莫诺苷对大鼠局灶性脑缺血再灌注损伤神经功能的影响

目的观察中药山茱萸的有效成分莫诺苷对大鼠局灶性脑缺血再灌注后神经功能的影响。方法Wistar雄性大鼠随机分为假手术组、模型组、莫诺苷小剂量组（30 mg/kg）、莫诺苷中剂量组（90 mg/kg）、莫诺苷大剂量组（270 mg/kg）、维生素E（VE）（35 mg/kg）,采用线栓法制作大鼠局灶性脑缺血再灌注模型,缺血30 min后再灌注3 d,应用Zea Longa法、爬网格、平行木、吊绳、Ludmila Belayev 12分评分法,观察莫诺苷对神经功能缺损的改善作用。结果与模型组比较,莫诺苷给药组（小、中、大剂量）Zea Longa法评分均差异极显著（P〈0.01）;与模型组比较,莫诺苷给药组（小、中、大剂量）吊绳法评分均差异极显著（P〈0.01）;Ludmila Belayev 12分评分法评分与模型组比较,莫诺苷大剂量组差异极显著（P〈0.001）,中剂量组差异极显著（P〈0.01）。结论莫诺苷对局灶性脑缺血再灌注模型大鼠有改善行为学评分的作用。     

插线法制作大鼠局灶性脑缺血再灌流模型方法的改进

目的 探索插线法制作大鼠局灶性脑缺血再灌流模型方法的改进。方法 在前人制作模型方法基础上进行部分改进,用提线法阻断血流和在颈外动脉上剪小口进行插线。结果 缩短了手术时间,简化了模型制作过程,提高模型成功率。结论 本法复制插线法制作大鼠局灶性脑缺血再灌流模型,省时,操作简便,提高成功率。     

AMD3100对局灶脑缺血/再灌注大鼠大脑皮质血管再生的影响

目的探讨AMD3100阻断SDF-1/CXCR4轴后,对局灶脑缺血/再灌注大鼠缺血半暗带血管再生的影响。方法将SD大鼠随机分为假手术组(S组)、模型组(IR组)、AMD3100组(IRA组)、生理盐水组(IRN组)。采用线栓法制备大鼠局灶脑缺血/再灌注模型,缺血2h后将IR、IRA和IRN组分为再灌注12h,1、3和7d四个亚组。HE染色观察局灶脑缺血/再灌注后大脑皮质病理变化。免疫组化法检测CD31在缺血半暗带表达。荧光定量PCR检测外周血中AC133mRNA表达。结果与IRN组比较,IRA 12h外周血中AC133mRNA显著升高,第1d升高达峰值(P0.01),IRA 3dAC133mRNA表达比IRA1d显著减少(P0.05);与IRN组比较,IRA组CD31阳性血管密度在第1d无显著变化(P0.05),第3和7d血管密度显著减少(P0.01);IRA 7d梗死区由大量坏死神经细胞和泡沫细胞填充,坏死较严重。结论持续注射AMD3100能动员干/祖细胞快速进入外周血,但可能抑制局灶脑缺血/再灌注大鼠缺血半暗带血管再生,加重梗死区坏死。     

昼夜节律对大鼠局灶性脑缺血再灌注损伤的影响

目的观察昼夜节律对大鼠局灶性脑缺血再灌注损伤的影响。方法将大鼠置人工光(6:00-18:00)暗(18:00-6:00)环境中,适应性饲养4周。然后将大鼠随机分为6:00组、12:00组、18:00组和24:00组,分别在相应的时间点采用线栓法制备局灶性脑缺血再灌注损伤大鼠模型。再灌注24 h后,观察昼夜节律对模型大鼠神经损伤症状,脑梗死范围,脑组织超氧化物歧化酶(SOD)活性和MDA含量,脑细胞内游离Ca2+浓度,血清中内皮素(ET)和血管内皮细胞生长因子(VEGF),以及脑组织病理变化的影响。结果各组大鼠均具有明显的神经损伤症状,其中24:00组大鼠的神经损伤症状明显轻于6:00组;6:00、12:00组大鼠脑梗死范围明显大于18:00、24:00组;18:00、24:00组大鼠SOD活性均显著高于6:00、12:00组,MDA含量均显著低于6:00组;24:00组大鼠脑细胞内游离Ca2+浓度显著低于6:00和12:00组,18:00组大鼠脑细胞内游离Ca2+浓度显著低于6:00;18:00、24:00组大鼠血清ET含量显著低于6:00组大鼠;24:00组大鼠血清VEGF含量显著低于6:00、12:00组;18:00、24:00组大鼠脑组织病理变化明显轻于6:00、12:00组。结论昼夜节律对大鼠脑缺血再灌注损伤具有显著影响。     

