针刺抗脑缺血性神经元凋亡作用与机制的研究

本研究在肯定针刺抗缺血性神经元凋亡的基础上,进一步探讨针刺对内源性促凋亡因素及抑制凋亡因素的调整作用,寻找针刺对元保护作用的切入点及作用机制。结果表明：（１）针刺能减轻脑缺血导致的神经缺损行为异常,缩小梗塞面积;（２）ＰＩ荧光染色及ＴＵＮＥＬ染以显示：大鼠缺血皮层梗塞区有大量凋亡阳性信号细胞,电针后细胞凋亡受到明显抑制测定ＮＯ含量及观察ｃＮＯＳ和ｉＮＯＳ免疫活性显示：大鼠脑缺血－再灌注后脑内ＮＯ水     

慢病毒载体介导Bdnf基因修饰的骨髓间质干细胞移植治疗脑梗死

为研究经Bdnf基因修饰的骨髓间质干细胞(Mesenchymal stem cells, MSCs)对脑梗死的协同治疗作用, 构建带有大鼠Bdnf基因之慢病毒载体, 并感染大鼠骨髓间质干细胞(Rat mesenchymal stem cells, rMSCs)。运用线栓法制备大鼠大脑中动脉栓塞模型, 经尾静脉注射移植, 对照组注射0.1 mol/L磷酸盐缓冲液(PBS)1 mL, Bdnf-rMSCs和Mock-rMSCs组分别注射Bdnf-rMSCs细胞悬液以及未插入目的基因的空病毒载体感染后的rMSCs细胞悬液各1 mL。各组大鼠分别于术后24 h、移植后2周及2月应用modified Neurological Severity Scores (mNSS)评价神经功能状况。结果显示, 与对照组相比, Mock-rMSCs及Bdnf-rMSCs移植组神经功能改善明显, mNSS评分差异有统计学意义(P<0.001), 而且Bdnf-rMSCs移植组明显优于Mock-rMSCs移植组(P<0.001)。移植后2周及2月, 与对照组相比两移植组梗死区脑组织结构恢复较好, 均可见EGFP阳性细胞在梗死区及其周边区聚集并存活, 并有部分细胞出现神经元样改变。Bdnf- rMSCs移植组中移植细胞大量表达BDNF, 两移植组中均有部分植入细胞表达神经细胞表面标志物。研究表明Bdnf基因修饰的rMSCs经静脉移植后可迁移至脑梗死灶周围, 向神经细胞分化并长期存活。移植后的干细胞可与其分泌的BDNF协同促进脑梗死后神经功能恢复, 这为将来基因工程干细胞移植治疗脑梗死提供了实验依据。     

Involvement of free radicals in cerebral vascular reperfusion injury evaluated in a transient focal cerebral ischemia model of rat

Free radicals have been suggested to be largely involved in the genesis of ischemic brain damage, as shown in the protective effects of alpha-phenyl-N-tert-butyl nitrone (PBN), a spin trapping agent, against ischemic cerebral injury. In the present study, the effects of PBN as well as MCI-186, a newly-developed free radical scavenger, and oxypurinol, an inhibitor of xanthine oxidase, were evaluated in a rat transient middle cerebral aretery (MCA) occlusion model to clarify the possible role of free radicals in the reperfusion injury of brain. The volume of cerebral infarction, induced by 2-h occlusion and subsequent 2-h reperfusion of MCA in Fisher-344 rats, was evaluated. The administration of PBN (100 mg/kg) and MCI-186 (100 mg/kg) just before reperfusion of MCA significantly reduced the infarction volume. In contrast, oxypurinol (100 mg/kg) failed to show any preventive effect on the infarction. These results suggest that free radical formation is involved in the cerebral damage induced by ischemia-reperfusion of MCA, and that hydroxyl radical is responsible for the reperfusion injury after transient focal brain ischemia. It is also suggested that xanthine oxidase is not a major source of free radicals.     

