ERK1/2通路在缺血预处理大鼠大脑皮质的表达和作用

目的 探讨ERK1/2在大鼠脑缺血预处理中的表达及作用.方法 采用大鼠大脑中动脉缺血再灌注损伤模型,随机分为假预处理组( Sham)和预处理组(BIP),各组又分4个亚组,每亚组6只动物.其中3个亚组分别于预处理或假预处理后15min、2h、24h用Western blot检测大脑皮质ERK1/2的表达.另外1个亚组在预处理或假预处理后24h栓塞左大脑中动脉2h,24h后行神经功能缺损评分,TTC染色法测脑梗死体积.结果 预处理后15min、2h大脑皮质p-ERK1/2表达较强,与假预处理组相比有统计学意义(P＜0.05),预处理后24h大脑皮质p-ERK1/2表达回落,与假预处理组相比无统计学差别(P＞0.05).两组预处理或假预处理后15min、2h、24h大脑皮质总ERK1/2表达无变化,组间相比无统计学差别(P＞0.05);与假预处理组相比,预处理显著减少大脑中动脉栓塞导致的脑梗死体积(P＜0.05).预处理减少神经功能缺损.结论 脑缺血预处理可能通过增强大脑皮质ERK1/2活性而保护皮质,减少脑梗死体积及神经功能缺损.     

Apelin-13对鼠脑缺血-再灌注后的作用及机制

目的探讨apelin对鼠脑缺血-再灌注后的保护作用及机制。方法改良线栓法制备CD-1小鼠脑缺血-再灌注模型。实验1:实验动物随机分为假手术(Sham)组、溶剂对照(Vehicle)组、缺血-再灌注(I/R)组、apelin-13小剂量(APLN-L)组、apelin-13中剂量(APLN-M)组及apelin-13大剂量(APLN-H)组;实验2:实验动物随机分为Sham组、Vehicle组、APLN-M组、APLN-M+PD98059(APLN+PD)组、PD98059+I/R(PD)组。再灌注后23h时,实验1检测各组神经功能评分、脑梗死体积、脑水肿、细胞凋亡及细胞外调节蛋白激酶(ERK1/2)的表达;实验2检测各组ERK1/2、Bax、Bcl-2、caspase-3的表达及cleaved caspase-3的活性。结果实验1:1APLN-H组神经功能评分明显低于Vehicle组(P<0.05);2梗死体积APLN-L、-M、-H组较Vehicle组减小;3脑组织含水量APLN-H、-M组明显低于Vehicle组(P<0.05);4TUNEL阳性细胞数APLN-H、-M组明显低于Vehicle组(P<0.05);5APLN-L、-M、-H组Bax、caspase-3、cleaved caspase-3表达明显低于Vehicle组(P<0.05);Bcl-2表达明显高于Vehicle组(P<0.05);6APLN-H、-M、-L组cleaved caspase-3活性明显低于Vehicle组(P<0.05);7 APLN-L、-M、-H组p-ERK1/2蛋白表达明显高于Vehicle组(P<0.05)。实验2:1APLN+PD组Bax、caspase-3、cleaved caspase-3表达明显高于APLN-M组,Bcl-2表达明显低于APLN-M组(P<0.05);2APLN+PD组cleaved caspase-3活性明显高于APLN-M组(P<0.05)。结论 Apelin-13对脑缺血-再灌注具有神经保护作用;ERK1/2信号通路参与了apelin-13的抗凋亡作用机制。     

