黄芪注射液对大鼠脑缺血再灌注损伤的保护作用研究

目的 探讨黄芪注射液对大鼠脑缺血/再灌注损伤的保护作用和机制.方法 应用线栓法经左侧颈外-颈内动脉插线建立大鼠大脑中动脉阻塞再灌注（MCAO/R）模型,经腹腔注射黄芪注射液（3 ml/kg）干预治疗.Longa法评价大鼠神经行为功能,氯化三苯基四氮唑（TTC）染色观察脑梗死体积,苏木精-伊红（HE）染色观察海马神经元形态结构,TUNEL法检测细胞凋亡,免疫组化检测c-jun氨基末端激酶（JNK3）蛋白表达水平.结果 经黄芪注射液治疗后大鼠海马神经元JNK3蛋白表达水平显著降低,凋亡细胞数量显著减少,神经元形态恢复,脑梗死体积缩小,大鼠神经行为功能显著改善.结论 黄芪注射液可通过下调JNK3表达水平而抑制细胞凋亡,缩小脑梗死体积,改善大鼠神经行为功能.     

缺血后处理对大鼠脑缺血再灌注损伤神经保护作用的研究

目的探讨大鼠脑缺血后处理对缺血再灌注损伤后神经元的保护作用。方法健康雄性SD大鼠30只,随机分为假手术组(SO组)、缺血再灌注对照组(MCAO组)、缺血后处理组(IPOC组)3组。采用线栓法制备大鼠MCAO模型及IPOC模型,分别用TTC染色法计算脑梗死体积、流式细胞术和ELISA法观察,对于大鼠缺血半暗带神经细胞凋亡率及血清神经元特异性烯醇化酶(NSE)含量的影响。结果 (1)大鼠脑缺血再灌注后24h,IPOC组较MCAO组梗死体积明显减小(P<0.05);(2)MCAO组大鼠脑缺血再灌注24h细胞凋亡发生率及血清中NSE的含量较SO组显著增加(P<0.01);(3)IPOC组神经元凋亡发生率及血清NSE较MCAO组显著降低(P<0.05或0.01)。结论大鼠脑缺血后处理对缺血再灌注神经元损伤有保护作用。     

大鼠局灶性脑缺血再灌注后LPA1的表达对神经元凋亡的影响

目的 探讨大鼠局灶性脑缺血再灌注后溶血磷脂酸受体1(LPA1)的表达对神经元凋亡的影响及其可能机制。方法 将24只SPF级SD雄性大鼠随机分为4组,每组各6只,分别为假手术组(A组)、大脑中动脉栓塞(MCAO)组(B组)、MCAO+溶剂组(C组)、MCAO+LPA1拮抗剂(Ki16425)组(D组); 4组均于手术后48 h取标本; 利用HE染色观察大鼠脑组织细胞形态的变化; 四氮唑红(TTC)染色观察大鼠脑梗死面积; 免疫荧光技术检测LPA1在大鼠皮层半暗带神经元表达水平; 免疫印迹、免疫组化技术检测大鼠脑组织中Caspase-3蛋白及p-Akt蛋白表达水平。结果 与A组比较,B组有明显的缺血再灌注损伤,表现为细胞肿胀,细胞溶解坏死,大鼠皮层半暗带神经元的LPA1表达水平较高; 与C组比较,D组大鼠脑梗死面积显著增大(P<0.05),缺血半暗带细胞肿胀更加明显,胞浆空泡区增大,细胞核固缩更加严重,细胞间隙增宽更明显; D组较C组缺血半暗带Caspase-3蛋白表达水平明显升高(P<0.05),而p-Akt蛋白表达水平明显降低(P<0.05)。结论 抑制大鼠局灶性脑缺血再灌后LPA1的表达可使大鼠脑梗死面积增大,细胞凋亡增加,同时p-Akt蛋白表达减少,这说明在大鼠局灶性缺血再灌注过程中LPA1对神经元具有保护作用,其机制可能是通过Akt途径来发挥保护作用的。     

