苷(BrdU),活体标记梗死灶周围皮层新生细胞.分别于造模后第9、15、30天取材.采用免疫荧光双标结合图像分析技术,检测各组大鼠梗死灶周围皮层BrdU阳性细胞、双标BrdU/胶质纤维酸性蛋白(GFAP)、BrdU/微管相关蛋白2(MAP-2)、BrdU/Wnt、BrdU/β-catenin、BrdU/散乱蛋白(Dishevelled)阳性细胞.结果 补阳还五汤组大鼠脑缺血再灌注9、15、30 d,梗死灶周围皮层BrdU+细胞数均较同时点模型组大鼠增加(P<0.01),双标BrdU+/GFAP+细胞数较模型组减少(P<0.01);缺血再灌注9、15 d,补阳还五汤组大鼠梗死灶周围皮层双标BrdU+/MAP-2+细胞数较模型组增加(P<0.01).与同时点模型组比较,脑缺血再灌注9 d,补阳还五汤组大鼠梗死灶周围皮层双标BrdU+/Wnt+细胞数增加(P<0.05);脑缺血再灌注15 d,补阳还五汤组大鼠梗死灶周围皮层双标BrdU+/Dishevelled+、BrdU+/β-catenin+细胞数增加(P<0.05);脑缺血再灌注30 d,补阳还五汤组大鼠梗死灶周围皮层双标BrdU+/Wnt+、BrdU+/Dishev-elled+、BrdU+/β-catenin+细胞数均增加(P<0.05或P<0.01).结论 补阳还五汤可激活脑缺血再灌注大鼠梗死灶周围皮层组织新生细胞增殖,抑制新生细胞向胶质细胞分化,促进新生细胞向神经元分化,并可增强新生细胞Wnt、β-catenin、Dishevelled蛋白的表达.     

麝香保心丸对小鼠缺血性脑卒中的保护作用及机制

 目的 探究麝香保心丸（Shexiang Baoxin Pill,SBP）在小鼠缺血性脑卒中的保护作用及机制。方法 通过大脑中动脉阻塞（middle cerebral artery occlusion,MCAO）构建脑缺血性卒中模型,将96只C57BL/6小鼠随机分成4组：假手术（Sham）组、模型（ischemia/reperfusion,I/R）组、低剂量麝香保心丸（LSBP,22.5 mg/kg）组和高剂量麝香保心丸组（HSBP,45 mg/kg）,每组24只。灌胃4周后制备小鼠MCAO模型。记录各组小鼠的大脑梗死体积及脑组织含水量,利用免疫荧光技术分析激活的小胶质细胞数量,采用qRT-PCR检测炎症因子的表达,通过Western blot法分析NF-κB信号通路相关蛋白质的表达情况。结果 与I/R组相比,LSBP组和HSBP组大脑梗死体积减少,脑组织含水量降低,激活的小胶质细胞数量减少,同时促进小胶质细胞由M1型向M2型极化,并抑制NF-κB信号通路的激活。结论 SBP可以减轻由缺血性脑卒中引起的小鼠脑组织损伤,其机制可能与改变小胶质细胞M1/M2的极化状态和抑制NF-κB信号通路的激活有关。     