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。     

Effect of dietary sesame oil as antioxidant on brain hippocampus of rat in focal cerebral ischemia

Oxidative stress may be regarded as an imbalance between free radical production and opposing antioxidant defenses. Free radical oxidative stress is implicated in rat cerebral ischemia and naturaceutical antioxidants are dietary supplements that have been reported to have neuroprotective activity. Many studies have reported dietary sesame oil (SO) as an effective antioxidant. In the present study the neuroprotective effect of dietary SO was evaluated against middle cerebral artery occlusion (MCAO)-induced cerebral ischemia injury in rats. Rats were fed on diet (20% SO) for 15 days. The middle cerebral artery of adult male Wistar rat was occluded for 2 h and reperfused for 22 h. The antioxidant properties of brain were measured as levels of reduced glutathione (GSH), glutathione-S-transferase (GST), glutathione peroxide (GPx), glutathione reductase (GR), catalase (CAT), superoxide dismutase (SOD) and thiobarbituric acid reactive substance (TBARS). A decrease in the activity of all the enzymatic and non-enzymatic antioxidants was observed along with an increase in lipid peroxidation (LPO) in MCAO group. The neurobehavioral activity of rats was also observed by using videopath analyzer. Dietary SO improved the antioxidant status in MCAO+SO group when compared with MCAO group. The results of neurobehavioral activity also support our biochemical data. The results obtained suggest protective effect of SO against cerebral ischemia in rat brain through their antioxidant properties.     

The protective effects and potential mechanism of Calpain inhibitor Calpeptin against focal cerebral ischemia–reperfusion injury in rats

To demonstrate the protective effects of Calpeptin as the Calpain inhibitor against focal cerebral ischemia–reperfusion injury in rats and to explore it’s possible mechanism. 96 rats were randomly divided into four groups. The model of middle cerebral artery occlusion was used for the research of focal cerebral ischemia. Using this animal model, the effects of Calpeptin on the neurological functions, infarction volume and infarction volume percentage of brain, Caspase-3 expression and neuronal apoptosis in hippocampal CA1 sector after focal cerebral ischemia–reperfusion injury in rats were investigated. The current results confirmed that Calpeptin as the Calpain inhibitor might paly an important role for neuroprotection against focal cerebral ischemia–reperfusion injury. Calpeptin could reduce the neuronal apoptosis in hippocampal CA1 sector when the rats was subjected to the focal cerebral ischemia–reperfusion, the potential mechanism might be related to the inhibition of the expression of Caspase-3 by Calpeptin. However, it is still unknown to what the exact mechanism of Calpeptin inhibits the activation of Caspase-3 in this process. Therefore, further research needs to be done to unravel the underlying mechanisms in the future.     

神经调节素对大鼠脑缺血再灌注损伤后炎症反应的抑制作用和机制研究

目的：研究神经调节素及基质金属蛋白酶-9对于小鼠大脑缺血再灌注损伤后炎症反应的抑制作用和机制。方法：选取100只成年雄性大鼠,随机分成对照和治疗组。采用线栓方法由颈内到颈外进行插线处理,造成大脑中动脉处于闭塞状态的再灌注动物模型。治疗组颈动脉进行注射少量NRG-1β干预性治疗,通过氯化三苯基四氮唑（TTC）检查脑梗塞范围,细胞凋亡采用原住脱氧核糖核苷酸末端转移酶介导缺口末端进行标记,采用免疫组织化学、免疫荧光双标记法及免疫印迹法观察脑组织基质金属蛋白酶-9（MMP-9）表达。结果：脑缺血再灌注损伤后,随时间延长及缺氧,对照组大鼠大脑皮质和纹状体区脑组织细胞凋亡,并且胶质细胞MMP-9蛋白表达逐渐增加。治疗组大鼠经注射NRG-1β干预性治疗后,缺血脑组织梗死范围及其细胞凋亡数量相对呈明显下降趋势。胶质细胞MMP-9表达呈降低趋势。结论：大鼠脑缺血再灌注损伤后体内NRG-1β抑制胶质细胞MMP-9的表达,控制缺血脑组织梗死的范围并抑制正常细胞的凋亡,发挥了重要的抗炎作用,可作为对于大脑缺血再灌注损伤的研究新靶点。     