线栓法制备不同体重KM小鼠局灶性脑缺血/再灌注模型的建立及评价

目的线栓法制备小鼠脑缺血/再灌注模型,并探讨影响该模型稳定性的因素。方法 KM小鼠100只,雌雄各半,体重20～39 g,分别将雌雄KM小鼠随机分为假手术组(n=20)和不同体重实验组(n=80)。其中实验组按小鼠体重分为以下8组:A组(♂,20～24 g)、B组(♂,25～29 g)、C组(♂,30～34 g)、D组(♂,35～39g),E组(♀,20～24 g)、F组(♀,25～29 g)、G组(♀,30～34 g)、H组(♀,35～39 g),每组10只。线栓栓塞法制备局灶性脑缺血/再灌注模型,选用单丝尼龙线(直径0.128～0.180 mm),液体石蜡处理浸泡后,从右侧颈总动脉进线,阻塞大脑中动脉血流,再灌注时拔出线栓,并对模型进行评价,采用的主要观察指标是神经功能损伤评分和2,3,5-三苯基氯化四氮唑(TTC)染色。结果同假手术相比,不同体重实验组小鼠脑缺血再灌注后,出现神经行为功能异常,TTC染色脑组织显示出明显的梗塞灶。与C、D组相比,A、B两组行为学评分以及梗死灶体积显著性增加(P<0.01);与G、H组相比,E、F两组的行为学评分以及梗死灶体积也显著性增加(P<0.01,P<0.05)。雄性KM小鼠与雌性相比,成模率较高且死亡率相对较低。此外,A组与E组相比,其行为学评分显著增加(P<0.01);B组与F组相比,小鼠行为学评分以及脑梗塞体积显著增加(P<0.01)。结论体重在25 g左右的雄性KM小鼠制备模型成功率较高,死亡率较低,适合建立小鼠局灶性脑缺血/再灌注模型。     

CGRP和NGF对局灶性脑缺血再灌注大鼠海马HSP70蛋白表达的影响

目的探讨外源性降钙素基因相关肽(CGRP)和神经生长因子(NGF)对局灶性脑缺血再灌注大鼠海马热休克蛋白70(HSP70)表达的影响.方法用线栓法制备大鼠大脑中动脉阻塞(MCAO)模型,应用免疫组化和显微图像分析方法检测局灶性脑缺血再灌注大鼠海马HSP70的表达.结果假手术组海马未见HSP70阳性细胞,缺血再灌注组海马HSP70阳性细胞数增多.分别注射CGRP或NGF后海马区HSP70阳性细胞平均光密度值明显高于缺血再灌注组(P<0.01),二者合用时平均光密度值较比单独应用高(P<0.05).结论CGRP和NGF上调缺血神经元HSP70的表达,二者合用作用更强,对缺血神经元恢复有促进作用.     

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。     

大鼠局灶性脑缺血后扩布性阻抑的发作及电针对其影响

目的：观察Wistar大鼠局灶性脑缺血后扩布性阻抑（SD）的发作情况及缺血后电针的影响。方法：线检法闭塞大鼠大脑中动脉,制备局灶性脑缺血模型。用神经电生理、神经病理等方法检测局灶性脑缺血后3h内SD发作情况及电针“合谷”穴（LI4）对SD的影响。结果：电针可减少局灶性脑缺血时SD的发作。结论：电针减少局灶性脑缺血时SD的发作,可能与电针缩小局灶性脑梗塞体积有关。     