远隔缺血预适应对大鼠局灶性脑缺血后诱导型一氧化氮合酶的影响

目的：研究远隔缺血预适应（ remote ischemic preconditioning,RIPC）对大鼠脑缺血再灌注后诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）表达的影响,以探讨 RIPC 保护脑缺血损伤的相关机制。方法应用线栓法制作大脑中动脉栓塞（middle cerebral artery occlusion,MCAO）模型,将21只健康雄性 SD 大鼠采用数字表法随机分为3组：假手术组（sham,n=7）,MCAO 组（n=7）,RIPC＋MCAO 组（n =7）。在 MCAO 手术前连续3 d,进行远隔肢体缺血预适应处理（双侧股动脉夹闭10 min/放开10 min,每天3次）。在缺血2 h-再灌注24 h 后进行神经功能评分,TTC 染色检测脑梗死体积,采用 Western blotting 方法检测梗死侧脑组织的 iNOS 蛋白表达水平。结果1）在 MCAO 手术过程中,3组实验动物的生理指标均在正常范围内,且组间差异无统计学意义。与 sham 组相比,MCAO 组大鼠再灌注24 h 时神经功能缺损评分显著升高、脑梗死体积明显增大（P〈0.05）。而 RIPC＋MCAO 组大鼠神经功能较 MCAO 组大鼠得到明显改善、脑梗死体积显著减少（P〈0.05）。2）再灌注24 h 时,与 sham 组相比,MCAO 组大鼠脑梗死侧 iNOS 蛋白表达显著增加（P〈0.05）。与 MCAO 组相比,RIPC＋MCAO 组大鼠脑梗死侧 iNOS 蛋白表达显著降低（P〈0.05）。结论 RIPC 处理对大鼠脑缺血损伤具有保护作用,其机制与降低脑缺血大鼠脑内 iNOS 蛋白表达有关。     

Electroacupuncture Attenuates Ischemic Brain Injury and Cellular Apoptosis via Mitochondrial Translocation of Cofilin

Objective: To investigate the potential mechanisms of electroacupuncture (EA) to prevent ischemic stroke. Methods: The method of middle cerebral artery occlusion (MCAO) was employed to establish a rat model of ischemic stroke. Seventy-eight Sprague-Dawley rats were divided into the sham group, MCAO + EA control (EC) group, and MCAO + EA (EA) group according to a random number table (n=26 per group). EA was applied to the acupoints of Baihui (DU 20) and Shenting (DU 24) 5 min and 6 h, respectively after the onset of MCAO for 30 min. Rats in the sham and EC groups received only light isoflurane anesthesia for 30 min after MCAO. The neuroprotective effects of EA were evaluated by rota-rod test, neurological deficit scores and infarct volumes. Additionally, Nissl staining and immunostaining were performed to examine brain damage, rod formation, cellular apoptosis, and neuronal loss induced by ischemia. The activities of caspase-3, and expression levels of cofilin and p-cofilin in mitochondria and cytoplasm after ischemic injury were determined by Western blot. Results: Compared with the EC group, EA significantly improved neuromotor function and cognitive ability after ischemic stroke (P<0.05 or P<0.01). Therapeutic use of EA also resulted in a significant decrease of cofilin rod formation and microtubule-associated protein-2 (MAP2) degradation in the cortical penumbra area compared with the EC rats (P<0.01). Furthermore, Western blot analysis showed that EA stimulation significantly inhibited mitochondrial translocation of cofilin and caspase-3 cleavage (P<0.05 or P<0.01). Additionally, brain damage (infarct volume and neuropathy), cellular apoptosis and neuronal loss induced by ischemia were remarkably suppressed by EA in the cortical penumbra of rats (P<0.05 or P<0.01). Conclusion: EA treatment after ischemic stroke may attenuate ischemic brain injury and cellular apoptosis through the regulation of mitochondrial translocation of cofilin, a novel mechanism of EA therapy.     

Neuroprotective Effects of Grape Seed Procyanidin Extract on Ischemia-Reperfusion Brain Injury