二烯丙基硫醚对大鼠脑缺血再灌注损伤后Nrf2、NQ01表达的影响

目的 探讨二烯丙基硫醚对大鼠局灶性脑缺血再灌注损伤后Nrf2、NQ01表达的影响.方法 实验动物随机分为假手术组、缺血再灌注组、200mg/kg二烯丙基硫醚预处理组.采用线栓法制备大鼠大脑中动脉缺血再灌注模型,缺血2h再灌注24h后进行神经行为学评分,测定脑梗死体积及脑组织中SOD、MDA活性,采用免疫荧光和Western Blot测定Nrf2、NQ01蛋白分子的表达.结果 与缺血再灌注组相比,大鼠经二烯丙基硫醚预处理后神经损害症状减轻,脑梗死体积缩小,SOD活性增强,同时MDA活性受到抑制,Nrf2、NQ01I蛋白分子表达上调.结论 二烯丙基硫醚对大鼠脑缺血再灌注损伤具有一定的神经保护作用,可能与其增强大鼠脑组织抗氧化酶活性和激活Nrf2/NQ01通路有关.     

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。     

桃仁红花煎剂对大鼠缺血再灌注后脑组织的保护作用

目的研究桃仁红花煎剂对大鼠局灶性脑缺血再灌注后脑组织的影响。方法 45只雄性SD大鼠随机平均为给药组、模型组和对照组。采用线栓法阻塞大鼠右侧大脑中动脉,使其缺血2h后再灌注24h建立局灶性脑缺血再灌注模型。术前2h和术后3、12h分3次灌胃给予桃仁红花煎剂,总剂量是40g/kg。通过神经行为评分评定大鼠神经功能变化,按干湿重法测定脑含水量,用氯化三苯基四氮唑法测定脑梗死范围,分光光度法测定缺血区脑组织中Na+-K+-ATP酶和Ca2+-ATP酶的活性。结果在缺血再灌注3h和24h后,给药组神经行为评分明显高于模型组(P<0.05)。缺血再灌注24h,给药组脑含水量和脑梗死体积明显少于模型组(P<0.05);给药组缺血脑皮层中Na+-K+-ATP酶和Ca2+-ATP酶活性明显高于模型组(P<0.05)。结论桃仁红花煎剂对大鼠缺血再灌注后脑组织有保护作用,其机制可能与其增强Na+-K+-ATP酶和Ca2+-ATP酶的活性、减轻脑水肿有关。     

丁苯酞预处理对大鼠脑缺血再灌注损伤的神经保护作用

目的研究丁苯酞预处理对大鼠局灶性脑缺血再灌注损伤的神经保护作用。方法健康成年SD雄性大鼠48只,随机分为假手术组、缺血再灌注组、丁苯酞预处理组,每组各16只。各组均灌胃5d后,采用线栓法制作大鼠局灶性脑缺血再灌注(MCAO)模型,缺血2h、再灌注24h,进行神经功能缺损评分,TTC染色及图像分析观察脑梗死体积,免疫组化法检测脑组织caspase-3、bcl-2表达的变化。结果与缺血再灌注组相比,丁苯酞预处理组神经缺损程度改善,梗死灶体积减少,caspase-3阳性细胞数量减少,bcl-2表达上调。结论丁苯酞可减轻缺血性脑血管病的发作,具有一定的神经保护作用。     

丁苯酞注射液预处理通过PI3K/Akt通路对大鼠脑缺血再灌注损伤的神经保护作用

目的研究丁苯酞预处理对大鼠脑缺血再灌注损伤的神经保护作用,并初步探讨PI3K/Akt信号通路在该过程中的作用。方法健康成年SD雄性大鼠随机原则分为假手术组、缺血再灌注组、丁苯酞预处理组(丁苯酞氯化钠注射液预处理后脑缺血再灌注组)。缺血2h再灌注24h后对各组大鼠进行神经功能缺损评分,并分别行TTC染色观察脑梗死体积,HE染色观察大鼠脑组织的病理形态,免疫组织化学检测方法观察caspase-3、p-Akt表达的变化。结果与假手术组相比缺血再灌注组有明显神经功能缺损,出现脑组织梗死,梗死区细胞受损,caspase-3、p-Akt阳性细胞表达增加;丁苯酞预处理组与缺血再灌注组相比神经功能缺损程度减轻,梗死灶体积减小,梗死区细胞损伤减轻,caspase-3阳性细胞表达减少,而p-Akt阳性细胞表达增加。结论丁苯酞预处理可以通过上调PI3K/Akt信号通路中p-Akt的表达,降低caspase-3的表达而起到神经保护作用。     