缺血后处理对大鼠脑缺血/再灌注损伤内质网应激通路相关分子的影响

目的探讨缺血后处理对大鼠局灶性脑缺血/再灌注损伤的保护作用及与内质网应激通路相关分子GRP78、caspase-12的关系。方法成年雄性Wistar大鼠58只,随机分为假手术组(sham组)、缺血/再灌注组(I/R组)和缺血后处理组(IP组),采用线栓法阻断大脑中动脉制备大鼠局灶性脑缺血/再灌注(MCAO)模型。大鼠脑缺血/再灌注后24 h进行神经行为学评分和脑梗死体积测定;脑缺血/再灌注6 h、12 h、24 h后免疫组织化学方法检测脑缺血侧半暗带区GRP78、caspase-12蛋白的表达。结果与缺血/再灌注组相比,后处理组再灌注24h神经行为学评分明显降低,脑梗死体积明显减少(P<0.05);后处理组再灌注12 h、24 h GRP78蛋白表达明显增加,再灌注24 h caspase-12蛋白表达明显减少。结论脑缺血后处理可能通过减弱内质网应激过程从而对随后发生的再灌注损伤起到了神经保护作用。其机制可能是增加GRP78蛋白表达、减少caspase-12蛋白表达而减轻神经细胞的凋亡。     

ERK和CREB磷酸化在硫氢化钠对大鼠脑缺血再灌注神经保护的机制探讨

目的探讨磷酸化细胞外调节蛋白激酶(p-ERK)和磷酸化cAMP反应元件结合蛋白(p-CREB)在外源性硫化氢(NaHS)干预下对大鼠脑缺血再灌注的神经保护作用及机制。方法将180只雄性SD大鼠随机分为3组:假手术组、模型组和NaHS干预组(100μmol/L),每组60只。线栓法制备大鼠大脑中动脉栓塞模型,2h后再灌注,灌注前20min给予生理盐水或NaHS腹腔注射,术后6h、1d、2d、5d、7d后行TTC染色观察脑梗死体积,尼氏染色检测神经元表达,WesternBlot检测海马p-ERK1/2、p-CREB表达,免疫组化检测Bcl-2表达。结果干预组脑梗死体积和神经元凋亡相对于模型组显著减少(P0.05)。与模型组相比,干预组各时间点p-ERK1/2、p-CREB蛋白表达均升高(P0.05),两者之间呈正相关;干预组Bcl-2相对于模型组升高(P0.05)。结论大鼠脑缺血再灌注损伤后NaHS可能通过诱导ERK1/2和CREB蛋白磷酸化,激活下游Bcl-2蛋白表达,起到抗凋亡作用,减少脑梗死体积,发挥神经保护作用。     

缺血后处理抑制神经元凋亡减轻大鼠脑缺血再灌注损伤

目的研究缺血后处理(Ipost C)对脑缺血再灌注损伤的神经保护作用及机制。方法通过手术前后局部脑血流多普勒检测,神经行为学测试和脑梗死面积评估等方法建立SD大鼠局灶性脑缺血再灌注模型,在大脑中动脉闭塞100 min后立即予Ipost C干预。使用改良神经损害严重程度评分和转棒实验评估大鼠神经功能损伤程度,TTC染色测量脑梗死面积。Western blot分析凋亡相关蛋白表达(Bax、细胞色素C),透射电镜观察线粒体损伤和TUNEL评估缺血周边皮质的凋亡。结果相对于脑缺血再灌注,Ipost C能显著减少脑梗死面积,降低神经功能受损,改善脑缺血再灌注后的局部脑血流量。同时,Ipost C能减少线粒体的Bax转位及细胞色素C的释放,减轻线粒体损伤及缺血侧大脑皮质神经元的凋亡。结论 Ipost C抑制神经元凋亡,减轻脑缺血再灌注损伤,有可能成为急性缺血性脑卒中临床治疗中的新靶点。     