高压氧对局灶性脑缺血后细胞凋亡的影响及其机制

目的：观察不同时间点高压氧（HBO）治疗对短暂性脑缺血的作用,并探讨其对细胞凋亡的影响。方法：在客观监测局部脑血流的条件下,大鼠经历短暂脑缺血后3h,6h,12h应用HBO治疗,24h后行神经功能评分和梗死体积测定,免疫组化染色各组Bcl-2、Bax、活性Caspase-3、活性Caspase-9以及TUNEL法检测细胞凋亡。结果：缺血后3h HBO治疗减少70％梗死体积,缺血后6hHBO治疗则减少梗死体积约44％,早期应用HBO治疗增加半暗带区细胞内Bcl-2的表达,减少活性Caspase-9和活性Caspase-3以及TUNEL阳性细胞数;缺血后12h应用HBO治疗却恶化神经功能,扩大梗死范围,而对上述凋亡各指标无影响。结论：HBO治疗短暂性局灶脑缺血具有时间窗,应争取在缺血后6h内应用HB0治疗,其早期治疗的神经保护作用与抑制细胞凋亡有关。     

The TRIF-dependent signaling pathway is not required for acute cerebral ischemia/reperfusion injury in mice

TIR domain-containing adaptor protein (TRIF) is an adaptor protein in Toll-like receptor (TLR) signaling pathways. Activation of TRIF leads to the activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-κB). While studies have shown that TLRs are implicated in cerebral ischemia/reperfusion (I/R) injury and in neuroprotection against ischemia afforded by preconditioning, little is known about TRIF’s role in the pathological process following cerebral I/R. The present study investigated the role that TRIF may play in acute cerebral I/R injury. In a mouse model of cerebral I/R induced by transient middle cerebral artery occlusion, we examined the activation of NF-κB and IRF3 signaling in ischemic cerebral tissue using ELISA and Western blots. Neurological function and cerebral infarct size were also evaluated 24 h after cerebral I/R. NF-κB activity and phosphorylation of the inhibitor of kappa B (IκBα) increased in ischemic brains, but IRF3, inhibitor of κB kinase complex-ε (IKKε), and TANK-binding kinase1 (TBK1) were not activated after cerebral I/R in wild-type (WT) mice. Interestingly, TRIF deficit did not inhibit NF-κB activity or p-IκBα induced by cerebral I/R. Moreover, although cerebral I/R induced neurological and functional impairments and brain infarction in WT mice, the deficits were not improved and brain infarct size was not reduced in TRIF knockout mice compared to WT mice. Our results demonstrate that the TRIF-dependent signaling pathway is not required for the activation of NF-κB signaling and brain injury after acute cerebral I/R.     

腺苷在脑缺血过程中的双重作用

随着内源性腺苷系统具有神经保护作用的提出,派生出新的课题——腺苷及其类似物能否治疗脑卒中等一系列神经系统疾病?随着研究的深入,这一问题已逐渐成为神经药理学研究的热点。大量的工作集中在腺苷及其类似物对脑卒中的治疗作用,但实验结果具有很大的不确定性。传统上认为系统性给予腺苷引起心率减慢、血压降低、脑供血减少,从而限制了腺苷的应用。因此,提出了合用外周腺苷受体拮抗剂、腺苷转运蛋白抑制剂及代谢阻断剂,这既能对抗其心血管副作用,又使得脑缺血区的内源性腺苷维持在较高水平,发挥神经保护作用。然而,在脑缺血的病理条件下,腺苷浓度已显著提升,逾越了其自调节的范围。在此情况下,继续强化腺苷的作用,是否有悖于机体自稳态的恢复?诚然,腺苷具有明显的神经保护作用,但近年的研究又显示腺苷及其某些代谢产物具有神经损伤作用,如何解释这些相互矛盾的现象?又如何评价腺苷在脑缺血过程中的作用?本文主要从作用机制上,综合评述腺苷在脑缺血过程中可能发挥的神经保护及损伤作用,以期为脑卒中的临床治疗和新药开发提供一定的参考。     