Nogo-A/NgR通路参与缺血预处理对局灶性脑缺血的神经保护作用

目的研究Nogo-A/NgR通路参与缺血预处理(IPC)对局灶性脑缺血的神经保护作用。方法雄性成年SD大鼠随机分为:对照组(C)、缺血预处理组(IPC)、Tat-NEP1-40组和Tat-β-Gal组。大脑中动脉栓塞(MCAO)10min作为IPC,IPC组在IPC后72h建立MCAO致局灶性脑缺血模型。再灌注24h后,对所有动物行神经功能缺损评分(NDS),并处死大鼠取脑,应用2%TTC染色测定梗死容积和免疫组化研究Nogo-A和NgR表达变化。结果与C组比较,IPC组显著改善局灶性脑缺血再灌注后24h大鼠的NDS[2(1.5~3)和1(0~2)](P<0.01)、减少脑梗死容积[(309.65±54.61)mm3和(65.09±26.06)mm3](P<0.01),NgR拮抗剂NEP1-40可部分逆转IPC的神经保护作用(P<0.01),而其溶剂β-Gal对IPC的神经保护作用无明显影响。免疫组化显示Nogo-A和NgR阳性细胞主要位于MCAO后导致的缺血区域,缺血后24h大鼠缺血区域Nogo-A和NgR表达明显增强,IPC可抑制缺血引起的Nogo-A和NgR表达增强。结论Nogo-A/NgR通路可能参与IPC诱导的脑缺血耐受,保护脑缺血性损伤。     

山楂总黄酮对实验性脑缺血再灌注损伤大鼠的保护作用

目的 研究山楂总黄酮（HFs）对实验性脑缺血再灌注损伤大鼠的保护作用.方法 SD雄性大鼠90只随机分为5组,各组按规定预防给药15 d后,按照Longa法制作大脑中动脉闭塞模型,假手术组除不插入栓线外其余相同,缺血2 h后再灌注.分别于再灌注3 h、24 h进行神经行为评分;再灌注24 h后断头取梗死侧大脑皮质匀浆,测定丙二醛（MDA）、一氧化氮（NO）含量及超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、一氧化氮合成酶（NOS）、诱导型一氧化氮合成酶（iNO）活性.结果 山楂总黄酮各剂量组再灌注3 h、24 h神经行为评分明显低于缺血再灌注组;与I/R组比较,再灌注24 h后,山楂总黄酮各剂量组缺血梗死侧大脑皮质丙二醛（MDA）、一氧化氮（NO）含量明显降低,超氧化物歧化酶（SOD）、过氧化氢酶（CAT）活性升高,一氧化氮合成酶（NOS）、诱导型一氧化氮合成酶（iNO）活性也明显低于I/R组,各组间差异有统计学意义.结论 山楂总黄酮对实验性脑缺血再灌注损伤大鼠有一定的保护作用,其作用机制可能与提高脑组织中SOD和CAT活性、抑制脂质过氧化及炎症反应有关.     