缺血后处理通过水通道蛋白-4对大鼠缺血再灌注损伤的脑保护作用及其机制

目的研究缺血后处理(IP)对大鼠脑缺血再灌注损伤的保护作用,探讨各组大鼠水通道蛋白-4(AQP4)的表达。方法实验分组:48只雄性SD大鼠,随机分为3组(n=16)。假手术组;对照组:行单纯缺血再灌注;缺血后处理组:IP组。采用线栓法阻断大脑中动脉制备大鼠局灶性脑缺血再灌注模型。大鼠脑缺血再灌注后24 h进行神经行为学评分和脑梗死体积测定,干湿重法测定脑水含量。并行免疫组织化学方法检测海马CA_1区细胞凋亡及AQP4表达的变化并行统计学分析。结果与对照组比较,IP组神经行为学评分明显降低,脑梗死体积、脑组织中的水分含量明显减少(P 0. 05)。再灌注24 h后,对照组海马CA_1区AQP4、凋亡细胞24 h表达量明显增加,IP组与对照组之间比较有差异(P 0. 05)。结论 IP组脑神经行为学评分、脑含水量、脑梗死体积均明显降低,提示缺血后处理可通过抑制AQP4的表达减轻缺血再灌注损伤后的脑水肿,起到脑保护作用。     

莱菔硫烷对大鼠局灶性脑缺血再灌注损伤的保护作用及机制研究

目的 探讨莱菔硫烷对大鼠局灶性脑缺血再灌注损伤的保护作用及机制.方法 采用线栓法制备大鼠大脑中动脉阻断局灶性脑缺血模型,分别于MCAO后1h腹腔注射莱菔硫烷2.5mg/kg、5mg/kg、10mg/kg.于缺血2h再灌注24h时进行神经行为缺损评分,TTC染色评价脑梗死体积,测定脑组织中超氧化物歧化酶(SOD)活力和丙二醛(MDA)含量.免疫荧光组织化学染色法检测黄核蛋白NQ01和脂质过氧化酶Prx6的表达.结果 莱菔硫烷给药组与对照组相比均能改善大鼠脑缺血再灌注后神经行为缺损评分,减少脑梗死体积.其中5mg/kg组能显著改善大鼠脑缺血再灌注后神经行为缺损评分,减少脑梗死体积,增强SOD活性,降低MDA含量.免疫荧光组织化学染色法提示NQ01和Prx6的表达明显增强.结论 莱菔硫烷对大鼠局灶性脑缺血再灌注损伤有神经保护作用,其机制可能与上调内源性抗氧化蛋白NQ01和Prx6的表达有关.     

iASPP在大鼠脑缺血再灌注中的表达及其意义

目的 观察p53凋亡刺激蛋白（apoptosis stimulating protein of p53,ASPP）家族的抑制因子（inhibitorymember of the ASPP family,iASPP）在大鼠脑缺血再灌注后的表达及其意义。方法 48只清洁级、成年健康雄性SD大鼠随机分为假手术组（8只）和脑缺血再灌注组（40只）。脑缺血再灌注组又分为缺血2h再灌注4h、12h、24h、48h和72h 5个时间点,其中每个时间点8只大鼠。苏木素-伊红染色测定脑梗死体积;原位末端转移酶标记技术检测半暗带细胞凋亡情况;免疫印迹法测定半暗带iASPP的表达变化。结果 脑缺血再灌注4h组和24h组的凋亡细胞阳性率与假手术组存在统计学差异（P <0.01）。脑缺血再灌注组的脑梗死体积百分比与假手术组相比具有统计学意义（P <0.05）。与假手术组相比,在脑缺血再灌注12h时iASPP表达开始下降（P <0.01）,再灌注24h降至最低（P <0.01）,再灌注48h开始回升（P <0.01）。结论 在脑缺血再灌注后,缺血半暗带凋亡细胞显著增多,iASPP表达下降,提示在脑缺血再灌注中iASPP的表达下降可能在细胞凋亡的发生中发挥重要作用,其表达上调可能具有神经保护作用。     