血栓通注射液对暂时性和永久性大鼠脑缺血模型的神经保护作用

血栓通注射液(冻干)(XST)是一种从三七中提取的中草药标准化产品,广泛用于临床治疗急性脑梗塞等脑血管疾病。本研究评估了XST在不同大鼠脑缺血模型中的急性和延长保护作用,并探讨了其对过氧化物酶(Prx) 6-toll样受体(TLR) 4信号通路的影响。用XST处理抑制过氧化物酶(Prx) 6-toll样受体(TLR) 4的蛋白质表达和p38的磷酸化水平,并且上调STAT3的磷酸化水平。XST治疗3 d可显著降低暂时性大脑中动脉阻塞(MCAO)诱导的梗死体积和肿胀百分比,并调节白细胞介素-1β(IL-1β)、IL-17、IL-23p19、肿瘤坏死因子-α(TNFα)和诱导型一氧化氮合酶(iNOS)。在永久MCAO大鼠中,XST可以减少梗死体积和肿胀百分比。XST治疗还可以增加大鼠的体重并改善一批功能结果。XST可以保护暂时性和永久性MCAO大鼠的缺血性损伤可能与Prx6-TLR4途径有关。     

Effects of cervical-lymphatic blockade on brain edema and infarction volume in cerebral ischemic rats

To observe the effects of cervical-lymphatic blockade (CLB) on brain edema and infarction volume of ischemic (MCAO) rat, we examined changes in cerebral water content, Ca2+ and glutamate concentrations, cerebral infarction volume and mRNA expression levels of N-methyl-D-aspartame receptor 1 (NMDA receptor 1) in the ischemic (left) hemisphere. The present results demonstrated that all the above indices in rats with middle cerebral artery occlusion plus cervical lymphatic blockade (MCAO+CLB) were markedly higher than those with only middle cerebral artery occlusion (MCAO) at different time points. These results indicated [corrected] that CLB can aggravate cerebral ischemia by increasing brain edema and infarction volume.     

Characterization of neuronal cell death in normal and diabetic rats following exprimental focal cerebral ischemia.

We have studied the forms of cell death following ischemia/reperfusion, and the influence of diabetes mellitus (DM) as an additional factor. Based on the models of diabetes and middle cerebral artery occlusion (MCAO), characteristics of cell death after ischemia/reperfusion were evaluated synthetically by different methods: pathology, FCM, TUNEL and DNA agarose electrophoresis. The results showed that the occurrence of cerebral injury after ischemia/reperfusion was accompanied by cell necrosis and cell apoptosis. Cell apoptosis was mainly located in the ischemic penumbral (IP) zone around the densely ischemic focus. The ischemic core was characterized by cell necrosis. At the same time, the results showed that the process of ischemic cerebral injury worsened by DM was related to inducing cell apoptosis in IP and mid zone. In conclusion, there existed not only cell apoptosis but cell necrosis in brain damage following focal cerebral ischemia/reperfusion and showed a close, internal relationship between them. Brain damage following cerebral ischemia/reperfusion was worsened distinctly under diabetic conditions.     