癫痫幼鼠海马丝裂酶原蛋白活化激酶表达的特点及其对巨噬细胞炎性蛋白-α/趋化因子受体5表达的影响

目的探讨癫痫幼鼠海马丝裂酶原蛋白活化激酶(MAPKs)表达的特点及其对巨噬细胞炎性蛋白-α(MIP-1α)/趋化因子受体5(CCR5)表达的影响。方法应用立体定向技术对侧脑室内注射海人酸(KA),建立幼鼠惊厥模型。将出生21 d的Wistar幼鼠分为空白对照组、磷酸盐缓冲液(PBS)对照组及KA 4h、8 h、16 h、24 h、3 d组,比较各组MAPKs表达的差异。另将出生21 d的幼鼠分为PBS+二甲亚砜(DMSO)组、KA+DMSO组、KA+PD98059(ERK1/2抑制剂)组和KA+SB203508(p38MAPK抑制剂)组,比较各组MIP-1α、CCR5表达的差异。采用Western Blot方法检测MAPKs蛋白水平及CCR5的表达。采用ELISA方法检测MIP-1α的表达。采用免疫组化染色方法检测各组大鼠海马P-ERK1/2、P-p38MAPKs等蛋白的表达。采用免疫荧光双标染色探讨P-ERK1/2和P-p38MAPK的胶质细胞来源。结果与空白对照组比较,KA 4 h、8h、16 h、24 h、3 d组P-ERK1/2水平明显增高;KA 8 h、16 h、24 h、3 d组P-P38MAPK水平明显增高(均P0.05)。侧脑室注射KA后大鼠P-ERK1/2与P-P38MAPK表达主要分布在齿状回门区和锥体细胞层等神经元损伤明显的海马组织中。在侧脑室注射KA后,部分P-ERK1/2来源于活化的小胶质细胞及星形胶质细胞,而P-p38MAPK仅在小胶质细胞中出现免疫活性表达。与PBS+DMSO组比较,KA+DMSO组、KA+PD98059组和KA+SB203508组大鼠海马组织中MIP-1α、CCR5蛋白水平均明显增高,OX-42阳性细胞数明显增加(均P0.05)。与KA+DMSO组比较,KA+PD98059组、KA+SB203508组大鼠海马组织中MIP-1α、CCR5蛋白水平均明显降低,OX-42阳性细胞数明显减少(均P0.05)。结论在幼鼠癫痫发生的早期阶段,MAPKs(ERK1/2、p38MAPK)可部分地调控MIP-1α/CCR5的表达。     

In vivo and in vitro characterization of a novel neuroprotective strategy for stroke: ischemic postconditioning.

As clinical trials of pharmacological neuroprotective strategies in stroke have been disappointing, attention has turned to the brain's own endogenous strategies for neuroprotection. Recently, a hypothesis has been offered that modified reperfusion subsequent to a prolonged ischemic episode may also confer ischemic neuroprotection, a phenomenon termed 'postconditioning'. Here we characterize both in vivo and in vitro models of postconditioning in the brain and offer data suggesting a biological mechanism for protection. Postconditioning treatment reduced infarct volume by up to 50% in vivo and by approximately 30% in vitro. A duration of 10 mins of postconditioning ischemia after 10 mins of reperfusion produced the most effective postconditioning condition both in vivo and in vitro. The degree of neuroprotection after postconditioning was equivalent to that observed in models of ischemic preconditioning. However, subjecting the brain to both preconditioning as well as postconditioning did not cause greater protection than each treatment alone. The prosurvival protein kinases extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), and Akt show prolonged phosphorylation in the cortex of postconditioned rats. Neuroprotection after postconditioning was inhibited only in the presence of LY294002, which blocks Akt activation, but not U0126 or SB203580, which block ERK and P38 MAP kinase activity. In contrast, preconditioning-induced protection was blocked by LY294002, U0126, and SB203580. Our data suggest that postconditioning may represent a novel neuroprotective approach for focal ischemia/reperfusion, and one that is mediated, at least in part, by the activation of the protein kinase Akt.     