Ucf-101对大鼠局灶性脑缺血再灌注后Caspase-3蛋白表达的影响

目的观察Ucf—101对大鼠脑缺血再灌注后神经元caspase-3蛋白表达及细胞凋亡的影响,研究其对缺血性脑损伤是否具有保护作用。方法将36只雄性WiStar大鼠随机分为3组：假手术组、缺血组及Ucf—101组,采用线栓法建立大鼠右侧大脑中动脉闭塞（middle cerebral artery occlusion,MCAO）2h再灌注模型,于再灌注后6h和24h断头取脑,采用TTC法测梗死体积,TUNEL法原位标记DNA片段,检测TUNEL阳性细胞的变化,免疫组化法观察脑皮质神经元caspase-3的表达。结果脑缺血再灌注后不同时间点（6h、24h）,Ucf-101组与缺血组相比梗死体积明显缩小,有显著性差异（P〈0．05）;假手术组未见梗死现象。缺血组TUNEL阳性细胞数较假手术组明显增多（P〈0．05）,脑皮质caspase-3的表达较假手术组亦显著增强（P〈0．05）,给予Ucf-101处理后,TUNEL阳性细胞数较缺血组明显减少（P〈0．05）,caspase-3的表达较缺血组亦明显减弱（P〈0．05）。结论Ucf-101能有效地抑制脑缺血再灌注损伤,下调脑皮质神经元Caspase-3蛋白的表达,抑制神经元的凋亡,发挥神经保护作用。     

大鼠局灶性脑缺血模型的有效制备

目的比较三种不同手术方法制作大鼠永久性脑缺血模型的效果,包括死亡率、神经功能评分、脑梗死体积、手术效率。方法将采用不同手术方法制备脑缺血模型的大鼠随机分为三组。1组在术中分别结扎颈总动脉（CCA）、颈外动脉（ECA）、枕动脉、翼腭动脉,并且用动脉夹对颈内动脉（ICA）进行临时夹闭;2组在术中分别结扎颈总动脉、颈外动脉,暴露枕动脉和翼腭动脉但不结扎,用丝线悬挂颈内动脉而不是用动脉夹夹闭,线栓在显微镜直视下插入颈内动脉越过翼腭动脉起始点至大脑中动脉分叉处;3组只暴露颈总动脉、颈外动脉和颈内动脉,结扎颈总动脉、颈外动脉,丝线悬挂颈内动脉,显微镜下将线栓盲插至颈内动脉大脑中动脉分叉处。分别检测三组模型的死亡率、神经功能评分、梗死体积、手术时间。结果第3组制作动物模型的方法所花费时间平均为17.5 min,死亡率较低,神经功能评分及梗死体积稳定。结论采用第3组手术方法可以缩短手术时间,提高手术效率,能够高效地制作出更加稳定的可用于临床实验的大鼠脑缺血模型。     

蕲蛇酶治疗脑梗死的临床研究

目的评价蕲蛇酶(Acutase)治疗脑梗死的疗效和安全性。方法选择符合AcutaseⅢ期临床试验方案的要求的脑梗死患者应用Acutase0．75u加人生理盐水250ml中,静脉滴注3h以上,每日1次,连用2周为1个疗程。在试验过程中,观察和如实记录各种不良反应及出现时间,严重程度及处理方法、效果。结果 Acutase治疗脑梗死的显效率为83．80％,总有效率为99．30％,不良反应少而短暂;同时,Acutase具有改善患者日常生活能力的作用。结论Acutase治疗脑梗死安全有效。     

龙津降纤酶治疗急性缺血性卒中临床疗效观察

目的 观察龙津降纤酶对急性缺血性情卒中的治疗效果。方法 选择诊断明确的急性因性卒中病人８０例,随机分为２组,对照组４０例,５％葡萄糖液５００ｍｌ加血塞通小射液０．４ｇ静脉滴注,连用１４天,降纤酶组４０例,入院后第１～３天分别给降酶１０ｕ加入生理盐水１００ｍｌ,静脉主１ｈ以上,按临床神经功能缺失程度评分标准于治疗前及治疗在进行疗效评定。结果 降纤酶组总有效率达９２．５％,显效率为７２．５％,明显优于     

臭氧大自血疗法治疗急性缺血性脑梗死患者的临床疗效评估

目的:研究臭氧大自血疗法治疗急性缺血性脑梗死患者的临床疗效,为临床治疗提供依据。方法:选取2014年10月到2015年8月我院收治的急性缺血性脑梗死患者210例,按照随机数字表法将患者分为研究组和对照组,每组105例,两组均给予常规治疗,研究组在常规治疗的基础上给予臭氧大自血疗法,应用barthel指数评定日常生活活动能力,应用美国国立卫生研究院卒中量表(NIHSS)评价神经功能缺损,比较两组临床疗效,治疗前后甘油三酯(TG)、总胆固醇(TC)、高密度脂蛋白(HLD-C)和低密度脂蛋白(LDL-C),并比较两组不良反应。结果:研究组总有效率为87.6%(92/105)显著高于对照组的73.3%(77/105),比较差异具有统计学意义(P0.05);治疗后两组NIHSS评分显著降低,barthel评分显著升高,且研究组NIHSS评分显著低于对照组,barthel评分显著高于对照组,比较差异具有统计学意义(P0.05);治疗后两组TG、TC和LDL-C均显著降低,HDL-C显著升高,且研究组TG、TC和LDL-C低于对照组,HDL-C高于对照组,比较差异具有统计学意义(P0.05);两组不良反应比较无统计学意义(P0.05)。结论:臭氧大自血疗法治疗急性缺血性脑梗死疗效较好,能明显改善患者的神经功能和日常生活。     