The Intrinsic PEDF is Regulated by PPARγ in Permanent Focal Cerebral Ischemia of Rat

Inflammatory damage plays a pivotal role in cerebral ischemia and may represent a target for treatment. Pigment epithelium-derived factor (PEDF) is proven to possess neuroprotective property. But there is little known about the intrinsic PEDF after cerebral ischemia. This study evaluated the time course expression of the intrinsic PEDF and its underlying regulation mechanisms after cerebral ischemia. Male Sprague-Dawley rats were subjected to permanent middle cerebral artery occlusion. Telmisartan (PPARγ agonist) and GW9662 (PPARγ antagonist) were systemically administered to explore the effect on PPARγ, PEDF, NF-κB and MMP-9 expression at 24?h after cerebral ischemia by western blot and qRT-PCR. The neurological deficits, brain water content and infarct volume were measured. Compared with normal group, the expressions of PEDF and PPARγ decreased, and the expression of NF-κB and MMP-9 increased at early stage after ischemia (P?相似文献   

尿激酶介入动脉溶栓治疗26例急性脑梗死临床分析

目的:观察缺血性脑梗死急性期尿激酶介入动脉溶栓治疗的临床疗效。方法:应用尿激酶早期动脉内溶栓治疗急性脑梗死26例。溶栓前及溶栓后2 h、30 d进行神经功能缺损评分,同时观察并发症及疗效。结果:颈内动脉闭塞9例,大脑中动脉主干(M1)段闭塞6例,大脑前动脉闭塞4例,椎基底动脉系统闭塞3例。溶栓治疗后10例获得完全再通,9例部分再通,3例未开通。临床症状完全恢复及明显好转16例,占总病例61.5%。术后NIHSS评分较术前明显提高。并发颅内出血1例。再通后发生再闭塞1例。结论:尿激酶动脉溶栓治疗急性脑梗死安全、有效、可行,但其远期疗效还需进一步观察。     

Comparative study of change in extracellular ascorbic acid in different brain ischemia/reperfusion models with in vivo microdialysis combined with on-line electrochemical detection

Information on the change in extracellular ascorbic acid (AA) during the acute period of cerebral ischemia is of great importance in the early therapeutic intervention of the cerebral ischemic injury since AA is known to be involved into most kinds of neurochemical changes in the cerebral ischemia. This study describes a fast and efficient method through integration of in vivo microdialysis with on-line electrochemical detection for continuous monitoring cerebral AA, allowing comparative study of the change in the extracellular AA level in different brain ischemia/reperfusion models. The method exhibits a high specificity for AA measurements, bearing a good tolerance against the fluctuation in the brain anoxia and acidity induced by cerebral ischemia/reperfusion. In the global two-vessel occlusion (2-VO) ischemia model, the striatum AA did not change with statistic significance until 60 min after occlusion and was decreased to be 91+/-3% (n=5, P<0.05) of the basal level (8.05+/-0.23 microM) at the time point of 60 min after occlusion. In the 2-VO ischemia/reperfusion model, AA remained unchanged during the 10 min of ischemia, and was sharply increased to be 267+/-74% (n=5, P<0.05) of the basal level after the initial 15 min of reperfusion, and then decreased to be 122+/-33% (n=5, P<0.05) of the basal level after 50 min of reperfusion. Extracellular AA was largely increased after 5 min of left middle cerebral artery occlusion (LMCAO) and was then gradually increased to be 257+/-49% (n=5, P<0.05) of the basal level after 60 min of LMCAO ischemia. In the LMCAO ischemia/reperfusion model, AA was greatly increased during 10 min of ischemia and then gradually increased to be 309+/-69% (n=5, P<0.05) of the basal level after the consecutive 50 min of reperfusion. The results demonstrated here may be useful for understanding the neurochemical processes in the acute period of cerebral ischemia and could thus be important for neuroprotective therapeutics for cerebral ischemic injury.     