缺血后处理对局灶脑缺血再灌注大鼠Toll样受体2表达的影响

目的 探讨缺血后处理对大鼠局灶性脑缺血再灌注时Toll样受体2(TLR2)表达的影响。方法 成年雄性SD大鼠90只,分为假手术组、缺血再灌注组、缺血后处理组各30只; 用线栓法建立局灶性大脑中动脉闭塞模型(MCAO),随机分为假手术组(sham)、缺血再灌注组(I/R)、缺血后处理组(IPC),分别于再灌注24、48、72 h后留取大脑皮质组织; 采用Longa的等级评分法进行神经行为学评分,免疫组化和蛋白质印记(Western blot)法检测TLR2蛋白的表达水平,逆转录-聚合酶链反应(RT-PCR)检测TLR2 mRNA表达水平。结果(1)缺血后处理组大鼠神经行为学评分明显改善;(2)缺血再灌注组TLR2蛋白在再灌注24、48、72 h表达水平明显升高(P<0.05); 缺血后处理组TLR2蛋白表达在各时间点均减少(P<0.05); TLR2 mRNA的表达趋势与蛋白表达基本一致。结论 缺血后处理可以降低TLR2表达水平,这可能是其脑保护作用的部分机制之一。     

Neuroprotection induced in vitro by ischemic preconditioning and postconditioning: modulation of apoptosis and PI3K-Akt pathways

Preconditioning and postconditioning are mild ischemic exposures before or after severe injurious ischemia, respectively, that elicit endogenous neuroprotective responses. Molecular mechanisms of neuroprotection through preconditioning and postconditioning are not completely understood. Here we optimized the in vitro oxygen and glucose deprivation (OGD) models of preconditioning and postconditioning in primary cortical neuron cultures that allow the studies of the corresponding molecular mechanisms of neuroprotection. We found that the cortical cells preconditioned with a single 45-min OGD treatment administered 24 h prior to injurious 2 h OGD were robustly protected after both 3 h and 16 h of reperfusion. For the postconditioning treatment, we found that three cycles of 15 min OGD followed by 15 min reperfusion, applied immediately after injurious 2 h OGD and prior to complete reperfusion, resulted in effective neuroprotection at both 3 h and 16 h of reperfusion. Using real-time RT–PCR arrays focused on genes of the apoptosis and PI3K–Akt pathways, we found that injurious OGD mainly induced apoptosis-related and repressed PI3K–Akt pathway-related genes after either 3 h or 16 h of reperfusion. Preconditioning treatment resulted in the activation of both pro-survival and anti-apoptotic pathways after 3 h of reperfusion and mainly anti-apoptotic pathway after 16 h of reperfusion. In contrast, the activation of PI3K–Akt pathway mainly contributed to the neuroprotective effect by the postconditioning treatment after 3 h of reperfusion, but differential gene expression likely contributed minimally, if at all, to the neuroprotection observed after 16 h of reperfusion. Among the novel markers of neuroprotection, Nol3 gene upregulation was observed after 3 h of reperfusion following either preconditioning or postconditioning treatments and after 16 h of reperfusion following preconditioning treatment.     

无创伤双后肢缺血后处理对移植胰腺缺血再灌注损伤的远隔保护

背景：缺血预处理及缺血后处理是近年来提出减轻缺血再灌注损伤有效方法。 目的：探讨无创伤双后肢缺血后处理对移植胰腺缺血再灌注损伤的影响及机制。 方法：18只糖尿病SD大鼠数字表法随机分为3组,对照组仅行开腹术;缺血再灌注组仅行胰腺移植;缺血后处理组,移植前行非创伤性双后肢缺血后处理。 结果与结论：缺血再灌注组血糖和胰腺组织中丙二醛水平均高于缺血后处理组(P < 0.01)、而超氧化物歧化酶活性低于缺血后处理组(P < 0.01);与缺血后处理组比较,缺血再灌注组胰腺组织凋亡指数明显增高(P < 0.01)。结果提示,无创伤双后肢缺血后处理对大鼠移植胰的缺血再灌注损伤具有保护作用,机制可能与可通过减少超氧化物歧化酶失活,从而清除氧自由基以及减少胰腺细胞凋亡等有关。     