Objective Oxidative stress (OS) plays a crucial role in ischemic stroke. Grape seed procyanidin extract (GSPE) was reported to be a critical regulator of OS. We hypothesized that GSPE might also be protective in ischemia-reperfusion brain injury. This study aimed to explore whether GSPE administration can protect mice from ischemia-reperfusion brain injury. Methods Transient middle cerebral artery occlusion (MCAO) was conducted followed by reperfusion for 24 hours to make ischemia-reperfusion brain injury in mice that received GSPE (MCAOG, n=60) or normal saline (MCAONS, n=60). Sham-operated mice (GSPE group and normal saline group) were set as controls. The neurological severity score (NSS) was used to evaluate neural function impairment 1 hour, 24 hour, 3 days and 7 days after MCAO. Mice underwent brain T2WI imaging with a 3T animal MRI scanner 24 hours after reperfusion, and the stroke volume of brains were calculated according to abnormal signal intensity. Immunohistopathological analysis of brain tissues at 24 h after reperfusion was performed for neuronal nuclear antigen (NeuN), CD34, Bcl-2, and Bax. Glutathione peroxidation (GSH-Px) activity and the level of malonaldehyde (MDA) of brain tissue were also examined. The above indexes were compared among the groups statistically. Results Significant functional improvement was observed 24 hours after MCAO in MCAOG group compared to MCAONS group (P<0.05). MCAOG group had smaller cerebral stroke volume (22.46 ± 11.45 mm3vs. 47.84±9.06 mm3,P<0.05) than MCAONS group 24 hours after MCAO. More mature NeuN-immunoreactive neurons and more CD34-positive cells in peri-infarct zones were observed in brain tissue of MCAOG mice 24 h after MCAO than that of MCAONS mice (bothP<0.05). MCAONS mice had significantly higher number of Bax-positive cells in brain tissue than MCAOG (P<0.05). The mean MDA level was significantly lower (P<0.05) and the GSH-Px activity was significantly higher (P<0.05) in brains of MCAOG mice compared to those of MCAONS mice. Conclusion GSPE administration protects mice from ischemia-reperfusion brain injury through attenuating oxidative stress and apoptosis, promoting angiogenesis, and activating antioxidant enzyme GSH-Px. GSPE may represent a new therapeutical direction for the treatment of ischemia-reperfusion brain injury.     

INTRANASAL DELIVERY OF NERVE GROWTH FACTOR TO PROTECT THE CENTRAL NERVOUS SYSTEM AGAINST ACUTE CEREBRAL INFARCTION

Objective To confirmed reliability and feasibility of intranasal nerve growth factor (NGF) bypassing the blood-brain barrier and its potential neuroprotective effects on acute cerebral ischemia. Methods (1) To assay NGF concentrations in different brain regions after middle cerebral artery occlusion (MCAO).Rats were randomly divided into intranasal (IN) NGF, intravenous (IV) NGF, and untreated group (n = 4). The concentrations of NGF of different brain regions in the three groups after MCAO were measured by ELISA. (2) To observe neuroprotective action of NGF on focal cerebral ischemic damage. Rats were randomly assigned to 4 groups: IN vehicle, 1N NGF,1V vehicle, 1V NGF (n = 8). Treatment was initiated 30 minutes after onset of MCAO and given again 24 hours later. Three neurologic behavioral tests were performed 24 and 48 hours following onset of MCAO. Corrected infarct volumes were determined 48 hours after onset of MCAO. Results The olfactory bulb in IN NGF group obtained the highest concentration (3252 pg/g) of NGF among all regions, followed by the hippocampus. The NGF concentrations in the olfactory bulb and hippocampus in IN NGF group were markedly higher than that in 1V NGF and control groups. The infarct volume in IN NGF group was markedly reduced by 38.8% compared with IN vehicle group. IN NGF group vestibulum function markedly improved compared with IN vehicle group at 24 and 48 hours after onset of MCAO (P24h = 0.02 and P48h = 0.04, respectively), Conolmion Intranasal NGF could pass through the blood-brain barrier, reach the central nervous system, reduce infarct volume, and improve neurologic function in rats following MCAO. Intranasal delivery of NGF may be a promising treatment for stroke.     