Free Fatty Acids, Neutral Glycerides, and Phosphoglycerides in Transient Focal Cerebral Ischemia

Abstract: Cerebral ischemia is known to cause an increase in levels of free fatty acids (FFAs) and diacylglycerols (DGs), although the mechanism(s) leading to these changes is not well understood. In this study, we examined FFA and DG levels along with those of other lipids in rats during and after transient focal cerebral ischemia induced by temporary occlusion of the right middle cerebral artery (MCA) and both common carotid arteries. During the duration of ischemia (15–60 min), there was a time-dependent increase (two- to 10-fold) in FFA levels in the right MCA cortex, whereas levels of DG and other lipids were not altered appreciably. FFA levels in right MCA cortex returned to near control values after reperfusion. However, following a 60-min ischemic insult, there was a second phase of FFA level increase that was evident after 16 h. The FFAs accumulated during the ischemia period were different from those after reperfusion, suggesting differences in mechanisms for their release. During the second phase of FFA release, there were increases in levels of DGs and triacylglycerols (TGs) with unusually high proportions of 20:4(n-6) and 22:6(n-3). The increases in FFA, DG, and TG levels were marked by a decrease in content of phosphoglycerides (PGs). It is interesting that the increases in levels of FFAs and neutral glycerides accounted only for 10% of the total PGs depleted. The lipid changes during this reperfusion period correlated well with the development of cortical infarct. Because FFAs are potent inhibitors of mitochondrial respiratory function, the time-dependent FFA accumulation during the ischemia period may be an important determinant for the extent of ischemia-induced injury after reperfusion.     

Activation of Calpain,Cathepsin-b and Caspase-3 during Transient Focal Cerebral Ischemia in Rat Model

Calpains, cathepsins and caspases play crucial role in mediating cell death. In the present study we observed a cascade of events involving the three proteases during middle cerebral artery occlusion (MCAo) in Wistar rats. The rats were MCA occluded and reperfused at various time points. We observed a maximal increase in the levels of calpains during 1h and 12 h after reperfusion than permanently occluded rats. Further, these levels were reduced by 1st and 3rd day of reperfusion. Similarly the cathepsin-b levels were significantly increased during 1h and 12 h, of reperfusion, followed by activation of caspase-3 which reached maximal levels by 1st and 3rd day of reperfusion. The sequential activation of calpains, cathepsin-b and cleaved caspase-3 is evident by the Western blot analysis which was further confirmed by the cleavage of substrates like PSD-95 and spectrin. The differences in the regional distribution and elevation of these proteases at different reperfusion time periods indicates that differential mode of cell death occur in the brain during cerebral ischemia in rat model.     

Effect of AMPA on Cerebral Cortical Oxygen Balance of Ischemic Rat Brain

We tested the hypothesis that the excitatory neurotransmitter receptor agonist, alpha amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), would worsen cerebral cortical oxygen supply/consumption balance during focal ischemia. In this study, we compared regional cerebral blood flow, arterial and venous O₂ saturation, O₂ extraction and oxygen consumption of ischemic and AMPA treated ischemic and control regions of rat brain. Ischemia was induced by middle cerebral artery (MCA) occlusion in isoflurane (1.4%) anesthetized Wistar rats. Twenty minutes after MCA occlusion, 10^–5 M AMPA was applied to the ischemic cortex (IC) for a period of 40 min; the fluid was changed every 10 min. After 1 hr of ischemia, animals were sacrificed and regional cerebral blood flow (rCBF) was determined using the C¹⁴-iodoantipyrine autoradiographic technique. Regional arterial and venous oxygen saturation were determined microspectrophotometrically. In control, the cerebral blood flow and oxygen consumption of the IC were significantly lower than the contralateral cortex (rCBF: 46 ± 20 vs. 81 ± 39 ml/min/100g, O₂ consumption: 2.8 ± 1.4 vs. 3.6 ± 1.4 ml O₂/min/100g). 10^–5 M AMPA did not significantly alter regional cerebral blood flow and oxygen consumption of the IC, but did decrease the average venous O₂ saturation of the IC from 50.2 ± 3.9% to 46.7 ± 1.6%. AMPA also significantly increased the frequency of small veins with less than 45% O₂ saturation in the IC (8 out of 56 veins in IC vs. 18 out of 56 veins in AMPA treated IC). Thus, topical application of 10^–5 M AMPA to the ischemic area worsens cerebral O₂ balance and suggests that excitatory amino acids contribute to the degree of cerebral ischemia.     