缝隙连接蛋白-43阻断剂辛醇对小鼠脑缺血再灌注损伤的神经保护作用

目的观察小鼠脑缺血再灌注后缝隙连接蛋白-43(Cx43)表达变化及缝隙蛋白阻断剂辛醇对小鼠梗死体积的影响。方法采用线栓法制备小鼠大脑中动脉阻塞模型(MCAO模型),应用免疫印迹(Western blot)方法观察脑缺血再灌注损伤前后Cx43表达变化和辛醇对Cx43表达的影响,并计算各组死亡率及脑梗死体积。结果小鼠脑缺血2h再灌注损伤24h条件下,缺血区Cx43表达在损伤前后无明显变化(P>0.05)。辛醇能显著抑制脑缺血再灌注损伤后Cx43表达,降低死亡率及减少脑梗死体积,具有统计学差异(P<0.05)。结论由Cx43组成的缝隙连接蛋白在脑缺血再灌注损伤过程中具有重要作用,缝隙蛋白-43阻断剂辛醇通过抑制海马及皮质Cx43表达,进而降低死亡率及梗死体积,从而发挥神经保护作用。     

Exendin-4, a glucagon-like peptide-1 receptor agonist, provides neuroprotection in mice transient focal cerebral ischemia

Glucagon-like peptide-1 (GLP-1) is an incretin hormone known to stimulate glucose-dependent insulin secretion. The GLP-1 receptor agonist, exendin-4, has similar properties to GLP-1 and is currently in clinical use for type 2 diabetes mellitus. As GLP-1 and exendin-4 confer cardioprotection after myocardial infarction, this study was designed to assess the neuroprotective effects of exendin-4 against cerebral ischemia–reperfusion injury. Mice received a transvenous injection of exendin-4, after a 60-minute focal cerebral ischemia. Exendin-4-treated vehicle and sham groups were evaluated for infarct volume, neurologic deficit score, various physiologic parameters, and immunohistochemical analyses at several time points after ischemia. Exendin-4 treatment significantly reduced infarct volume and improved functional deficit. It also significantly suppressed oxidative stress, inflammatory response, and cell death after reperfusion. Furthermore, intracellular cyclic AMP (cAMP) levels were slightly higher in the exendin-4 group than in the vehicle group. No serial changes were noted in insulin and glucose levels in both groups. This study suggested that exendin-4 provides neuroprotection against ischemic injury and that this action is probably mediated through increased intracellular cAMP levels. Exendin-4 is potentially useful in the treatment of acute ischemic stroke.     

神经节苷脂对大鼠脑缺血再灌注损伤的脑保护作用

目的探讨神经节苷脂对大鼠脑缺血再灌注损伤的脑保护作用。方法采用线栓法制作缺血再灌注大鼠模型,分别用神经节苷脂(治疗组)和生理盐水(对照组)腹腔注射。观察两组大鼠缺血90min、缺血90min再灌注24h的脑梗死面积、神经功能缺损程度、细胞凋亡数、细胞凋亡率。结果治疗组大鼠于相同时间点脑梗死面积较对照组明显减小,仅表现轻度的神经功能缺损,且神经细胞的凋亡数较对照组显著减少(均P<0.01)。结论神经节苷脂能明显减小大鼠实验性脑缺血的脑梗死面积,减轻脑缺血再灌注后神经功能缺损程度,显著减轻缺血区神经元损害,具有显著的脑保护作用。     

Neuroprotective effects of total saponins from Rubus parvifolius L. on cerebral ischemia/ reperfusion injury in rats

This study examines the neuroprotective effects and mechanisms of action of total saponins from Rubus parvifolius L. (TSRP) on focal cerebral ischemia and reperfusion injury in rats. Focal cerebral ischemia and reperfusion injury was performed in rats using the suture method. The results indicate that intragastric injection of TSRP, at 5, 10 and 20 mg/kg, could decrease neurological impairment, reduce cerebral infarct volume, diminish pathological changes, and significantly inhibit the apoptosis of neurons surrounding the ischemic area. In addition, TSRP upregulated the expression of the anti-apoptotic factor Bcl-2, at the protein and mRNA levels, and it downregulated the expression of the pro-apoptotic factor Bax, at the protein and mRNA levels. These findings indicate that TSRP protects against cerebral ischemia/reperfusion injury, and that it may do so by regulating the expression of Bcl-2 and Bax.     