白细胞介素-18在小鼠缺血性脑卒中的损伤作用及其机制

目的:探讨白细胞介素（IL）-18在小鼠缺血性脑卒中的损伤作用及其机制。方法:采用C57BL/6J成年雄性小鼠构建光化学法诱导局灶性大脑皮层缺血性脑卒中模型,随机分为对照组、脑梗死组、IL-18分泌型结合蛋白（IL-18BP）治疗组,每组18只。术后14 d进行神经功能评分并进行HE染色测量小鼠脑梗死面积。术后3 d实时荧光定量PCR检测小鼠脑组织中肿瘤坏死因子-α（TNF-α）、IL-1β、IL-6和IL-10的表达,流式细胞术检测小鼠脑组织中小胶质细胞表型。结果:与脑梗死组相比,IL-18BP治疗组神经功能显著改善并且脑梗死面积显著降低（t=2.750、2.235,均P<0.05）。IL-18BP治疗组脑组织中TNF-α、IL-1β和IL-6的表达显著降低（t=3.091、2.328、3.443,均P<0.05）,IL-10的表达升高（t=4.997,P<0.05）。与脑梗死组相比,IL-18BP治疗组脑组织中M1型小胶质细胞数量明显降低（t=4.779,P<0.05）,M2型小胶质细胞数量明显升高（t=2.619,P<0.05）。结论:在缺血性脑卒中,IL-18可介导免疫炎性损伤并发挥调节作用。     

大黄酚对局灶性脑缺血再灌注小鼠缺血半暗带区HIF-1α与VEGF表达的影响

目的 研究大黄酚(chrysophanol, CHR)对局灶性脑缺血再灌注小鼠缺血半暗带区缺氧诱导因子-1α(hypoxia inducible factor-1α, HIF-1α)及血管内皮生长因子(vascular endothelial growth factor, VEGF)表达的影响,探究CHR对脑缺血再灌注损伤的长期脑保护机制。方法 采用数字表法随机将18只健康雄性2月龄C57BL小鼠分为3组:假手术(Sham)组、大脑中动脉梗塞(middle cerebral artery occlusion, MCAO)组、CHR组(自造模当日至脑缺血再灌注后14 d按0.1 mg·kg^-1·d^-1腹腔注射CHR),每组6只。线栓法制作小鼠MCAO模型,缺血45 min后拔出线栓进行再灌注。于再灌注14 d将小鼠处死、取脑,通过免疫荧光染色方法检测脑组织冰冻切片缺血半暗带区内HIF-1α及VEGF水平,并通过免疫荧光双标染色方法确定HIF-1α、VEGF是否在神经元中表达。结果 1)Sham组小鼠脑内偶见HIF-1α阳性细胞。MCAO组小鼠脑缺血半暗带区HIF-1α的表达水平比Sham组显著升高(P<0.05)。经CHR治疗14 d的脑缺血再灌注小鼠半暗带区内HIF-1α的表达水平比MCAO组显著减少(P<0.05)。2)Sham组小鼠脑内可见大量的VEGF阳性细胞。MCAO组小鼠脑缺血半暗带区VEGF的表达水平比Sham组显著降低(P<0.05)。经CHR治疗14 d的脑缺血再灌注小鼠半暗带区内VEGF的表达水平比MCAO组显著增加(P<0.05)。3)在小鼠脑缺血半暗带区,HIF-1α或VEGF分别与神经元标志物NeuN共定位。结论 CHR可能通过下调HIF-1α的表达水平、上调VEGF的表达水平,减少神经元损伤,从而对脑缺血再灌注损伤发挥长期神经保护作用。     

Neuroprotective Effects of Grape Seed Procyanidin Extract on Ischemia-Reperfusion Brain Injury△