MAPK信号转导通路中ERK、JNK和P38在大鼠肝脏缺血再灌注和缺血后处理中表达的变化

背景：近年来,随着器官移植的不断开展和深入研究,人们逐渐认识到供肝原发性无功能是引起肝移植患者早期死亡的主要原因,而缺血再灌注损伤是导致供肝功能不良的重要因素。如何减轻或消除缺血再灌注损伤一直是临床研究的热点。 目的：观察肝缺血再灌注损伤和缺血后处理后MAPK级联通路中P38,JNK和ERK活化的情况。 设计、时间及地点：随机对照动物实验,于2007-05/10在中南大学湘雅医院动物实验中心完成。 材料：102只Wistar大鼠随机分为假手术组6只,缺血30 min再灌注组48只和缺血后处理组48只,后2组又分为0,0.5,1,2,4,8,12,24 h不同时间处理亚组,每亚组6只。 方法：建立大鼠肝脏体内局部缺血再灌注-缺血后处理模型,于复灌后0,0.5,1,2,4,8,12,24 h取肝脏组织。 主要观察指标：应用免疫组织化学的方法对磷酸化的p-P38、p-JNK和p-ERK进行免疫组化检测并作半定量分析。 结果：①p-ERK,p-JNK在缺血后即有轻度增高,但在再灌注后30 min开始增高明显,持续到再灌注后4 h,高峰出现在再灌注后2 h。缺血后处理组p-ERK,p-JNK的表达在再灌注后1,2,4 h较缺血再灌注组增高(P < 0.05)。②p-P38在缺血后即有轻度地增高,但在再灌注后30 min开始增高明显,高峰出现在再灌注后1 h,2 h后开始下降,表达程度维持在缺血后水平。与缺血再灌注组相比,缺血后处理组p-P38的表达增高,但仅在再灌注后1 h明显增高(P < 0.05)。 结论：缺血后处理可通过增高ERK和P38的磷酸化水平,降低JNK的磷酸化水平减轻缺血再灌注导致的肝脏损伤。     

Electroacupuncture Alleviated Neuronal Apoptosis Following Ischemic Stroke in Rats via Midkine and ERK/JNK/p38 Signaling Pathway

This study aimed to evaluate the effects of electroacupuncture (EA) intervention administered at rats of middle cerebral artery occlusion (MCAO)/reperfusion. Fifty-four male Sprague-Dawley rats were divided into three groups, consisting of sham group, MCAO/R group, and EA group. EA treatment at Quchi and Zusanli acupoints was applied in rats of EA group at 24 h after MCAO once per day for 3 days. Our results indicated that EA treatment reduced infarct volumes and neurological deficits, as well alleviated the apoptotic cells in peri-infarct cortex, indicating that EA exerted neuroprotective effect in cerebral ischemic rats. Moreover, EA treatment may effectively reverse the upregulation of caspase-3 and Bim and alleviate the inhibition of Bcl-2 following 72-h ischemic stroke. EA may significantly reverse the promoted relative density level of p-ERK1/2, p-JNK, and p-p38 in the EA group compared with the MCAO/R group. In addition, the growth factor midkine (MK) was upregulated at 72 h after MCAO/R, and EA treatment may significantly prompt expression of MK. Our study demonstrated that EA exerted neuroprotective effect against neuronal apoptosis and the mechanism might involve in upregulation of MK and mediation of ERK/JNK/p38 signal pathway.     

Neuroprotective effect of postischemic administration of sodium orthovanadate in rats with transient middle cerebral artery occlusion.