颈交感神经干离断对局灶性脑缺血再灌注后大鼠海马iNOS表达的影响

目的采用颈交感干离断（TCST）模拟星状神经节阻滞，观察其对局灶性脑缺血再灌注损伤（CIRI）大鼠脑梗死容积及海马诱导型一氧化氮合酶（iNOS）表达等的影响，并探讨其脑保护作用的机制。方法将大鼠随机分成实验组（A组）、对照组（B组）和假手术组（C组）；采用线栓法行大脑中动脉栓塞（MCAO）制作大鼠局灶性CIRI模型，A组于TCST后即行MCAO，2h后再恢复灌注；B组为单纯CIRI组；C组仅完成与A组相似的手术步骤但不造成MCAO、不行TCST；再灌注24h后观察各组大鼠神经行为学评分、脑梗死容积及海马iNOs的表达变化。结果A组大鼠脑梗死容积和神经行为学评分均低于B组（P〈0．05）；与A组、C组相比，B组大鼠海马iNOS的表达增加（P〈0．05），而A组与C组间无显著差异（P〉0．05）。结论TCST可通过下调大鼠海马iNOS的表达而对局灶性CIRI发挥脑保护作用。     

褪黑素对SD大鼠脑缺血再灌注损伤后c-fos表达的影响

目的探讨褪黑素在大鼠脑缺血再灌注损伤中的神经保护作用及可能机制。方法选取45只雄性SD大鼠,分为假手术组(5只)、脑缺血再灌注组(20只)、褪黑素干预组(20只);脑缺血再灌注组和褪黑素干预组根据时间点第6小时、第1天、第3天、第7天分为4个组,每组5只。采用Longa线栓法建立大鼠左侧大脑中动脉栓塞(MCAO)模型,采用HE染色检测脑组织的病理改变,TUNEL染色检测神经细胞的凋亡,免疫组织化学(免疫组化)法及蛋白质印迹法(Western Blotting)观察大鼠脑组织内c-fos表达情况。结果在脑缺血再灌注组的各时间点的HE染色显示,胶质细胞呈现程度不一的增生,神经元出现坏死;褪黑素干预能减轻脑缺血再灌注后胶质细胞增生及神经元的坏死。在TUNEL染色凋亡检测中,脑缺血再灌注组各时间点的神经细胞凋亡升高;褪黑素干预组各时间点的细胞凋亡数低于脑缺血再灌注组(P <0.05)。在免疫组化及蛋白质印迹检测中,脑缺血再灌注组c-fos表达增加,在第1天时达到高峰,之后表达逐步降低;在褪黑素干预组,c-fos表达趋势与缺血再灌注组一致,但表达水平比缺血再灌注组相应时间点低,差异有统计学意义(P <0.05)。结论褪黑素能够减轻脑缺血再灌注后神经元的损伤,降低c-fos的表达,表明褪黑素可能通过调控c-fos的表达在脑缺血再灌注中发挥神经保护作用。     

动员自体骨髓干细胞对大鼠脑缺血／再灌注损伤及细胞凋亡的影响

目的 探讨应用G-CSF动员自体骨髓干细胞对大鼠脑缺血／再灌注损伤及细胞凋亡的影响。方法 应用线栓法制备大鼠局灶性大脑中动脉栓塞／再灌注(MCAO／R)模型，应用粒细胞集落刺激因子(G-GSF)刺激自体骨髓干细胞分裂增殖，并用5-溴脱氧尿核苷(Brdu)标记。观察大鼠神经病学评分，HE染色和免疫组化检测脑缺血区病理改变及CD34和Brdu阳性细胞，原位末端标记法(TUNEL法)观察细胞凋亡。结果 模型动员组大鼠脑缺血／再灌注后24h，大量炎症细胞浸润。再灌注后48h，缺血区可见CD34和Brdu阳性细胞；72h后CD34阳性细胞消失，而Brdu阳性细胞持续存在；模型未动员组缺血区无CD34和很少Brdu阳性细胞表达。48h缺血区新生毛细血管密度明显高于对照组。再灌注后24h细胞凋亡显著，1周时达高峰；与模型非动员组比较，模型动员组48h后细胞凋亡改善明显。结论 自体骨髓干细胞经G-CSF动员后可向大鼠脑缺血区趋化并可分化为神经元前体细胞，显著促进脑缺血区血管再生，降低脑神经功能评分，降低细胞凋亡率。     