Objective Oxidative stress (OS) plays a crucial role in ischemic stroke. Grape seed procyanidin extract (GSPE) was reported to be a critical regulator of OS. We hypothesized that GSPE might also be protective in ischemia-reperfusion brain injury. This study aimed to explore whether GSPE administration can protect mice from ischemia-reperfusion brain injury.Methods Transient middle cerebral artery occlusion (MCAO) was conducted followed by reperfusion for 24 hours to make ischemia-reperfusion brain injury in mice that received GSPE (MCAOG, n=60) or normal saline (MCAONS, n=60). Sham-operated mice (GSPE group and normal saline group) were set as controls. The neurological severity score (NSS) was used to evaluate neural function impairment 1 hour, 24 hour, 3 days and 7 days after MCAO. Mice underwent brain T2WI imaging with a 3T animal MRI scanner 24 hours after reperfusion, and the stroke volume of brains were calculated according to abnormal signal intensity. Immunohistopathological analysis of brain tissues at 24 h after reperfusion was performed for neuronal nuclear antigen (NeuN), CD34, Bcl-2, and Bax. Glutathione peroxidation (GSH-Px) activity and the level of malonaldehyde (MDA) of brain tissue were also examined. The above indexes were compared among the groups statistically.Results Significant functional improvement was observed 24 hours after MCAO in MCAOG group compared to MCAONS group (P<0.05). MCAOG group had smaller cerebral stroke volume (22.46 ± 11.45 mm³ vs. 47.84±9.06 mm³, P<0.05) than MCAONS group 24 hours after MCAO. More mature NeuN-immunoreactive neurons and more CD34-positive cells in peri-infarct zones were observed in brain tissue of MCAOG mice 24 h after MCAO than that of MCAONS mice (both P<0.05). MCAONS mice had significantly higher number of Bax-positive cells in brain tissue than MCAOG (P<0.05). The mean MDA level was significantly lower (P<0.05) and the GSH-Px activity was significantly higher (P<0.05) in brains of MCAOG mice compared to those of MCAONS mice.Conclusion GSPE administration protects mice from ischemia-reperfusion brain injury through attenuating oxidative stress and apoptosis, promoting angiogenesis, and activating antioxidant enzyme GSH-Px. GSPE may represent a new therapeutical direction for the treatment of ischemia-reperfusion brain injury.     

米诺环素介导小胶质细胞形态转换减轻缺血性脑卒中早期脑损伤的机制研究

目的 观察大鼠缺血性脑损伤早期的病理生理特点,探讨米诺环素在缺血性脑损伤早期的神经保护效应及可能机制.方法 线栓法制备大鼠缺血性脑损伤(middle cerebral artery occlusion,MCAO)模型,在MCAO 6、24 h分别给予米诺环素腹腔注射,通过TTC染色观察脑梗死面积,TUNEL染色及尼氏染色检测神经元凋亡情况;免疫荧光染色了解小胶质细胞形态变化以及Western blot检测凋亡通路NF-κB表达情况.使用Morris水迷宫检测手术28 d后大鼠学习记忆能力.结果 米诺环素减少MCAO 6 h后大鼠脑梗死面积[(43.0±5.3)cm3 vs (36.0±6.8)cm3, P<0.01],减少脑神经元凋亡数量并促进M2型(神经保护型)小胶质细胞增殖,MCAO 24 h后米诺环素治疗不能改善脑梗死以及神经元细胞凋亡(P>0.05).米诺环素显著减少MCAO 6 h诱导的凋亡信号通路蛋白NF-κB的表达(P<0.01).MCAO 28 d后大鼠学习记忆能力较假手术组显著降低;米诺环素显著改善MCAO后学习记忆能力(P<0.05),而MCAO 24 h后米诺环素不能改善大鼠神经功能障碍,差异无统计学意义(P>0.05).结论 米诺环素能促进缺血性脑卒中早期活化小胶质细胞向M2型转化并抑制凋亡信号通路,减轻脑缺血诱导的神经炎症及细胞凋亡,改善大鼠学习记忆功能.     

Protective Effects of Focal Ischemic Preconditioning and HSP70 Expression on Middle Cerebral Artery Occlusion in Rats

A brief ischemic event (ie, ischemic preconditioning, (PC) can result in a subsequent resistance to severe ischemic tissue injury, i.e., ischemic tolerance (IT). The phenomenon has been described in several organs (espe- cially in brain and heart). Several studies on brain toler- ance have identified the protective effects of transient focal ischemia to preceding permanent focal stroke and its consequences on neurological deficits. However, little is known about the molecular underpinning of…