Effect of Resveratrol on Antioxidant Enzyme Activities in the Brain of Healthy Rat

We have studied the effect of resveratrol on lipoperoxidation and antioxidant enzyme activity level in the brain of healthy rats. When intraperitoneally administered, resveratrol significantly and dose dependently decreased brain malondialdehyde level. Resveratrol also increased in a dose-dependent way brain superoxide dismutase, catalase and peroxidase activities. Optimal effect on antioxidant enzyme and lipoperoxidation products were obtained with resveratrol concentration of 12.5 mg/kg body wt. Native polyacrylamide gel electrophoresis analysis of antioxidant isoenzymes revealed that resveratrol up regulated at least two acidic superoxide dismutase isoforms called A₁ and A₂, two basic isoforms called B₁ and B₂. Resveratrol also up regulated two catalase isoforms and a broad peroxidase band corresponding to several isoforms. All these findings suggest that resveratrol is able to cross the blood brain barrier and exerts potent antioxidant features. Resveratrol also exerts neuroprotective properties by up regulating several detoxifying enzymes, most of which are iron proteins.     

Reduction of Mitochondrial Electron Transport Complex Activity Is Restricted to the Ischemic Focus After Transient Focal Cerebral Ischemia in Rats: A Histochemical Volumetric Analysis

Using histochemical methods offering high topographical resolution for evaluation of changes in the ischemic focus and the penumbra, the mitochondrial electron transport chain (ETC) complexes I, II, and IV were examined in rats subjected to 2 h of proximal occlusion of the middle cerebral artery (MCAO) followed by no reperfusion, 1 h reperfusion, 4 h reperfusion, or 4 h reperfusion plus treatment with the free radical scavenger -PBN. Serial brain cryosections were histochemically stained to visualize activity of complexes I, II, and IV, and the volumes of tissue with reduced activity in the ipsilateral cortex and caudate putamen were measured by densitometric image analysis. Reductions in complex I, II, and IV activity were restricted to areas in the ischemic foci in cortex and caudate putamen, which microscopically displayed signs of early morphological damage. In cortex, the tissue volume with reduced activity did not change significantly during reperfusion but progressively increased in the caudate putamen, possibly reflecting a faster maturation of morphological damage in this region. Treatment with -PBN did not affect the observed reductions in activities. We deduce that inhibition of mitochondrial ETC complex activity does not play a critical role for recruitment of the penumbra in the infarction process.     

Delayed Treatment with Carboxy-PTIO Permits a 4-h Therapeutic Window of Opportunity and Prevents Against Ischemia-Induced Energy Depletion Following Permanent Focal Cerebral Ischemia in Mice

We examined whether a nitric oxide scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-l-oxyl-3-oxide (carboxy-PTIO), could offer neuroprotective actions and improve cerebral energy metabolism in a model of stroke. Sixty C57BL/10J mice were given either carboxy-PTIO (0.3–1.2 mg/kg) or vehicle intraperitoneally, 0.5 h after permanent middle cerebral artery occlusion, to evaluate the dose–response effects. An additional 70 animals received carboxy-PTIO (0.6 mg/kg) or vehicle, 2–6 h post-ischemia, for establishing the therapeutic window. Subgroups of animals, treated with carboxy-PTIO (0.6 mg/kg) or vehicle, were used for measuring cerebral bioenergetic metabolites (ATP, ADP, AMP, adenosine). Mice treated with carboxy-PTIO (0.6 mg/kg) had dose-specifically reduced brain infarction, significantly by 27–30% (P < 0.05), even when therapy was delayed up to 4 h after the ischemic insult (P < 0.05). Four hour post-ischemia, ATP depleted in the ischemic hemisphere (P < 0.05). Administration with carboxy-PTIO not only improved the recovery of ATP in the ischemic hemisphere (P < 0.05), but also enhanced adenosine content across the ischemic and non-ischemic hemispheres (P < 0.05). The neuroprotection of carboxy-PTIO may be partly attributed to the beneficial effects of improving cerebral energy metabolism.