Orthovanadate is a competitive inhibitor of protein tyrosine phosphatases. Some of its reported biologic effects are its insulin mimetic property and its activation of phosphoinositide 3-kinase and extracellular-signal regulated kinase (ERK). The authors previously reported its neuroprotective effect on delayed neuronal death of gerbil hippocampal CA1 neurons via Akt and ERK activation after transient forebrain ischemia. In the present study, the neuroprotective effect of postischemic intraperitoneal administration of sodium orthovanadate (2 l/kg of 50-mmol/l sodium orthovanadate in saline) was investigated in rats with transient middle cerebral artery occlusion. Ischemic neuronal injury was evaluated 1 day and 28 days after ischemia. The neuroprotective effect of orthovanadate was significant in the cortex but not the caudate putamen (ischemic core) at both 1 and 28 days after ischemia. In orthovanadate group, the activities of Akt and ERK were maintained after reperfusion; they were decreased in saline group. Blood glucose level decreased but within normal range. Regional cerebral blood flow was lower than that of saline group only at 0 hours after reperfusion. These data suggest that orthovanadate has neuroprotective effects in rats with transient middle cerebral artery occlusion and that these effects are mediated by Akt and ERK activation. Furthermore, low blood glucose levels and gradual recovery of regional cerebral blood flow may contribute to neuroprotection.     

Multiple modes of action of tacrolimus (FK506) for neuroprotective action on ischemic damage after transient focal cerebral ischemia in rats

While the immunosuppressant tacrolimus (FK506) is known to be neuroprotective following cerebral ischemia, the mechanisms underlying its neuroprotective properties are not fully understood. To determine the mode of action by which tacrolimus ameliorates neurodegeneration after transient focal ischemia, we therefore evaluated the effect of tacrolimus on DNA damage, release of cytochrome c, activation of microglia and infiltration of neutrophils following a 60-min occlusion of the middle cerebral artery (MCA) in rats. In this model, cortical brain damage gradually expanded until 24 h after reperfusion, whereas brain damage in the caudate putamen was fully developed within 5 h. Tacrolimus (1 mg/kg) administered immediately after MCA occlusion significantly reduced ischemic damage in the cerebral cortex, but not in the caudate putamen. Tacrolimus decreased both apoptotic and necrotic cell death at 24 h and reduced the number of cytochrome c immunoreactive cells at 8 h after reperfusion in the ischemic penumbra in the cerebral cortex. In contrast, tacrolimus did not show significant neuroprotection for necrotic cell death and reduction of cytochrome c immunoreactive cells in the caudate putamen. Tacrolimus also significantly decreased microglial activation at 8 h and inflammatory markers (cytokine-induced neutrophil chemoattractant and myeloperoxidase [MPO] activity) at 24 h after reperfusion in the ischemic cortex but not in the caudate putamen. These results collectively suggest that tacrolimus ameliorates the gradually expanded brain damage by inhibiting both apoptotic and necrotic cell death, as well as suppressing inflammatory reactions.     

Protective effect of delayed remote limb ischemic postconditioning: role of mitochondrial K(ATP) channels in a rat model of focal cerebral ischemic reperfusion injury

Delayed remote ischemic postconditioning (DRIPost) has been shown to protect the rat brain from ischemic injury. However, extremely short therapeutic time windows hinder its translational use and the mechanism of action remains elusive. Because opening of the mitochondria K(ATP) channel is crucial for cell apoptosis, we hypothesized that the neuroprotective effect of DRIPost may be associated with K(ATP) channels. In the present study, the neuroprotective effects of DRIPost were investigated using adult male Sprague-Dawley rats. Rats were exposed to 90 minutes of middle cerebral artery occlusion followed by 72 hours of reperfusion. Delayed remote ischemic postconditioning was performed with three cycles of bilateral femoral artery occlusion/reperfusion for 5 minutes at 3 or 6 hours after reperfusion. Neurologic deficit scores and infarct volumes were assessed, and cellular apoptosis was monitored by terminal deoxynucleotidyl transferase nick-end labeling. Our results showed that DRIPost applied at 6 hours after reperfusion exerted neuroprotective effects. The K(ATP) opener, diazoxide, protected rat brains from ischemic injury, while the K(ATP) blocker, 5-hydroxydecanote, reversed the neuroprotective effects of DRIPost. These findings indicate that DRIPost reduces focal cerebral ischemic injury and that the neuroprotective effects of DRIPost may be achieved through opening of K(ATP) channels.