Lipoxin A4 Inhibits 5-Lipoxygenase Translocation and Leukotrienes Biosynthesis to Exert a Neuroprotective Effect in Cerebral Ischemia/Reperfusion Injury

Lipoxin A(4) (LXA(4)), a biologically active eicosanoid with anti-inflammatory and pro-resolution properties, was recently found to have neuroprotective effects in brain ischemia. As 5-lipoxygenase (5-LOX) and leukotrienes are generally considered to aggravate cerebral ischemia/reperfusion (I/R) injury, we investigated their effects on LXA(4)-mediated neuroprotection by studying middle cerebral artery occlusion (MCAO)/reperfusion in rats and oxygen-glucose deprivation (OGD)/recovery in neonatal rat astrocyte primary cultures. LXA(4) effectively reduced infarct volumes and brain edema, and improved neurological scores in the MCAO/reperfusion experiments; this effect was partially blocked by butoxycarbonyl-Phe-Leu-Phe-Leu-Phe (Boc2), a specific antagonist of the LXA(4) receptor (ALXR). Total 5-LOX expression did not change, regardless of treatment, but LXA(4) could inhibit nuclear translocation induced by MCAO or OGD. We also found that LXA(4) inhibits the upregulation of both leukotriene B(4) (LTB(4)) and leukotriene C(4) (LTC(4)) and the phosphorylation of extracellular signal-regulated kinase (ERK) induced by MCAO or OGD. The phosphorylation of the 38-kDa protein kinase (p38) and c-Jun N-terminal kinase (JNK) was not altered throughout the experiment. These results suggest that the neuroprotective effects of LXA(4) are probably achieved by anti-inflammatory mechanisms that are partly mediated by ALXR and through an ERK signal transduction pathway.     

厄贝沙坦对大鼠局灶性脑缺血再灌注后炎症反应的影响

目的观察厄贝沙坦对大鼠局灶性脑缺血再灌注后脑内及外周炎症反应的影响。方法采用改良Longa方法制备大鼠大脑中动脉阻塞(middle cerebralartery occlusion,MCAO)模型,于缺血90min再灌注后24h和72h进行梗死体积的测量,采用免疫组化和ELISA方法测量脑内和外周血的粘附分子。结果厄贝沙坦可以显著减少局灶性脑缺血再灌注后24h和72h的梗死体积(均P<0.01),改善神经功能(均P<0.01);降低脑内ICAM-1、VCAM-1的表达及其外周血浆中可溶性的形式sICAM-1、sVCAM-1蛋白的水平(均P<0.05)。结论厄贝沙坦可以降低粘附分子的表达,减少梗死体积,改善神经功能,对脑缺血再灌注起保护作用。     

Ziprasidone attenuates brain injury after focal cerebral ischemia induced by middle cerebral artery occlusion in rats

Ziprasidone is an atypical antipsychotic drug used for the treatment of schizophrenia. Recent studies have reported that atypical antipsychotics have neuroprotective effects against brain injury. In the present study, the effect of ziprasidone on ischemic brain injury was investigated. Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in rats. All the animals experienced ischemia for 1h and then underwent reperfusion. The infarct size induced by MCAO was significantly reduced in the animals that received acute treatment with 5mg/kg ziprasidone and subchronic treatment with 2.5mg/kg ziprasidone for 7 days compared with that in the vehicle-treated animals. The acute treatment with ziprasidone significantly improved neurological functions, as measured by the modified neurological severity score, in a dose-dependent manner. The subchronic treatment produced more rapid recovery from functional deficits than the vehicle treatment. The immunohistochemical investigation revealed that the subchronic treatment prevented severe loss of neuronal marker intensity and attenuated the increased in microglial marker intensity in the infarcted cortical area. These results suggest that ziprasidone has neuroprotective effects in a rat model of ischemic stroke and provide new insight for its clinical applications.