Delayed activin A administration attenuates tissue death after transient focal cerebral ischemia and is associated with decreased stress-responsive kinase activation

Focal cerebral ischemia and reperfusion initiates complex cellular and molecular interactions that lead to either cell repair or destruction. In earlier work, we found that activin A is an early gene response to cerebral ischemia and supports cortical neuron survival in vitro. In this study, the ability of exogenous activin A to attenuate injury from transient middle cerebral artery occlusion was tested in adult mice. Intracerebroventricular administration of activin A prior to middle cerebral artery occlusion reduced infarct volume apparent 1 day after experimental stroke. A single activin A administration at 6 h following ischemia/reperfusion reduced lesion volumes at 1 and 3 days and led to improved neurobehavior. Moreover, activin A treatment spared neurons within the ischemic hemisphere and led to a concomitant reduction in microglial activation. Activation of the stress-responsive kinases p38 and c- jun N-terminal kinase implicated in neuronal apoptosis after stroke was reduced following activin A treatment. Together these findings suggest that activin A promotes tissue survival after focal cerebral ischemia/reperfusion with an extended therapeutic window.     

The Neuroprotective Effects of Coccomyxa Gloeobotrydiformis on the Ischemic Stroke in a Rat Model

Stroke is a major cause of mortality and the leading cause of permanent disability. In this study, we adopted the classic middle cerebral artery occlusion(MCAO) stroke model to observe the therapeutic effects of coccomyxa gloeobotrydiformis(CGD) on ischemic stroke, and discuss the underlying mechanisms. Low dose (50 mg/kg.day) and high dose (100 mg/kg.day) concentrations of the drug CGD were intragastrically administrated separately for 8 weeks. Infarct volumes, neurologic deficits and degree of stroke-induced brain edema were measured 24 hours after reperfusion. Furthermore, oxidative stress related factors (SOD and MDA), mitochondrial membrane potential, and apoptosis regulatory factors (mitochondrial Cyt-C, Bcl-2, Bax, and caspase-3) were all investigated in this research. We found that CGD attenuated cerebral infarction, brain edema and neurologic deficits; CGD maintained the mitochondrial membrane potential and decreased mitochondrial swelling. It also prevented oxidative damage by reducing MDA and increasing SOD. In addition, CGD could effectively attenuate apoptosis by restoring the level of mitochondrial Cyt C and regulating the expression of Bcl-2, Bax and caspase 3. These results revealed that CGD has a therapeutic effect on ischemic stroke, possibly by inducing mitochondrial protection and anti-apoptotic mechanisms.     

电针和碱性成纤维生长因子(bFGF)对脑缺血时神经细胞的保护作用

采用暂时性脑缺血再灌注大鼠模型,及H＆E、TUNEL细胞染色等实验技术,观察电针或碱性成纤维生长因子,以及两者合用对缺血性神经细胞死亡的影响。实验结果表明,电针与碱性成纤维生长因子合用与单纯使用电针或碱性成纤维生长因子相比,可明显减少暂时性脑缺血再灌注后神经细胞坏死和凋亡。提示碱性成纤维生长因子与电针可具有互补或加强的神经保护作用。两者合用具有一定的临床实际价值。     

Caffeic acid phenethyl ester ameliorates cerebral infarction in rats subjected to focal cerebral ischemia

The effects of caffeic acid phenethyl ester (CAPE), an antioxidant derived from propolis, on the infarct volume elicited by focal cerebral ischemia were studied on Long-Evans rats. Cerebral infarction was induced by microsurgical procedures with ligation of the right middle cerebral artery (MCA) and clipping of bilateral common carotid arteries (CCA) for 60 min. The rats were sacrificed 24 h later and serial brain slices of 2 mm thickness were taken and stained for the measurement of infarct area. CAPE was administered intravenously 15 min before MCA occlusion. Pretreatment of CAPE (0.1, 1 and 10 microg/kg) significantly reduced the total infarct volume from 169.6 +/- 14.5 mm3 (control) to 61.0 +/- 24.1 mm3 (0.1 microg/kg CAPE), 47.4 +/- 9.1 mm3 (1 microg/kg CAPE), and 42.4 +/- 8.7 mm3 (10 microg/kg CAPE), respectively. Plasma nitric oxide (NO) content was significantly increased in rats subjected to focal cerebral ischemia. It is concluded that CAPE possesses neuroprotective properties in focal cerebral ischemia injury in rats possibly through its antioxidant effect and/or via the upregulation of NO production.